1 Rheumatology A and INSERM U1016, Paris Descartes University, Cochin
Hospital, Paris, France
2 Inventiva, Daix, France
3 INSERM UMR_S 999, Le Plessis Robinson, France
4 Université Paris Descartes, Montrouge, France
5 INSERM U1016, Paris, France
Background: The pan-PPAR agonist IVA337 recently demonstrated efficacy
in experimental skin fibrosis.
Objectives: To evaluate the antifibrotic effects of IVA337 in
preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension
(PH).
Methods: IVA337 has been evaluated in the mouse model of
bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being
characterized by non-specific interstitial pneumonia and severe vascular remodeling
of pulmonary arteries leading to PH. Mice received 2 doses of IVA337 (30 mg/kg or
100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.
Results: Both doses of IVA337 were well tolerated. IVA337 demonstrated
at a dose of 100 mg/kg a marked protection from the development of lung fibrosis
induced by bleomycin compared to mice receiving 30 mg/kg of IVA337 or vehicle.
Indeed, IVA337 (100 mg/kg) reduced by 61% and 28% tissue density on histological
measurements and total lung hydroxyproline concentrations, respectively, as compared
to vehicle.
Similarly, Fra-2 transgenic mice treated with 100 mg/kg of IVA337 displayed reduced
lung density (20% vs. vehicle) and significant increase of functional residual
capacity (30% vs. vehicle) when assessed by chest micro-CT imaging. These results
were emphasized by a 50% reduction of the Ashcroft fibrosis score and by a 48%
reduction of hydroxyproline concentrations upon IVA337 (100 mg/kg) compared to
vehicle treated mice.
Successful targeting of the TGF-b signaling axis was observed in both mouse models
upon treatment with 100 mg/kg of IVA337 and concomitantly reduced T cell and B cell
infiltration was observed. Regarding vessel remodeling and related PH, treatment
with 100 mg/kg of IVA337 led to a substantial attenuation of right ventricular
systolic pressure and right ventricular hypertrophy compared to mice receiving the
vehicle, it also markedly reduced medial wall thickness and the number of
muscularized distal pulmonary arteries. In vitro in primary human lung fibroblasts,
IVA337 inhibited TGFβ-mediated fibroblasts to myofibroblasts transition and
PDGF-mediated proliferation.
Conclusions: We demonstrate that 100 mg/kg IVA337 prevents lung fibrosis
in two complementary animal models and substantially attenuates PH in the Fra-2
mouse model. The pan-PPAR agonist IVA337 is an appealing therapeutic candidate for
SSc, both, for skin and key complications.
2. CHANGES IN GENE EXPRESSION IN PATIENTS WITH SYSTEMIC SCLEROSIS TREATED WITH
MYCOPHENOLATE MOFETIL (MMF) PLUS BELIMUMAB OR MMF PLUS PLACEBO
1 Department of Molecular and Systems Biology, Geisel School of Medicine
at Dartmouth, Hanover, NH, USA
2 Department of Rheumatology, Hospital for Special Surgery, New York, NY,
USA
Background: Belimumab (Benlysta™) is a monoclonal antibody that inhibits
B cell survival and that is approved for the treatment of systemic lupus
erythematosus. We investigated gene expression changes in the context of a pilot
trial assessing safety and efficacy of treatment with belimumab in patients with
early diffuse cutaneous systemic sclerosis (dcSSc) on background mycophenolate
mofetil (MMF) therapy.
Methods: We analyzed gene expression in skin biopsies from a 52-week,
investigator-initiated, single-center, double-blind, placebo-controlled pilot study
(NCT01670565). 20 patients that had recently started on MMF were randomized 1:1 to
receive monthly belimumab or placebo, 10 mg/kg intravenously. We compared treatment
groups in terms of differential gene expression, pathway enrichment, molecular
intrinsic subsets (inflammatory, proliferative and normal-like) as well as safety
and efficacy. Clinical improvers were defined as patients with ≥20% decrease in
modified Rodnan skin score (MRSS) post-treatment.
Results: MRSS decreased in both groups as follows (median [interquartile
range]): in the belimumab group, from 27 [26.5, 31] to 18 [11, 23], p = 0.039; in
the placebo group, from 28 [22, 28] to 21 [14, 25], p = 0.023. In the belimumab
group, 7/9 were clinical improvers compared to 3/9 in the placebo group (p = 0.153).
Clinical improvers on belimumab were characterized by significantly decreased
expression of genes (e.g. IL6, CCL2, THBS1 and TNFRS-F12A) and pathways (e.g.
extracellular matrix organization, TGFB signaling, B cell receptor signaling and
NFKB signaling) relevant to SSc pathogenesis. The majority of patients classified as
either inflammatory or proliferative at baseline displayed a decrease in MRSS and
became normal-like post-treatment. The improvement in MRSS correlated significantly
with decrease in the inflammatory signature in patients assigned to the inflammatory
subset at baseline (r = 0.81, p = 0.008).
Conclusions: Patients in both treatment groups experienced significant
improvements in MRSS. Significant gene expression changes were observed only in
improvers in the belimumab group. They were consistent with mechanism of action and
show that clinical response to belimumab and improvement of skin fibrosis is
associated with significant decrease in B cell signaling and profibrotic genes and
pathways. Intrinsic subset analyses suggest that movement towards the normal-like
subset coincides with a trajectory of MRSS improvement.
3. SIMULTANEOUS HIGH THROUGHPUT RNA-SEQ PROFILING OF SKIN AND DERMAL FAT IN
SSC
2 Washington University School of Medicine, Department of Internal
Medicine, Division of Rheumatology, St. Louis, USA
3 Washington University School of Medicine, Department of Genetics, St.
Louis, USA
Background: Systemic sclerosis (SSc) is a complex and highly
heterogeneous disease with multi-organ involvement. Tools for accurate disease
sub-classification and validated biomarkers are still lacking. Skin and subcutaneous
tissue are affected, and circulating immune cells are activated. We performed
RNA-Seq analyses on simultaneously obtained skin and peripheral blood mononuclear
cells (PBMCs) from patients with well-characterized SSc.
Methods: We obtained PBMCs and skin biopsies from healthy controls and
individuals with limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous
systemic sclerosis (dcSSc), scleroderma sine scleroderma (SSS) and a Very Early
Diagnosis of Systemic Sclerosis (VEDOSS). When available, intradermal adipose tissue
was isolated. Patients were followed longitudinally, and PBMCs and skin were
obtained serially. We then performed stranded RNA-Seq for transcriptome
profiling.
Results: In skin the most significant increases in gene expression were
in COMP, COL4A4, and IGFBP4. Genes increased in
SSc skin are significantly enriched for promoter AP-2 alpha transcription factor
site. Differentially-expressed functional categories were enriched for extracellular
matrix and immune system processes. Remarkably, several genes involved in
consumption of NAD+ (PARP1, PARP9, PARP12, PARP14, and
SIRT2) were increased in SSc, while NAD+ biosynthetic genes
were not elevated. In contrast, we detected a marked decreased in
SPAG17, part of the ciliary axoneme central pair complex, in
SSc skin. Decreased genes were significantly enriched for POU4F1 binding sites, and
modestly enriched for selenocysteine synthesis. PBMC transcriptomes showed only a
relatively modest pattern of altered gene expression.
Conclusions: In SSc patients, skin offers the optimum view of disease
activity with ~ order of magnitude more genes showing differential expression
compared to PBMCs. However, the presence of DE genes in the blood gives hope for the
possibility for developing blood biomarkers of disease activity. Current studies are
actively assessing differential splice junction usage to identify genes that switch
isoforms, but do not exhibit gene level differential expression. The changes in
adipose tissue are of high interest.
Acknowledgments: JV, BK, MC - SPARC; EDOR - SPARC, RDCC; DM-H -
Rheumatology training grant.
4. EFFICACY OF M10, A CASPACE CLEAVAGE PRODUCT OF THE HGF RECEPTOR, IN
BMP9-INDUCED FIBROSIS IN SCLERODERMA LUNG FIBROBLASTS
1 Division of Rheumatology and Immunology, Department of Medicine, Medical
University of South Carolina, Charleston, South Carolina, USA
2 Division of Rheumatology, Endocrinology and Nephrology, Hokkaido
University Graduate School of Medicine, Sapporo, Japan
Background: We recently identified M10, a caspase cleavage product of
the HGF receptor MET, as an anti-fibrotic peptide that interacts with Smad2 and
inhibits TGFβ-induced Smad2 phosphorylation. In several fibrotic diseases, including
systemic sclerosis (SSc), not only Smad2/3 but also Smad1/5 plays an important role
in the regulation of fibrosis. Among the TGFβ superfamily members, TGFβ1 activates
Smad2/3 signaling mainly through the type I receptor ALK5, whereas bone
morphogenetic proteins (BMPs) transduce Smad1/5/8 signaling mainly through
ALK1/2/3/6 receptors. BMP9, the most recently discovered member of the BMP family,
has been identified as a pro-fibrotic factor in mouse embryo fibroblasts. The aims
of this study are to investigate the role of BMP9 in human fibroblasts and to
further explore the anti-fibrotic mechanisms of the M10 peptide.
Methods: Fibroblasts were derived from lung tissues obtained at autopsy
from SSc patients and from age- race-, and sex-matched normal subjects. MRC5 human
fetal lung fibroblasts were purchased from Sigma. Potential peptide-protein
interactions were modeled in-silico using PepSite. Smad
phosphorylation, type I collagen, and smooth muscle α-actin (α-SMA) expression were
determined by immunoblotting and RT-PCR.
Results: Using computational modeling from PepSite we found a
statistically significant (p<0.0001) interaction of M10 with the
BMP9/ALK1/ActRIIB complex. The most probable binding sites were located at β-turn
motifs in BMP9. We demonstrate that when recombinant BMP9 is added to medium,
phosphorylation of Smad1/5/8 is rapidly induced in MRC5 and SSc lung fibroblasts.
This is accompanied by an increase in expression of type I collagen in MRC5 cells
and SSc lung fibroblasts as well as in normal lung fibroblasts treated with BMP9.
The expression of α-SMA is increased in normal lung fibroblasts after BMP9 treatment
in a dose-dependent manner. Notably, the anti-fibrotic peptide M10 was shown to
downregulate BMP9-induced type I collagen and to reduce BMP9-upregulated α-SMA
expression and Smad1/5/8 phosphorylation in lung fibroblasts.
Conclusions: BMP9 stimulates collagen production and Smad1/5/8
phosphorylation in human fetal lung fibroblasts and in adult lung fibroblasts
obtained from SSc patients and matched normal subjects. M10 peptide binds to BMP9
and inhibits BMP9's pro-fibrotic effects through the Smad1/5/8 signaling
pathway.
Acknowledgments: Funded by NIH/NIAMS P60 AR062755, Scleroderma
Foundation and SC SmartState®.
1 Department of Dermatology, University of Tokyo Graduate School of
Medicine, Tokyo, Japan
2 Department of Dermatology, Graduate School of Medical Sciences,
International University of Health and Welfare, Tokyo, Japan
3 Arthritis Center, Rheumatology, Boston University School of Medicine,
Boston, USA
Background: Systemic sclerosis (SSc) is a multisystem autoimmune disease
characterized by vascular injury and fibrosis development. In SSc lesional skin,
transcription factor Friend leukemia virus integration 1 (Fli1) is constitutively
down-regulated in various cell types, and, in part, by the epigenetic mechanism in
dermal fibroblasts, and Fli1 deficiency is deeply related to the pathogenesis of
SSc. Although Fli1 deficiency in dermal fibroblasts, dermal microvascular
endothelial cells, and epithelial cells induces an SSc-like phenotype in each cell
type, the impact of Fli1 deficiency in immune cells on the pathogenesis of SSc
remains to be elucidated. On the other hand, recent clinical trial of tocilizumab,
an anti-IL-6 receptor antibody, identified M2 macrophages as a major driver of this
disease. Based on these findings, we focused our interests on myeloid cells and
evaluated the impact of Fli1 deficiency in myeloid cells on the pathogenesis of
SSc.
Methods and Results: We generated myeloid cell-specific Fli1 knockout
(Fli1 MyeKO) mice by crossing
Fli1flox/flox mice with LysM-Cre
mice
(Fli1flox/flox;LysM-Cre+/-)
and compared their skin features with control littermates
(Fli1flox/flox;Lys-Cre-/-).
In in vivo Matrigel plug assay, a well-established method to investigate postnatal
vasculogenesis, Fli1 MyeKO mice exhibited the differentiation of
F4/80-positive monocytic lineage cells into pericytes poorly expressing a-smooth
muscle actin, followed by the formation of enlarged and immature blood vessels. In
addition, mesenchymal stem cells, a potential source of mural cells in postnatal
vasculogenesis, isolated from Fli1 MyeKO mice manifested
de-differentiated phenotype. Under a physiological condition, disintegrity of dermal
vasculature, which is characterized by dilated capillaries and arteriolar stenosis,
and loss of capillaries became evident at the age of 2 months and further
exacerbated along with aging in Fli1 MyeKO mice. Notably,
endothelial-to-mesenchymal transition occurred in parallel with microangiopathy,
resulting in the development of dermal fibrosis.
Conclusions: Myeloid Fli1 deficiency induces destabilization and
impaired remodeling of vasculature and endothelial-to-mesenchymal transition due to
dysregulated postnatal vasculogenesis, leading to the development of vasculopathy
and dermal fibrosis, which are reminiscent of SSc.
6. AUTOANTIGEN-REACTIVE B CELLS CONTRIBUTE TO PROGRESSION OF SYSTEMIC
SCLEROSIS THROUGH B CELL AFFINITY MATURATION-DEPENDENT IL-6 AND IL-10
PRODUCTION
1 Department of Dermatology, The University of Tokyo Graduate School of
Medicine, Tokyo, Japan
2 Department of Applied Chemistry, The University of Tokyo Graduate School
of Engineering, Tokyo, Japan
Purpose: Autoantigen-reactive B cells have the ability to produce
inflammatory cytokines independently of an antibody-producing function, which
results in the development of autoimmune diseases. Indeed, Rituximab, B cell
depleting antibody, can ameliorate several autoimmune diseases, such as rheumatoid
arthritis, systemic lupus erythematosus, vasculitis, and systemic sclerosis (SSc).
However, the role of autoantigen-reactive B cells is still not clear, because the
number of autoantigen-reactive B cells is too small to study their functions
directly. Although several studies have revealed that B cells play crucial roles in
SSc development, how autoantigen-reactive B cells contribute to disease progression
of SSc remains totally unclear. In this study, we investigated the role of
autoantigen-reactive B cells directly using our original microfluidic-enzyme linked
immunosorbent assay (ELISA) system, which integrates immunoassay into a microchip in
order to detect extremely small amounts of analytes and can study single
autoreactive B cells.
Methods: After topoisomerase I (topo I)-specific B cells were purified
from SSc patients, their antibody affinity and cytokine production were assessed by
the micro-ELISA system. We also studied autoantigen-reactive B cell function using
topo I and Freund's adjuvant-induced SSc model mice, which were previously
established by us. The affinity against topo-I protein and cytokine production
ability of topo I-specific B cells were assessed by the micro-ELISA system. In
addition, to directly analyze topo I-specific B cell function, we adoptively
transferred topo I-specific B cells to topo I and Freund's adjuvant-induced SSc
model mice.
Results: The micro-ELISA system revealed that high affinity topo
I-specific B cells obtained from SSc patients produced a higher amount of IL-6
compared with low affinity B cells. In addition, IL-10 production of high affinity B
cells was lower than those of low affinity B cells. Similar results were obtained in
SSc model mice. In addition, adoptively transferred low affinity topo I-specific B
cells ameliorate disease progression of SSc model mice, though high affinity topo
I-specific B cells increased disease activity.
Conclusions: These results suggest that distinct B cell cytokine
production is determined by B cell autoantigen affinity. Autoreactive B cells with
higher autoantigen affinity could be a novel therapeutic target in SSc.
ADDITIONAL ABSTRACTS LISTED ALPHABETICALLY
MICROVASCULAR FLOW ASSESSED BY DYNAMIC OPTICAL COHERENCE TOMOGRAPHY: FIRST
NON-INVASIVE QUANTITATIVE OUTCOME MEASURE OF MICROVASCULAR DISEASE IN SYSTEMIC
SCLEROSIS
Abignano G1-3, Daniel A1,4, Green L1,2, Eng
S1,2, Del Galdo F1,2
1 Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of
Leeds, Leeds, UK
2 NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching
Hospitals NHS Trust, Leeds, UK
3 Rheumatology Department of Lucania, Rheumatology Institute of Lucania
(IReL), San Carlo Hospital of Potenza and Madonna delle Grazie Hospital of Matera,
Potenza, Italy
4 Rheumatology Unit, Hospitals of the University of Coimbra, Coimbra,
Portugal
Background: Virtual skin biopsy by Optical Coherence Tomography (OCT)
has been proposed as quantitative outcome measure of fibrosis in Systemic Sclerosis
(SSc). Dynamic OCT (D-OCT) is a newly developed technology that allows
quantification of blood flow in vivo during OCT scans.
Objectives: Here we aimed to determine the validity of skin D-OCT as
outcome measure of the skin microvascular disease, employing as comparator nailfold
video-capillaroscopy (NVC) capillary patterns in SSc patients, and as clinical gold
standard the presence of digital ulcers (DU).
Methods: 104 subjects were enrolled in this study in two independent
cohorts. In cohort 1 (Criterion Validity), 40 SSc patients fulfilling the ACR/EULAR
2013 criteria with different NVC pattern (10 for each normal/non- specific, early,
active, ad late NVC pattern) and 10 healthy volunteers (HV), underwent nailfold
D-OCT. In cohort 2 (Face/Content Validity) 36 SSc patients with (18) or without (18)
DU and 18 patients with Raynaud's phenomenon (RP) and SSc specific ANA, who did not
fulfill ACR/EULAR 2013 criteria (SRP group) underwent D-OCT of index and middle
fingers on the proximal phalanx. Microvascular flow (MVF) was analyzed using the
proprietary software from Michelson Diagnostics Ltd.
Results: In cohort 1 nailfold D-OCT visualized the corresponding SSc
patterns seen at NVC. Within these images MVF measurements were significantly
different between HV and SSc patients with any specific capillary pattern (0.16 ±
0.02 vs 0.10 ± 0.01, p = 0.0028) and between SSc patients without and with specific
capillary pattern (0.14 ± 0.01 vs 0.10 ± 0.01, p = 0.02). Further, MVF was
significantly lower in patients displaying capillary loss as main feature compared
with those with remarkable angiogenesis (p = 0.03).
In Cohort 2 the median MVF was significantly different among the 3 groups: 0.134 (DU)
vs 0.153 (no DU) vs 0.167 (SRP) (p<0.0001), and in the DU vs no DU (p<0.001)
or vs SRP (p<0.001). Further, DU patients on Sildenafil (n = 6) had a
significantly higher MVF than DU patients on no Sildenafil (p<0.01).
Conclusions: MVF assessed by D-OCT shows face/content and criterion
validity as biomarker of skin microvascular disease in SSc. Future longitudinal
studies are needed to evaluate its sensitivity to change over time.
USE OF PATTERNED COLLAGEN COATED SLIDES TO STUDY NORMAL AND SCLERODERMA LUNG
AND SKIN FIBROBLAST MIGRATION
Ahmed Abdi B1, Lopez L2, Karrar S1, Renzoni
E3, Wells A3, Tam A1, Etomi O1,
Hsuan JJ4, Martin GR2, Shiwen X1, Denton
CP1, Abraham D1, Stratton R1
1 Centre for Rheumatology and Connective Tissue Disease, Royal Free
Hospital Campus, University, College Medical School, London, UK
2 MuriGenics, Inc., Vallejo, CA, USA
3 Imperial College London, Royal Brompton Campus, London, UK
4 Institute for Liver and Digestive Health, Royal Free Hospital Campus,
University College Medical School, London, UK
Introduction: Systemic sclerosis (SSc) is a spreading fibrotic disease
affecting the skin and internal organs, most notably the lungs. In this study, we
aimed to model pathogenic fibroblast migration in SSc by using novel patterned
collagen substrates to identify enhancing factors, measure the effect of migrating
cells on underlying extracellular matrix (ECM) and test therapeutic inhibitors. A
possible autocrine effect of stem cell factor (SCF), in maintaining SSc fibroblast
migration was explored.
Methods: Woven and aligned pattern collagen substrates were used to
investigate alignment and migration of skin and lung fibroblasts from SSc patients
and healthy controls. Proteomic analysis as well as phosphorylation arrays were used
to determine differences in ECM protein modification and phosphorylation events
between SSc and HC. Phenotypic differences were confirmed by Western blotting and
qPCR. Cellular subpopulations in lung fibroblasts were analysed by flow
cytometry.
Results: Normal lung but not skin fibroblasts consistently elongated and
aligned with aligned but not woven collagen and migrated dependent on PDGF or serum.
SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly
but were less restricted to alignment of the collagen. Multiple collagen proline and
lysine-modifying enzymes were identified in SSc but not control fibroblast ECM
preparations, indicating differential levels of modification by the diseased cells.
Phosphorylation profiling of migrating cells revealed a SCF/c-Kit paracrine
mechanism contributing to migration via a subpopulation of c-Kit positive cells.
Heparin and imatininib which target PDGF, SCF, and c-Kit, both inhibited lung
fibroblast migration individually, showing synergy in SSc cells. Pathologic lung
fibroblasts from SSc patients modify ECM during migration, remain growth factor
dependent and are sensitive to inhibitors.
Conclusions: The use of collagen substrate slides has enabled profiling
of cellular populations undergoing migration. Lung fibroblasts were found to be
dependent on growth factors including PDGF and SCF for migratory potential, which
was blocked by existing drug inhibitors more effective in combination. These data
also indicate autocrine function for SCF/c-Kit in scleroderma fibroblasts acting to
maintain migratory potential, which could be targeted by specific therapies.
PHARMACOKINETIC AND PHARMACODYNAMIC STUDIES OF “M10” - A NEWLY DISCOVERED
ANTI-FIBROTIC PEPTIDE
1 Division of Rheumatology and Immunology, Department of Medicine, Medical
University of South Carolina, Charleston, South Carolina, USA
2 Division of Rheumatology, Endocrinology and Nephrology, Hokkaido
University Graduate School of Medicine, Sapporo, Japan
Background: The pathogenesis of scleroderma-associated interstitial lung
disease (SSc-ILD) remains poorly understood and lung fibrosis often leads to
respiratory failure and death. The molecular mechanisms underlying the pathogenesis
of lung fibrosis are not well understood, and there is a great need for more
effective treatments for this potentially fatal complication. We recently identified
a small fragment of the c-Met receptor, designated as “M10”, as a peptide with
strong anti-fibrotic properties. The current study was undertaken to study the
pharmacokinetics and pharmacodynamics of M10 in mice with bleomycin-induced
pulmonary fibrosis.
Methods: Pulmonary fibrosis was induced in male C57BL6 mice by
intra-tracheal instillation of bleomycin (2.0 U/kg). M10 was administrated by
intraperitoneal injection in a dose of 1mg/kg of mouse weight. Blood was collected
at different time points (15 min, 30 min, 1hr, 2hr, 4hr, 24hr and 48hr) after M10
injection in the presence of 6% (v/v) 0.1 M sodium citrate. Concentration of M10 in
mouse plasma was measured using an indirect Enzyme-Linked Immunosorbent Assay
(ELISA) developed in our laboratory. Pharmaceutical grade synthetic M10 was used as
a calibrator, and commercially available anti-c-Met C12 antibody was used to capture
the M10 antigen in the ELISA.
Results: The detection limit of M10 by ELISA is 10ng/ml with a measuring
limit of 250ng/ml. Recovery limits of M10 were 80%-120%. Intra-assay coefficient of
variation was 5.3%-6.3% and inter-assay coefficient of variation was 5.0%-8.0% over
a buffer concentration of 15-250 ng/ml. Peak M10 concentration following a single
intraperitoneal injection was achieved within 6 hours and declined to minimal levels
by 48 hours. Our experimentally obtained half-life for M10 is comparable to the
theoretical half-life for M10 predicted using ProtLifePred® software.
Conclusions: We present pharmacokinetic and pharmacodynamic data
demonstrating that the anti-fibrotic peptide M10 has very good stability and
sustainability in plasma, which should facilitate its consideration as a potential
therapeutic peptide in the treatment of SSc-ILD and other fibrosing diseases.
Acknowledgments: Funded by NIH/NIAMS P60 AR062755, Scleroderma
Foundation and SC SmartState®.
IS SYSTEMIC SCLEROSIS RELATED PULMONARY ARTERIAL HYPERTENSION A DISTINCT
PHENOTYPE? A LUNG MORPHOMETRIC ANALYSIS OF SYSTEMIC SCLEROSIS ASSOCIATED VS
IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION
1 Division of Pulmonary and Critical Care, Medical University of South
Carolina, Charleston, South Carolina, USA
2 Division of Pathology and Laboratory Medicine, Medical University of
South Carolina, Charleston, South Carolina, USA
3 Division of Rheumatology, Medical University of South Carolina,
Charleston, South Carolina, USA
4 Department of Public Health Sciences, Medical University of South
Carolina, Charleston, South Carolina, USA
Introduction: Systemic sclerosis associated pulmonary arterial
hypertension (SSc-PAH) patients have a significantly higher mortality when compared
to patients with idiopathic pulmonary arterial hypertension (IPAH). The goal of this
pilot project was to understand the pathological and biological differences between
the pulmonary vasculopathies of SSc-PAH and IPAH using quantitative lung
morphometry.
Methods: We utilized bio-banked SSc-PAH (n = 24) and IPAH (n = 9) lung
tissue specimens and conducted a comparative quantitative lung morphometry study. H
& E (Hematoxylin and Eosin) and VVG (Verhoeff Van Gieson) stained lung sections
from the specimens were analyzed for the following: 1. Plexiform lesions, 2.
Interstitial cellularity, 3. Insterstitial fibrosis, 4. Small vessel intimal
proliferation, 5. Pulmonary vein intimal proliferation, 6. Smooth muscle
hypertrophy. Based on a post-hoc comparison using a tukey adjustment from an ANOVA,
we compared the SSc-PAH and IPAH specimens for all the above mentioned variables
(continuously distributed). We used Bonferroni correction for multiple comparisons.
For the plexiform lesions (categorical variable), we used a Fisher's exact test to
compare the SSc-PAH and IPAH groups.
Results: The specimens available to us were small lung sections so a
comprehensive evaluation of pulmonary vascular and parenchymal lesions was not
possible. 3/9(33.3%) IPAH lungs had notable plexiform lesions while none of the
SSc-PAH specimens had plexiform lesions. The SSc-PAH lung specimens were also
significantly different when compared to the IPAH lung specimens with respect to: a)
interstitial cellularity score (5.4 vs 2.1, p<0.0001), b) interstitial fibrosis
score (8.4 vs 0.7, p<0.0001). The SSc-PAH and IPAH lung specimens did not differ
significantly with respect to pulmonary arterial intimal proliferation score (7.3 vs
7.1, p = 0.9) or arteriolar smooth muscle hypertrophy score (4.3 vs 5.8, p = 0.23).
The pulmonary vein intimal proliferation score trended worse for the SSc-PAH group
when compared to the IPAH group (4.1 vs 2.3, p = 0.09).
Conclusions: Our analysis suggests that SSc-PAH patients have a distinct
vasculopathy when compared to IPAH patients. These differences could have
therapeutic implications with regard to their response to pulmonary arterial
vasodilators and potentially, disease management.
CELL TYPE SPECIFIC GENE EXPRESSION ANALYSIS OF EARLY SYSTEMIC SCLEROSIS SKIN
SHOWS A PROMINENT ACTIVATION PATTERN OF INNATE AND ADAPTIVE IMMUNE SYSTEM
1 University of Texas Health Science Center at Houston, Houston, TX,
USA
2 University of Michigan, Ann Arbor, MI, USA
3 Northwestern University, Chicago, IL, USA
4 Georgetown University, Washington, DC, USA
5 Medical University of South Carolina, Charleston, SC, USA
6 Hospital for Special Surgery, New York City, NY, USA
7 Johns Hopkins University, Baltimore, MD, USA
8 George Washington University, Washington, DC, USA
9 Pittsburgh University, Pittsburgh, PA, USA
10 Harvard University, Boston, MA, USA
11 Columbia University, New York City, NY, USA
12 University of Utah, Salt Lake City, UT, USA
Objective: To examine the global gene expression profile in patients
with very early diffuse systemic sclerosis (SSc).
Methods: Skin biopsies were obtained from patients enrolled in the
Prospective Registry for Early Systemic Sclerosis (PRESS). All patients had diffuse
cutaneous involvement and disease duration <2 years. Fifty-seven patients and
age-, gender-matched controls were examined by nextGen RNA Sequencing (Depth: 50
million reads, 75 bp length) on Illumina HiSeq 3000 platform. Cell type-specific
signature scores were calculated for each patient sample compared to the average
score in controls as previously described (Assassi et al. Arthritis Rheum 2015).
Gene signatures for 15 cell types present in skin were calculated including:
Fibroblasts, keratinocytes, CD4+ and DC8+ T-cells, NK cells, dendritic cells, M1 and
M2 macrophages, and B-cells.
Results: The median disease duration was only 1.1 years while median
mRSS score was 22. In comparison to controls, 2537 transcripts were differentially
expressed (FDR <5%).
The cell-type specific signature score analysis revealed a prominent up-regulation of
innate and adaptive cell types. The three most frequent activated cell-type
signatures were M2 Macrophage (present in 96% of patient samples), fibroblast (93%),
and microvascular endothelial cells (91%). Interestingly, the proportion of samples
with an adaptive immune system signature was much higher than the frequencies
observed in our previously published data set (61 SSc patients with mean disease
duration of 7.7 years and 36 matched controls). Specifically, CD8+ T cell, CD4+ T
cell, and B-cell signatures were present in 67%, 61%, and 67% of samples in the
present study whereas those signatures were present in less than 25% of SSc samples
with established disease in the previous data set. Consistent with this finding, an
Ingenuity Pathway Analysis indicated that Th1 and Th2 Activation Pathway was among
the top over-represented pathways in the present data set. The analysis to determine
the predictive significance of these cell specific signatures for response to
immunosuppression is ongoing.
Conclusions: Skin samples of patients with early diffuse involvement
have a prominent adaptive immune signature, which is not present in established
disease. This finding can have important implications for clinical trials targeting
adaptive immune system.
DISTINCT MECHANISMS OF POLYMERIZATION OF SMOOTH MUSCLE α-ACTIN AND β/γ-ACTIN
ISOFORMS IN SCLERODERMA LUNG FIBROBLASTS
1 Division of Rheumatology, Department of Medicine, Medical University of
South Carolina, Charleston, SC, USA
2 Division of Rheumatology, Endocrinology and Nephrology, Hokkaido
University Graduate School of Medicine, Sapporo, Japan
3 Nanjing Medical University, Nanjing, China
4 Hospital Universitario 12 de Octubre, Madrid, Spain
5 Department of Surgery, Medical University of South Carolina, Charleston,
SC, USA
Background: The actin cytoskeleton of scleroderma lung fibroblasts
consists of three different isoforms: smooth muscle α-actin (α-SMA), β-actin and
γ-actin. All actin isoforms are present in cells in either a monomeric state
(G-actin) or a polymeric state (F-actin). G-actin is localized mainly to the cytosol
(cytosolic actin), while bundles of polymerized F-actin form an actin stress fiber
network. We previously demonstrated that scleroderma lung fibroblasts exhibit a
predominantly contractile phenotype while expressing large amounts of the IQ motif
containing GTPase activating protein (IQGAP1). The objective of this study was to
examine effects of IQGAP1 on expression, polymerization, and functions of α-SMA in
scleroderma lung fibroblasts.
Methods: Fibroblasts were derived from lung tissues obtained at autopsy
from SSc patients and from age- race-, and sex-matched normal subjects. Contractile
activity of lung fibroblasts was measured using a collagen gel contraction assay.
Expression and organization of α-SMA was analyzed by immunoblotting,
immunofluorescent staining, and polymerization assay. Transcriptional activity of
α-SMA promoter was studied by luciferase assay.
Results: We demonstrate that IQGAP1 mediates α-SMA expression and
contractile activity of scleroderma lung fibroblasts as well as normal lung
fibroblasts stimulated with thrombin, TGFβ1 or CTGF. We also show that IQGAP1 binds
directly to polymerized β- and γ-actin isoforms and facilitates polymerization of β-
and γ-actin by cross-linking filaments into interconnected bundles. IQGAP1 does not,
however, appear to bind polymerized α-SMA in scleroderma lung fibroblasts,
suggesting that a different mechanism of α-SMA polymerization in scleroderma lung
fibroblasts involves stimulation of actin assembly through the myosin light chain
(MLC) and the neuronal Wiskott-Aldrich syndrome protein-actin-related proteins 2 and
3 (N-WASP-Arp2/3) pathways.
Conclusions: We speculate that unique mechanisms of α-SMA polymerization
in scleroderma lung fibroblasts results in the development of a highly contractile
myofibroblast phenotype as seen in fibroblasts from patients with
scleroderma-associated interstitial lung disease.
Acknowledgments: Funded by Scleroderma Foundation and SC
SmartState®.
1 Department of Rheumatology, Leiden University Medical Center, Leiden,
The Netherlands
2 Department of Clinical Chemistry and Laboratory Medicine, Leiden
University Medical Center, Leiden, The Netherlands
3 Department of Pulmonology, Leiden University Medical Center, Leiden, The
Netherlands
4 Department of Cardiology, Leiden University Medical Center, Leiden, The
Netherlands
Background: IgA en IgG anti-topoisomerase I antibody levels were
associated with skin score in Systemic Sclerosis (SSc). Additionally, a study in 28
SSc patients suggested a more favourable disease course in IgM anti-topoisomerase
negative patients. So far, these data await replication. No large studies evaluating
IgG, IgA and IgM levels in association with SSc disease course have been
performed.
Objectives: To evaluate whether isotype specific levels of
anti-topoisomerase I antibodies are associated with disease course in SSc.
Methods: Serial levels of IgG, IgM and IgA anti-topoisomerase I
antibodies were assessed in IgG anti-topoisomerase positive patients included in the
Leiden Systemic Sclerosis cohort. Patients were grouped in quartiles based on IgG,
IgM and IgA levels (Q1 = lowest quartile, Q4 = highest quartile) at inclusion
(baseline). Clinical characteristics were compared between groups. Severe disease
was defined as need for aggressive immunosuppressive therapy (mycophenolate,
cyclophosphamide or autologous hematopoietic stem cell transplantation) within 1
year from inclusion.
Results: In total, 105 baseline serum samples positive for IgG
anti-topoisomerase were evaluated in which IgA and IgM levels were detectable in
100% (n = 105) and 94% (n = 99) respectively. Time since first non-Raynaud
phenomenon was 3 years (IQR 1-11); 45% (n = 47) had diffuse cutaneous involvement;
78% (n = 82) were naïve for aggressive immunosuppression. High baseline IgG and IgM
anti-topoisomerase levels, but not IgA, were significantly associated with severe
disease (Odds ratios, IgG: Q1: ref, Q2: OR 2.7 [95% CI 0.8-9.6], Q3: OR 1.1 [95% CI
0.3-4.1], Q4: OR 5.1 [95% CI 1.5-17.7], IgM: Q1: ref, Q2: OR 1.7 [95% CI 0.5-5.6],
Q3: OR 1.0 [95% CI 0.3-3.6], Q4: OR 3.3 [95% CI 1.0-10.9], IgA: Q1: ref, Q2: OR 1.4
[95% CI 0.4-4.4], Q3: OR 0.8 [95% CI 0.2-2.6], Q4: OR 1.1 [95% CI 0.3-3.8]).
Conclusions: We here present an in-depth analysis of IgG, IgA and IgM
anti-topoisomerase levels in a relatively large-size cohort of SSc patients. Our
first results indicate that high IgG and IgM anti-topoisomerase I antibody levels
associate with severe disease in the following 12 months. This finding suggests that
an active immune response against topoisomerase I, as indicated especially by the
presence of high levels of IgM, could be a relevant driving factor in SSc
pathogenesis. Evaluations of anti-topoisomerase I antibody levels during follow-up
is ongoing.
TO WHAT EXTENT DO AUTO-ANTIBODIES HELP TO IDENTIFY HIGH-RISK PATIENTS IN
SYSTEMIC SCLEROSIS?
1 Department of Rheumatology, Leiden University Medical Center, Leiden,
The Netherlands
2 Department of Clinical Chemistry and Laboratory Medicine, Leiden
University Medical Center, Leiden, The Netherlands
3 Department of Pulmonology, Leiden University Medical Center, Leiden, The
Netherlands
4 Department of Cardiology, Leiden University Medical Center, Leiden, The
Netherlands
Background: In Systemic Sclerosis tight monitoring during the first
years of disease is required in order to detect organ complications timely. Although
a clear pathophysiologic role of disease specific auto-antibodies has never been
confirmed, these antibodies are associated with distinct clinical futures, strongly
indicating that their prevalence is of relevance.
Objectives: To evaluate to what extent disease specific auto-antibodies
in Systemic Sclerosis improve clinical subsetting, identifying high-risk
disease.
Methods: Clinical clusters of patients were determined, using data from
the Combined Care In Systemic Sclerosis cohort, Leiden University Medical Center.
Hierarchical clustering based on Ward Method was performed on Principal Component
Analysis scores of solely baseline clinical variables. To determine disease-risk,
5-year mortality rates since first non-Raynaud phenomenon were assessed. Prevalence
of disease specific auto-antibodies in each cluster was studied. Second, the cluster
process was repeated, taking auto-antibodies into account. Clinical and
auto-antibody characteristics of obtained clusters were compared to clustering based
on clinical variables alone.
Results: Of 407 patients, 91% (n = 371) fulfilled ACR/EULAR 2013
criteria. Prevalence of auto-antibodies was: anti-centromere 37%, anti-topoisomerase
24%, anti-RNA polymerase III 5%, anti-fibrillarin 4% and anti-Pm/Scl 5%. Clinical
cluster analysis identified 4 different clusters with two clusters showing higher
than average mortality (resp. 17% and 7% vs. total group mortality of 4% (n = 15)).
Adding auto-antibody status to the cluster process resulted in 5 clusters, with an
additional cluster 5 with frequent RNA polymerase III (18%, n = 14; mortality rate
8%). The total number of patients clustered to a cluster with higher than average
mortality-risk increased indicating the need of more intensive screening in a larger
group of patients.
Conclusions: Auto-antibodies partially contribute to risk-stratification
and clinical subsetting in Systemic Sclerosis. We hypothesize that additional
characteristics of auto-antibodies like isotypes might reflect their
pathophysiological role, and that the value of autoantibodies as biomarkers for
severe disease might increase when adding these characteristics.
DISEASE SPECIFIC AUTO-ANTIBODY PREVALENCES IN SYSTEMIC SCLEROSIS PATIENTS WITH
AND WITHOUT MALIGNANCY
Boonstra M1, van Wijngaarden SE2, Ajmone Marsan
N2, Ninaber MK3, Huizinga TWJ1, de
Vries-Bouwstra JK1
1 Department of Rheumatology, Leiden University Medical Center, Leiden,
The Netherlands
2 Department of Cardiology, Leiden University Medical Center, Leiden, The
Netherlands
3 Department of Pulmonology, Leiden University Medical Center, Leiden, The
Netherlands
Background: Systemic Sclerosis is an auto-immune disease characterised
by presence of disease specific auto-antibodies in a majority of patients. Cancer
risk in Systemic Sclerosis is increased, and concomitant malignancy significantly
worsens prognosis. RNApIII auto-antibodies are associated with an immunologic immune
response to cancer and recently this has also been suggested for Pm/Scl
antibodies.
Objectives: To compare prevalence of different disease specific
autoantibodies in Systemic Sclerosis between patients with and without
malignancies.
Methods: Baseline clinical findings and auto-antibody prevalences were
compared between patients with a clinical diagnosis of Systemic Sclerosis in the
Leiden Combined Care in Systemic Sclerosis (CCISS) cohort with and without a
malignancy. For inclusion, recent follow-up data, auto-antibody status and at least
2 visits to the Leiden care pathway had to be available.
Results: Of the 305 patients (94.4% fulfilled ACR/EULAR 2013 SSc
criteria) included, 46 patients (15.1%) had a history of cancer. Prevalence of ACA
was 38.0% (n = 116), Anti-topoisomerase 25.9% (n = 79), RNA polymerase III 6.6% (n =
20), U3RNP 4.3% (n = 13), U1RNP 9.2% (n = 28), Pm/Scl 6.9% (n = 21), anti-ThTo 1.6%
(n = 5), anti-Ku 2.0% (n = 6), ANA-ENA- 4.3% (n = 13), ANA+/ ENA+, no disease
specific antibodies 9.5% (n = 29), >1 disease specific antibodies 8.9% (n = 27).
Patients with a malignancy at baseline, were older (age 61 vs. 53yr, p 0.001), had
less often diffuse cutaneous disease (13 vs. 27%, p 0.043) and more often pulmonary
arterial hypertension (13 vs. 3%, p 0.001). There were no significant differences in
auto-antibody prevalence between patients with and without a malignancy, although
numerically prevalence of anti-topoisomerase was less common (17.4%, n = 8 vs.
27.4%, n = 71) and prevalence of RNA polymerase III (10.9%, n = 5 vs. 5.8%, n = 15)
was more common in patients with a malignancy.
Conclusions: The recently observed association between cancer and Pm/Scl
positivity could not be confirmed in our prospective cohort. Though nonsignificant,
numerically RNApIII antibodies were more prevalent. Based on our data, extensive
screening for malignancies can only be justified guided by clinical findings. A link
between cancer auto-immunity and Systemic Sclerosis may be present, more research in
this field is warranted, as this may shine light on disease pathogenesis of both
diseases.
PREDICTORS OF HAND CONTRACTURE IN EARLY SSC AND THE EFFECT ON HAND FUNCTION: A
PROSPECTIVE LONGITUDINAL STUDY OF THE GENISOS COHORT
Buni M1, Joseph J2, Pedroza C3,4, Theodore
S5, Nair D5, McNearney TA6, Draeger
HT6, Reveille JD5, Assassi S5, Mayes
MD5
1 Department of Internal Medicine, McGovern Medical School, University of
Texas, Houston, TX, USA
2 VA Medical Center, Washington, DC, USA
3 Medical School, Center for Clinical Research and Evidence-Based Medicine
and Department of Pediatrics, University of Texas McGovern, Houston, TX, USA
4 Department of Internal Medicine, Division of Rheumatology and
Immunogenetics, McGovern Medical School, University of Texas, Houston, TX, USA
5 Eli Lilly and Company, Indianapolis, IN, USA
6 University Health System, University Medicine Associates, San Antonio,
TX, USA
Objectives: To identify baseline disease features that predict
progression of hand contractures and to assess the effect of contractures on
functional status in the prospective GENISOS cohort.
Methods: Rate of decline in hand extension, as an indicator of hand
contracture, was the primary outcome. We assessed longitudinal hand extension
measurements, modified Health Assessment Questionnaire (MHAQ) score, demographic,
clinical and serological variables. Subjects with 2 or more hand measurements at
least 6 months apart were included.
Results: A total of 1087 hand measurements for 219 patients were
available over an average of 8.1 years. The hand extension decreased on average by
0.11 cm/year. Anti-topoisomerase I (ATA) positivity and higher modified Rodnan Skin
Score were predictive of faster decline in hand extension (p = 0.009 and p = 0.046,
respectively).
In a subgroup analysis of 62 patients with ≤2 years from SSc onset, ATA and diffuse
disease type were associated with faster decline in hand extension; conversely,
anti-centromere positivity was associated with slower rate of decline. Although the
rate of decline in patients with diseases duration ≤2 years was numerically higher,
the difference was not statistically significant. Hand extension continued to
decline in a linear fashion over time and was inversely related to overall
functional status.
Conclusion: ATA was predictive of hand contracture development in both
early disease (≤2 years) and in the overall cohort. Hand extension continued to
decline over time in a linear fashion and was inversely associated with MHAQ
scores.
Acknowledgements: The National Institute of Arthritis, Musculoskeletal
and Skin Diseases (NIAMS) of the National Institutes of Health (NIH) Centers of
Research Translation (CORT) P50-AR054144, NIH grant N01-AR-02251, R01-AR-055258, and
K23AR061436; by the University of Texas Health Science Center NIH funded Clinical
and Translational Science Award (CTSA) Clinical Research Unit UL1 RR024148; and by
the Department of Defense (DoD) Congressionally Directed Medical Research Program
W81XWH-07-1-011 and WX81XWH-13-1-0452.
Cai G1, Mehta BK1, Li Z1, Johnson
ME1, Huang M1, Franks JM1,2, Wood
TA1, Kolstad KD3, Stark M3, Valenzuela
A3, Fiorentino D3, Simms RW4, Orzechowski
N5, Chung L3, Whitfield ML1,2
1 Department of Molecular and Systems Biology, Geisel School of Medicine
at Dartmouth, Hanover, NH, USA
2 Program in Quantitative Biomedical Science, Geisel School of Medicine at
Dartmouth, Hanover, NH, USA
3 Department of Immunology and Rheumatology, Stanford University School of
Medicine, Palo Alto, CA, USA
4 Boston University School of Medicine, Boston, MA, USA
5 Dartmouth-Hitchcock Medical Center, Hanover, NH, USA
Background: Systemic sclerosis (SSc) is a complex disease characterized
by substantial genotypic and phenotypic heterogeneity. Four molecular gene
expression subsets have been identified from SSc skin, including inflammatory,
fibroproliferative, normal-like, and limited. Genome-wide association studies have
identified genetic variants associated with disease risk. However, these studies
were not designed to interrogate genetic changes underlying specific gene expression
subtypes. Here, our objective was, by RNA-seq in SSc skin, to identify potential
genetic variants associated with specific gene expression subtypes and disease
phenotypes.
Methods: We generated RNA-seq data from skin biopsies of 42 patients
with SSc and 14 healthy controls. Variants were detected and filtered by quality,
sequencing depth, minor allele frequency, and potential deleterious effects on gene
function. Gene burden tests were used to identify susceptibility loci in cases
compared to healthy controls, as well as in healthy controls of a European
population from the phase III 1000 Genomes Project (N = 503). Furthermore, we
quantitated gene expression and used an additive genetic model to identify gene
expression associated variants. Variants enriched in each of the SSc molecular
subsets were detected by Fisher's exact test.
Results: We identified 267 potentially deleterious variants in 253 genes
in patients with SSc, including 30 genes (e.g. IRF6, NCF2, IL37)
enriched in immunological processes and 10 genes (e.g. ITGB4, COL5A1,
LAMA3) significantly enriched in extracellular matrix-related pathways.
The variant in LAMA3 was significantly associated with its corresponding mRNA
expression levels. We found variants enriched in each of the inflammatory (35
variants), fibroproliferative (7 variants), and normal-like (45 variants) subsets.
Potentially deleterious variants in IL37 were enriched in patients with diffuse skin
involvement and were significantly correlated with increased gene expression of IL6
and STAT3 in patient samples. Consistently, HapMap B cell lines harboring
deleterious IL37 variants showed increased expression of IL6 upon immune
stimulation.
Conclusions: This study demonstrates the value of RNA-seq for
identifying genetic variants associated with SSc gene expression subsets. Results
indicate genetic variants are specific to each gene expression subset and may
influence innate immune responses and ECM deposition. The identified IL37 variant
may modulate SSc pathogenesis by increasing expression of the IL-6 pathway.
SERUM TRYPTOPHAN AND KYNURENINE LEVELS ARE ALTERED IN SSC AND SHOW DISTINCT
CLINICAL AND AUTOANTIBODY ASSOCIATIONS SUGGESTING POTENTIAL ROLE IN
PATHOGENESIS
Campochiaro C1, Lytten SD2, Nihtyanova S1,
Ong VH1, Denton CP1
1 Centre for Rheumatology and Connective Tissue Diseases, Royal Free
Hospital, London, UK
2 SeraDiaLogistics, Munich, Germany
Introduction: The enzyme Indoleamine dioxygenase (IDO) degrades Trp into
Kyn and is induced by IFN-gamma/IL-1. The Trp pathway may play an important role in
immune regulation. We measured serum level of Kyn, Trp and Kyn/Trp ratio in SSc and
explored clinical and ANA associations.
Methods: Serum levels of Kyn and Trp were measured in 97 SSc pts and 10
healthy controls (HC). Kyn/Trp ratio was calculated. Serial serum samples were
obtained for 40 pts. Association with disease characteristics were evaluated using
mixed effects models.
Results: Kyn mean levels were 1.3 μmol/L (95% CI 0.7,2.0) in HCs. They
were significantly higher among SSc, in lcSSc 0.8 μmol/L higher (p = 0.036) and in
dcSSc 1.1 μmol/L higher (p = 0.003). ARA + pts had significantly higher mean Kyn
levels (2.7 μmol/L; 95% CI 2.4,3.0), compared to ACA + pts and ATA + pts (0.6
μmol/L, p = 0.034 and 0.9 μmol/L, p = 0.001 respectively).
Mean Trp level in HCs was 59.2 μmol/L (95% CI52.8,65.6). Compared to HCs, SSc Trp
levels were significantly reduced (by 8.5 μmol/L in lcSSc, p = 0.020 and 13.4 μmol/L
in dcSSc, p<0.001). Difference between the two subsets was significant (p =
0.009). Trp levels were lowest among ARA+pts (45.2μmol/L), followed by ATA +
(48.4μmol/L) and ACA + (52.8μmol/L), with significant difference between ACA + and
ARA + (p = 0.001).
Mean Kyn/Trp ratio for HCs was 22.6 μmol/mmol (95% CI 2.8,42.4). This was
significantly lower than SSc (lcSSc 44.1 μmol/mmol, p = 0.056 and dcSSc 56.8
μmol/mmol, p = 0.002) with a difference between the two subsets of 12.7 μmol/mmol (p
= 0.039). ARA + pts had a higher ratio (62.9 μmol/mmol) compared to ATA + and ACA +
(difference of 22.1 and 22.3 respectively p = 0.002).
On average, there was no significant change of Kyn, Trp or Kyn/Trp over time.
Conclusions: These data suggest dysregulation in tryptophan metabolism
that could influence immune cell function and fibrosis in SSc. This may be
particularly relevant in the dcSSc subset, and in ARA+ patients. As well as being of
pathogenetic relevance, stability of these analytes over time may make these markers
of Trp metabolism useful in SSc stratification.
INTEGRATING ANALYSIS OF SKIN RNA IN SITU HYBRIDIZATION USING RNASCOPE AND
WHOLE SKIN GENE EXPRESSION IN SYSTEMIC SCLEROSIS SKIN TO LOCALIZE KEY PATHOGENIC
DRIVERS OF SKIN FIBROSIS
1 Centre for Rheumatology and Connective Tissue Diseases, Royal Free
Hospital, London, UK
2 GlaxoSmithKline, Stevenage, UK
Introduction: Skin gene expression profiling has been demonstrated to
distinguish scleroderma from normal skin and has also been used to detect different
subsets of disease. The aim of our study was to compare gene expression in early
versus late dcSSc and define cells most likely responsible for the upregulation of
candidate genes in early patients.
Methods: Total RNA was extracted from forearm skin biopsies of 3 early
ARA + dcSSc patients and 3 late ARA + dcSSc patients. COMP, CCL2, PDGFA, COL1A1,
IFI44, IL-6, MMP3, SERPINE, TGFb, THBS1 were measured using quantitative PCR
analysis. MRSS was assessed in all patients and was correlated with normalized gene
expression. RNA in situ hybridization for ACTA2, CCL2, COL1A1, COL3A1, COMP, SERPINE
and THBS1 mRNA was performed.
Results: Whole skin relative normalized gene expression of COMP, CCL2,
IL-6, SERPINE and THBS1 was significantly (p<0.05) higher in early compared to
late dcSSc patients (COMP 25049.15 ± 10862.16 vs 5256.47 ± 2380.53; CCL2 1722.04 ±
598.93 vs 638.25 ± 279.96; IL-6 43.98 ± 40.76 vs 11.91 ± 5.44; SERPINE 1770.32 ±
704.46 vs 184.48 ± 61.37; THBS1 7190.75 ± 309.89 vs 1176.81 ± 235.35). A positive
correlation between SERPINE and THBS1 and MRSS (SERPINE R2 = 0.493, p =
0.09; THBS1 R2 = 0.91, p = 0.02) was found. RNAscope analysis
demonstrated SERPINE+ staining in fibroblast-like cells in the mid-dermis only in
early dcSSc compared to HCs. An increased number of THBS1+ and COMP+ fibroblast-like
cells (α-SMA+) were present in the deep dermis of early dcSSc.
Conclusions: Our data show upregulated SERPINE, THBS1, COMP, CCL2 and
IL-6 in early compared to late dcSSc patients. RNAscope technology allowed us to
localise expression of these genes specifically in the dermis of patients in subsets
of cells. These findings highlight how specific cells could be responsible for the
pro-fibrotic and inflammatory changes typical of the early stages of the disease.
Our data suggest that the upregulation of fundamental pro-fibrotic and inflammatory
genes could be crucial in the early stages of SSc and specific subsets of cells in
the dermis could be the responsible for this deregulation.
14-3-3Z SEQUESTERS CYTOSOLIC T-BET, UP-REGULATING IL-13 IN TC2 AND SCLERODERMA
CD8+ LYMPHOCYTES
1 Department of Immunology, University of Pittsburgh School of Medicine,
Pittsburgh, USA
2 RiMED Foundation, Palermo, Italy
3 Department of Medicine, Division of Rheumatology and Clinical
Immunology, University of Pittsburgh School of Medicine, Pittsburgh, USA
4 Department of Cell Biology, University of Pittsburgh School of Medicine,
Pittsburgh, USA
Background: Interleukin (IL)-13-producing CD8+ T cells have been
implicated in the pathogenesis of type-2 driven inflammatory human conditions. We
have shown that CD8+IL-13+ cells play a critical role in cutaneous fibrosis, the
most characteristic feature of systemic sclerosis (scleroderma; SSc). However, the
molecular mechanisms underlying IL-13 and other type-2 cytokine production by CD8+ T
cells remain unclear.
Objective: Establish the molecular basis of IL-13 over-production by SSc
CD8+ T cells, focusing on T-bet modulation of GATA-3 activity, which we showed to
underlie IL-13 over-production in SSc CD8+IL-13+ cells.
Methods: Biochemical and biophysical methods were employed to determine
expression and association of T-bet, GATA-3 and regulatory factors in CD8+ T cells
isolated from the blood and lesional skin of SSc patients with severe skin
thickening. ChIP analysis determined GATA-3 binding to the IL-13 promoter.
ImageStream analysis and confocal microscopy visualized the subcellular localization
of T-bet and GATA-3. Transcript levels were decreased by small interfering RNAs.
Results: The interaction of T-bet with the adaptor protein 14-3-3z in
the cytosol of SSc CD8+ T cells reduces T-bet translocation into the nucleus and its
ability to associate with GATA-3, allowing more GATA-3 to bind to the IL-13 promoter
and inducing IL-13 up-regulation. Strikingly, we show that this mechanism is also
found during Tc2 polarization of healthy donor CD8+ T cells.
Conclusions: We identified a novel molecular mechanism underlying type-2
cytokine production by CD8+ T cells revealing a more complete picture of the complex
pathway leading to SSc disease pathogenesis.
PROTEOMIC ANALYSIS OF HUMAN FIBROBLASTS IN SYSTEMIC SCLEROSIS REINFORCES THE
ROLE OF TRANSFORMING GROWTH FACTOR-β AND POINTS TOWARD EPIDERMAL GROWTH FACTOR
RECEPTOR/PHOSPHATIDYLINOSITOL PATHWAY INHIBITION
Chaigne B1-4, Clary G1,2,5, Le Gall M1,2,5,
Dumoitier N1-3, Lofek S1,2, Chafey P1,2,5,
Moinzadeh P6, Krieg T6, Denton CP7, Mouthon
L1-4
1 INSERM U1016, Cochin Institute, Paris, France
2 CNRS UMR 8104, Paris, France
3 Paris Descartes University, Sorbonne Paris Cité, Paris, France
4 Department of Internal Medicine, Cochin Hospital, Paris, France
5 Proteomic core facility of Paris Descartes University (3P5), Paris,
France
6 Department of Dermatology, University of Cologne, Cologne, Germany
7 Institute of Immunity and Transplantation, Centre for Rheumatology and
Connective Tissue Diseases, Royal Free Hospital, London, UK
Systemic sclerosis (SSc) is a rare autoimmune connective tissue disorder
characterized by autoimmunity, vasculopathy and fibrosis. Fibrosis is due to an
exaggerated activation of fibroblasts by the transforming growth factor-β (TGF-β).
In this study we investigated the proteomic response of skin fibroblasts to TGF-β in
patients with SSc.
Skin fibroblasts from 4 patients with SSc and 3 healthy controls (HC) were cultured
in the presence or in the absence of TGF-β. Two-dimensional gel electrophoresis and
mass spectrometry (MS) were used to identify proteins differentially expressed
between groups. Ingenuity Pathway analysis (IPA) and Panther softwares were used to
analyse identified proteins. Finally real-time cell analyser (RTCA) was used to
assess fibroblast proliferation and viability in order to validate proteins of
interest.
We identified 687 protein spots differentially expressed between groups. 297 protein
spots were analysed by MS. Principal component analysis revealed significant
differences between fibroblasts of patients with SSc and HC when cultured in the
presence or in the absence of TGF-β. IPA revealed specific process networks such as
actin cytoskeleton signalling, integrin signalling, and remodelling of epithelial
adherens junctions. Panther revealed predominant biological processes such as
cellular process, metabolic process and cellular component organization. TGF-β
enhanced the synthesis of fibroblasts' proteins involved in actin cytoskeleton
signalling and integrin signalling. Using IPA and RTCA we identified and validated
the involvement of epidermal growth factor receptor (EGFR) and phosphatidylinositol
3 kinase (Pi3K) in the proliferation and the viability of fibroblasts from patients
with SSc.
In conclusion, we confirmed that the proteome profile of fibroblasts differs between
patients with SSc and HC and demonstrated that fibroblasts enhance their proteomic
phenotype upon stimulation with TGF-β. We finally highlighted that EGFR and Pi3K are
proteins of interest in fibroblasts from patients with SSc and should be considered
as potential targets to inhibit fibrosis in SSc.
BASOPHILS ARE ACTIVATED AND ARE ABLE TO STIMULATE BOTH B CELLS AND FIBROBLASTS
IN SYSTEMIC SCEROSIS
3 Paris Descartes University, Sorbonne Paris Cité, Paris, France
4 Department of Internal Medicine, Cochin Hospital, Paris, France
5 LABEX Inflamex, Paris-Sorbonne University, Paris, France
Recent studies have emphasized the fibrotic and immunological roles of basophils. In
this work, we studied their role in patients with systemic sclerosis (SSc).
Using flow cytometry and xCelligence assay we characterized surface expression of
basophil markers and assessed their ability to stimulate B cells and activate
fibroblasts.
Sixty-five patients with SSc who had never received steroids or immunosup-pressant
treatment and 38 healthy controls (HC) were included. Expression levels of CD203c
and CCR3 on basophils were significantly higher and lower in SSc patients than in
HC, respectively. Patients with SSc had a lower proportion of CRTH2+ basophils than
HC and CRTH2 expression on basophils negatively correlated with disease
characteristics including pulmonary hypertension and modified Rodnan skin score.
Activated basophils produced more BAFF, and increased IL-6 and TGF-β production by B
cells. Lastly, we demonstrated basophil profibrotic function in SSc by showing
higher proportion of TGF-β-secreting basophils and enhanced proliferation of
fibroblasts induced by activated basophils in a TGF-β-dependent manner in SSc.
This study shows that peripheral basophils from SSc patients are activated, are able
to simulate B cells, and enhance fibroblast proliferation through the production of
TGF-β, supporting a key role of basophils in the pathophysiology of SSc.
POLYMORPHISMS IN NKX2-5 ASSOCIATED WITH SCLERODERMA AND PULMONARY HYPERTENSION
REVEAL REGULATORY MECHANISMS FOR EXPRESSION IN DISEASED VESSELS
Dritsoula A1, Papaioannou I1, Guerra SG1,
Fonseca C1, Martin J2, Herrick AL3, Abraham
DJ1, Denton CP1, Ponticos M1
1 Centre for Rheumatology and Connective Tissue Diseases, Division of
Medicine, University College London, London, UK
2 Institute of Parasitology and Biomedicine López-Neyra, IPBLN-CSIC, PTS
Granada, Granada, Spain
3 Centre for Musculoskeletal Research, University of Manchester,
Manchester, UK
Objectives: NKX2-5 is a transcription factor required for the formation
of the heart and vessels during development. Postnatal expression is significantly
downregulated, then re-activated in disease during conditions characterized by
vascular remodelling. Our aim is to investigate the mechanisms that activate NKX2-5
expression in diseased vessels, such as scleroderma associated pulmonary
hypertension.
Methods: A case-control genetic association study was performed in two
independent cohorts of scleroderma patients. Associated Single nucleotide
polymorphisms (SNP) were cloned into reporter vectors and transcriptional activity
was assessed by reporter-gene assays. SNP function was further evaluated through
protein-DNA binding assays, chromatin immunoprecipitation and RNA silencing.
Results: Meta-analysis of two independent cohorts revealed association
of rs3131917 with scleroderma (p = 0.029). Furthermore, rs3132139, located
downstream of NKX2-5, was significantly associated with pulmonary
hypertension (p = 0.01); the association was replicated in the independent cohort (p
= 0.045). We demonstrated that the region where rs3132139 and rs3131917 are located,
is a novel functional enhancer which increases NKX2-5 transcriptional activity
through the binding of GATA6, c-JUN, and MEF-2c. We also identified that an
activator/co-activator TEAD/YAP1 complex is bound at another functional SNP,
rs3095870, which showed marginal association with scleroderma (p = 0.03). TEAD1
binds the risk allele of rs3095870 and activates NKX2-5
transcription.
Conclusions:NKX2-5 is genetically associated with scleroderma and pulmonary
hypertension. Functional evidence revealed a regulatory mechanism which results in
NKX2-5 transcriptional activation in human vascular smooth
muscle cells through the interaction of an upstream promoter and a novel downstream
enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular
diseases characterized by vascular remodelling.
PATIENT REPORTED OUTCOMES AND THE PATULOUS ESOPHAGUS IN PATIENTS SYSTEMIC
SCLEROSIS?
1 Department of Internal Medicine, University of Utah, Salt Lake City, UT,
USA
2 Feinberg School of Medicine, Department of Medicine, Division of
Rheumatology, Northwestern University, Chicago, IL, USA
3 Feinberg School of Medicine, Department of Medicine, Division of
Gastroenterology and Hepatology, Northwestern University, Chicago, IL, USA
4 Division of Rheumatology, Department of Internal Medicine, University of
Utah, Salt Lake City, UT, USA
5 Division of Gastroenterology and Hepatology, Department of Internal
Medicine, University of Utah, Salt Lake City, UT, USA
Background: A patulous esophagus is a well-recognized systemic sclerosis
(SSc) feature. Symptoms related to esophageal dysfunction impact health-related
quality of life. The project goal was to determine the association between
intraluminal esophageal diameter on high-resolution computed tomography of the chest
(HRCT) and SSc disease burden as assessed by a patient-reported outcome (PRO)
instrument that has been validated in SSc patients.
Methods: A cross-sectional, retrospective analysis of Northwestern
Scleroderma Registry (NSR) patients with available HRCT exams and PRO instruments
completed within 6 months were analyzed. Anxiety, depression, fatigue, pain
interference, physical function, sleep disturbance, ability to participate in social
roles and activities domains from the Patient-Reported Outcomes Measurement
Information System (PROMIS)-29 Profile were analyzed. Patients were categorized into
two esophageal diameter groups (normal; <10 mm vs. dilated; defined as >10mm
in the proximal and mid esophagus, or >15 mm in the distal esophagus). Analysis
was performed using SAS with significance set at p<0.05.
Results: There were 151 NSR SSc patients with HRCT and PROMIS-29 (mean,
SD) for duration between HRCT and PRO completion was 1.9 mo, 1.8). No significant
differences in age (p = 0.85), sex (p = 0.03), race (p = 0.47), tobacco use (p =
0.20), body mass index (p = 0.84), presence of SSc-specific antibodies (p = 0.11
SCL70; 0.135 centromere; 0.78 RNApol3), modified Rodnan Skin Score (mRSS) (p =
0.81), and disease duration (p = 0.08) were identified in the normal vs. dilated
esophageal diameter populations. Anxiety (p = 0.24), depression (p = 0.26), fatigue
(0.34), pain interference (p = 0.26), physical function (p = 0.71), sleep
disturbance (p = 0.18) and satisfaction (p = 0.29) were not significantly different
between the two groups. There was significantly more proton pump inhibitor (PPI) use
(p<0.0001) in the dilated esophagus group (n = 108, 88%) compared to normal
caliber group (n = 14, 54%).
Conclusions: In patients with SSc, esophageal diameter on HRCT does not
appear to be associated with demographic or SSc disease-specific clinical
parameters. Proton pump inhibitor use is more common in SSc patients with a dilated
vs. normal esophageal diameter. Proton pump inhibitor use may improve health-related
quality of life in patients with SSc as assessed by PROMIS-29. Future studies should
be designed to determine whether initiation of PPI is appropriate in asymptomatic
patients with SSc.
IL7 AXIS IN SYSTEMIC SCLEROSIS: ANALYSIS OF LIGAND AND RECEPTOR POLYMORPHISMS
AND SERUM IL7 LEVELS
Fonseca C1*, Moinzadeh P2*, Host L1,
Riemekasten S1, Guerra SG1, Khan K1, Abraham
D1, Krieg T2, Ong V1, Denton
CP1
1 Centre for Rheumatology and Connective Tissue Diseases, University
College London, London, UK
2 Department if Dermatology, University of Cologne, Cologne, Germany
*Fonseca C and Moinzadeh P contributed equally to this work.
Background: Systemic sclerosis (SSc) is characterized by vascular
damage, autoimmunity and fibrosis. Differences in expression and signaling through
IL7 receptor (IL7R) have been identified as factors determining clinical activity in
other autoimmune diseases. We conducted a candidate gene association study of tagged
polymorphisms in the IL7/IL7R in SSc and also we investigated whether differences in
the IL7 serum level exist among different SSc subsets.
Methods: Initially seven IL7 and 15 ILR SNPs were genotyped in 728 SSc
patients and 260 healthy controls. Four SNPs with significant association were
tested in an additional 257 SSc and 106 controls. All patients and controls were UK
Caucasian and classified according the autoantibody status and organ involvement.
Furthermore, serum of 130 SSc patients and 44 healthy controls were used to
determine the IL7 level, using a high sensitivity Human IL7 assay. The distribution
of IL7 values by groups were analyzed using the non-parametric Mann-Whitney
test.
Results: There was a significant difference between SSc patient's
positive for anti-topoisomerase I antibody (ATA) versus ATA negative in four SNPs
located in the IL7R region (rs11567685 rs11567751, rs987107 and rs3194051) and one
in IL7 gene (rs2717548). The association with ATA remained significant for
rs11567751 and rs3194051 when both groups were combined. Association of rs987107
with pulmonary hypertension was found in the second stage. Serum levels of IL7 were
increased in patients diffuse SSc (14.90 ± 7.48 ng/ml) compared to patients with the
limited form (8.95 ± 5.27 ng/ml; p<0.0001. ACA positive had lower median IL7
serum level (p<0.005), whereas patients with anti-RNAP antibodies showed a
significant higher IL7 serum level (p<0.007).
Conclusions: Together, these results suggest potential genetic and
functional significance of IL7 in determining sub phenotype in SSc. An association
of SNPs in the IL7R region with the presence of ATA and significant differences of
IL7 serum levels, IL7 expression may suggest potential as a candidate marker in
disease assessment.
NOVEL MACHINE LEARNING CLASSIFIER ACCURATELY PREDICTS INTRINSIC MOLECULAR
SUBSETS FOR PATIENTS WITH SYSTEMIC SCLEROSIS
Franks JM1,2, Martyanov V1, Cai G1,
Whitfield ML1,2
1 Department of Molecular and Systems Biology, Geisel School of Medicine
at Dartmouth, Hanover, NH, USA
2 Quantitative Biomedical Sciences, Geisel School of Medicine at
Dartmouth, Hanover, NH, USA
Background: High-throughput gene expression profiling of skin biopsies
from patients with systemic sclerosis (SSc) has identified four “intrinsic” gene
expression subsets conserved across multiple cohorts and tissues: inflammatory,
fibroproliferative, normal-like, and limited. In order to classify patients in
clinical trials or for diagnostic purposes, supervised methods that can assign a
single sample to a molecular subset are required. Here, we introduce a novel machine
learning classifier, which is a robust predictor of intrinsic subset.
Methods: Three independent SSc cohorts (Milano et al. 2008, Pendergrass
et al. 2012, Hinchcliff et al. 2013) with gene expression data and intrinsic subset
assignments were carefully curated and merged to create a training dataset covering
a broad set of 297 skin biopsies representing 97 unique patients. Supervised machine
learning algorithms were rigorously trained and internally evaluated using repeated
three-fold cross-validation. We performed external validation using two independent
SSc datasets: Chakravarty et al. 2015, which contains 16 samples/8 patients and
Gordon et al. 2015, which contains 12 samples/6 patients. Additionally, we validated
the classifier on a cohort of SSc patients with gene expression data independently
generated by Assassi et al. 2015 (102 samples/97 patients).
Results: Over repeated cross-fold validation, gene expression features
were selected using multinomial elastic net and incorporated into the final model
which achieved an average classification accuracy of 88%. All molecular subsets were
classified with high average sensitivity and specificity, particularly inflammatory
(83.3% sensitivity, 95.8% specificity) and fibroproliferative (89.7% sensitivity,
94.1% specificity). Through multiple rounds of external validation, the classifier
maintained an accuracy ranging from 70% to 85%. In a thorough re-analysis of gene
expression data from Assassi et al., we identified subsets of patients that
represent the canonical inflammatory, fibroproliferative, and normal-like
subsets.
Discussion: In this study, we have developed a highly accurate and
reliable classifier for SSc molecular subsets for single samples trained and tested
on diverse cohorts comprised of 427 skin biopsies from 208 independent patients.
Internal and external validation of the supervised classifier suggests that machine
learning methods are powerful and robust for stratifying intrinsic subsets and may
have the potential to aid clinical decision-making for patients with SSc.
ELEVATED MECP2 EXPRESSION IN DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS DERMAL
FIBROBLASTS IS ASSOCIATED WITH ANTI-FIBROTIC EFFECTS
He Y, Tsou PS, Khanna D, Sawalha AH
Division of Rheumatology, Department of Internal Medicine, University of Michigan,
Ann Arbor, Michigan, USA
Background: Systemic Sclerosis (SSc) is a multisystem autoimmune
connective tissue disorder characterized by vascular injury and fibrosis of the skin
and internal organs. Methyl-CpG-binding protein 2 (MeCP2) is a transcription
activator or repressor depending on its interacting complexes. Increasing evidence
shows that MeCP2 is closely involved in lung, liver, and kidney fibrosis. In this
study, we aim to elucidate the role of MeCP2 in dermal fibroblasts from patients
with SSc.
Methods: Dermal fibroblasts were isolated from healthy controls or
patients with diffuse cutaneous SSc (dcSSc). MeCP2 expression was analyzed by
qRT-PCR and western blotting. A scratch wound healing assay was used to evaluate
fibroblast migration in vitro. MeCP2 overexpression was achieved by transfecting
fibroblasts with MeCP2 DNA plasmids, while transfection with empty vectors was used
as controls. MeCP2 knockdown was done using MeCP2 siRNA.
Results: MeCP2 expression was increased by 1.8-fold (P = 0.02) in dcSSc
fibroblasts (n = 6) compared to normal fibroblasts (n = 7) at the protein level.
However, no change was observed at the mRNA level, suggesting that dysregulation of
MeCP2 in dcSSc occurs at the post-transcriptional level. To investigate functional
implications of elevated MeCP2 in dcSSc fibroblasts, we performed a wound healing
assay. Overexpression of MeCP2 in normal fibroblasts inhibited cell migration at
24hrs post scratch (n = 4, p = 0.02) while MeCP2 knocked down dcSSc fibroblasts
showed higher wound repair ability than negative controls at 24hrs (n = 5,
p<0.01). In addition, the mRNA levels of pro-fibrotic genes like α-smooth muscle
actin and collagen 1a1 were significantly downregulated in MeCP2 overexpressing
normal fibroblasts (n = 6, p = 0.03) while anti-fibrotic PPAR-γ was upregulated (n =
6, p<0.01), suggesting a potential role of MeCP2 in myofibroblast
differentiation.
Conclusions: MeCP2 exerts anti-fibrotic effects in dcSSc by reducing
cell migration and myofibroblast differentiation in dermal fibroblasts. MeCP2
overexpression in dcSSc fibroblasts might be a defense mechanism to counteract the
pro-fibrotic nature of the disease. Identifying genome-wide transcriptional targets
of MeCP2 in fibroblasts via ChIP-seq and RNA-seq, which is ongoing in our
laboratory, will help comprehensively demonstrate the role of MeCP2 in SSc. MeCP2
might be a novel target to restore normal skin function in this devastating
disease.
IN VITRO SKIN MODELS MIMIC FIBROGENIC SIGNATURES OF SYSTEMIC SCLEROSIS
Huang M1, Baugh L2, Popovich D1, Cai
G1, Kosarek NN1, Black LD2, Garlick
J3, Whitfield ML1
1 Department of Molecular Systems Biology, Geisel School of Medicine at
Dartmouth, Hanover, NH, USA
2 Department of Biomedical Engineering, Tufts University School of Dental
Medicine, Boston, MA, USA
3 Department of Diagnostic Sciences, Tufts University School of Dental
Medicine, Boston, MA, USA
Skin fibrosis is a common feature of systemic sclerosis (SSc). Skin equivalent
tissues incorporating multiple cells types have not been previously reported for
SSc. We have previously shown that human skin equivalents (hSE) are morphologically
similar to human skin and can be used to study fibroblast behavior in a realistic
3D, tissue microenvironment in which epithelial-dermal cross-talk is indispensable
for recreating cell phenotypes observed in vivo. To understand if fibroblasts from
SSc patients (SScDF) preserve their in vivo-fibrotic phenotypes in a 3D skin-like
tissue in vitro, we incorporated SScDF isolated from skin biopsies of SSc patients
and site-, age-matched normal control dermal fibroblasts (NDF) into bovine Type I
collagen to fabricate hSE. We then evaluated their ability to modify the properties
of the ECM. Atomic force microscopy (AFM) was used to evaluate stromal rigidity.
SScDF hSE showed a 4-fold higher stiffness than in NDF hSE. This suggested that
SScDF modified the collagen matrix to increase stiffness similar to what is observed
in SSc cutaneous fibrosis in patients. Since excess collagen production is a
hallmark of SSc, de novo ECM production of fibroblasts was
stimulated with ascorbic acid for five weeks to secrete and assemble a granulation
tissue-like self-assembly (SA) tissue. SScDF produced thicker ECM compared to NDF
assessed by histological stain. Consistent with the observation in hSE, SScDF SA
showed an increased rigidity of de novo ECM deposition by AFM in
comparison with NDF SA. SScDF-derived ECM contained more collagen than NDF-derived
ECM as determined by hydroxyproline assay. Additionally, DNA microarray analyses of
SA tissues showed inflammatory innate signaling genes observed in SSc skin biopsies
were increased in SScDF SA tissues. These data demonstrate that relative to NDF,
SScDF can reproducibly generate in vitro skin-like tissues with the thicker dermis,
increased ECM stiffness, and show increased expression of innate immune signaling
genes in the absence of other cell types. Over all, this study suggests that primary
dermal fibroblasts from SSc patients can consistently mimic their in vivo fibrotic
phenotypes when grown in skin-like 3D tissues.
TYPE VI COLLAGEN FORMATION: A NEW OBJECTIVE BLOOD-BASED MARKER REFLECTING
FIBROSIS OF THE SKIN IN SYSTEMIC SCLEROSIS
2 Department of Dermatology, Bispebjerg Hospital, Copenhagen, Denmark
Background: Systemic sclerosis (SSc) is characterized by fibrosis of the
skin. The dermis of the skin is rich in type I and III collagen, but other collagens
as type VI collagen are present in the skin. There is a lack of objective disease
activity markers in SSc for frequent assessment of patients.
Objectives: The objective is to examine blood-based markers of type I,
III, and VI collagen formation as surrogates of disease activity and skin fibrosis
in SSc.
Methods: SSc patients fulfilling the ACR criteria (n = 121) were
included and divided into limited SSc (lSSc, n = 79) and diffuse SSc (dSSc, n = 42).
Markers of type I, III and VI collagen formation (P1NP, PRO-C3 and PRO-C6,
respectively) were measured in serum by ELISA.
Results: PRO-C3 and PRO-C6 were significantly higher early (<4 years)
dSSc compared to early lSSc patients (both p = 0.006). PRO-C6 was significantly
elevated in early dSSc compared to late dSSc (p = 0.04) and increased in late dSSc
compared to late lSSc (p = 0.02). Both PRO-C3 and PRO-C6 correlated with mRSS when
adjusted for age, gender, and BMI with a R-partial of 0.36 (p = 0.0001) and 0.29 (p
= 0.002), respectively.
Both PRO-C3, and PRO-C6 could separate the patient group having the highest tertile
of mRSS from the rest with an AUC (95%CI) of 0.73 (0.62-0.82), and 0.74 (0.63-0.83),
respectively.
PRO-C3 and PRO-C6 performed equally well for identification of patients with the
highest skin score (highest tertile vs. the middle and lower tertile) with a
relative risk of 2.3. The risk of being in the highest tertile of mRSS compared to
the lowest were significantly higher with a high PRO-C6 (relative risk 2.6) compared
to PRO-C3 (relative risk: 2.2).
Conclusions: Markers reflecting fibrosis (measures of type III and VI
collagen formation) could be novel objective, and potentially predictive, biomarkers
of disease activity and severity.
Approval number: H-B-2008-131.
LONG-TERM OUTCOMES IN SYSTEMIC SCLEROSIS ASSSOCIATED PULMONARY ARTERIAL
HYPERTENSION FROM THE PULMONARY HYPERTENSION ASSESSMENT AND RECOGNITION OF
OUTCOMES IN SCLERODERMA REGISTRY (PHAROS)
Kolstad KD1, Li S1, Steen V2, Chung
L1 on behalf of the PHAROS investigators
1 Division of Immunology and Rheumatology, Department of Medicine,
Stanford University School of Medicine, Palo Alto, CA, USA
2 Division of Rheumatology, Georgetown University Medical Center,
Washington DC, USA
Background: Pulmonary arterial hypertension (PAH) is a leading cause of
death in patients with systemic sclerosis (SSc). We sought to examine long-term
survival and associated clinical features in patients with SSc-PAH.
Methods: Pulmonary Hypertension Assessment and Recognition of Outcomes
in Scleroderma is a prospective registry of SSc patients at high risk for PAH or
with incident PH based on right heart catheterization (RHC) at enrollment. Incident
World Health Organization Group I PAH patients were included in this analysis.
Baseline characteristics and cause of death in the short (<4 year) and long (≥4
years) term were assessed. Kaplan-Meier survival curves were generated for the
overall cohort, restricted to PAH-related deaths, and patient groups stratified by
initial PAH therapy. Baseline characteristics were compared between long-term
survivors and patients who died of PAH-related causes in the short-term.
Results: A total of 160 incident SSc-PAH patients were included. The
overall 1, 3, 5, and 8-year cumulative survival rates were 95%, 75%, 63%, and 49%,
respectively. When events were restricted to deaths due to PAH-related causes, the
1, 3, 5, and 8- year survival rates were 97%, 83%, 76%, and 76%, respectively.
Long-term survivors were more likely to die of causes unrelated to SSc and those who
died in the short-term were more likely to die of PAH related causes. The group of
patients with initial single or combination oral therapy had improved survival
compared to those started on systemic prostacyclin either as monotherapy or in
combination, likely related to more severe disease at diagnosis in the latter group
(p<0.0001). At baseline, long-term survivors had significantly longer 6-minute
walk distance (p = 0.022), higher% predicted diffusion capacity (DLCO) (0.009),
lower forced vital capacity to DLCO ratio (p = 0.008), and lower systolic pulmonary
artery pressure on echocardiogram (p = 0.0001). They also had significantly better
hemodynamics.
Conclusions: These data suggest that if PAH is well controlled in the
first several years after diagnosis, SSc-PAH patients have favorable outcomes.
However, there is a sub-group of patients who quickly progress and die of PAH
related causes in the short-term. Further research to identify biomarkers of this
poor prognostic sub-group is ongoing.
SEROLOGICAL BIOMARKERS OF ECM TURNOVER ARE ASSOCIATED WITH SKIN FIBROSIS AND
LUNG INVOLVEMENT IN SYSTEMIC SCLEROSIS
1 The First Department of Internal Medicine, University of Occupational
and Environmental Health, Kitakyushu, Japan
2 Nordic Bioscience, Biomarkers and Research, Herlev, Denmark
Background: Extracellular matrix (ECM) unbalance of the skin is a
hallmark of systemic sclerosis (SSc). However, currently there is no objective tool
to monitor the ECM unbalance in SSc patients allowing for better understanding of
the disease stage and activity.
Objectives: We investigated the potential of serological biomarkers of
ECM turnover (collagen formation and degradation) as biomarkers of skin fibrosis and
internal organ involvement in SSc patients.
Methods: Peripheral blood obtained from 79 SSc patients and 19 healthy
subjects was included in the study. Type I, III, IV, V and VI collagen formation
(P1NP, PRO-C3, P4NP7S, PRO-C5, PRO-C6) and degradation (C3M, C4M2, C5M, C6M)
biomarkers were measured in serum by ELISA. The extent of internal organ involvement
(renal, lung, vasculopathy and gastrointestinal) were recorded.
Results: SSc patients compared to healthy individuals had higher levels
of C5M, C6M and PRO-C6 (p = 0.0001, p<0.0001, p<0.0001, respectively). C4M2,
PRO-C3 and PRO-C6 was associated with skin fibrosis assessed by mRSS (Spearman's rho
= 0.24, 0.39 and 0.29, respectively). Patients with signs and manifestation of lower
gastrointestinal lesion compared to patients without lesion had higher levels of C3M
(median 10.7 vs 13.8ng/mL, p = 0.017). PRO-C6 was higher in patients with pulmonary
hypertension compared to patients without any signs of pulmonary hypertension
(median 9.6 vs 14.3ng/mL, p = 0.006) with a Spearman's correlation coefficient of
0.31. C6M was higher in patients with definite pulmonary hypertension (>1)
compared to patients with signs of or no pulmonary hypertension (median 29.9 vs
22.1, p = 0.026).
Conclusions: Serological biomarkers of type V and VI turnover (C5M, C6M,
PRO-C6) were associated with SSc. Especially, turnover of type VI collagen was
associated with skin sclerosis, pulmonary hypertension, interstitial pneumonia and
renal dysfunction. This study indicates that biomarkers of collagen turnover have a
potential as new objective tool of skin fibrosis and internal organ involvement in
SSc.
LOW CONCENTRATIONS OF S-NITROSOTHIOLS IN THE PLASMA OF PATIENTS WITH RAYNAUD'S
PHENOMENON (RP) AND SYSTEMIC SCLEROSIS (SSc)
Kundu D, Abraham D, Black CM, Denton CP, Bruckdorfer KR
Centre for Rheumatology and Connective Tissue Diseases, UCL Division of Medicine,
London, UK
Background: Systemic sclerosis (SSc) is an autoimmune, connective tissue
disease of skin and internal organs leading to extensive fibrosis. The free radical,
nitric oxide (NO) is suggested in the pathogenesis of SSc. NO has multiple roles in
signal transduction. S-nitrosothiols (RSNOs) are long-lived bioactive forms of NO
involved in redox regulation of protein by S-nitrosylation.
Objective: In this study we have measured plasma concentrations of
S-nitrosothiols in RP and SSc patients.
Methods: Venous blood was collected from 16 Raynaud's phenomenon (RP)
patients, 45, systemic sclerosis (SSc): 34 limited SSc (lcSSc) and 11 with diffuse
cutaneous disease (dSSc). Twenty six healthy subjects were used as controls. Plasma
S-nitrosothiol concentrations were measured by chemiluminescence. The measurements
were related to the extent of biological age, capillary skin scores and disease
duration.
Results: Plasma RSNO levels in patients with RP and in those with SSc
was significantly lower compared to the concentrations in control subjects. In SSc,
plasma S-nitrosothiols were often below the level of detection (1nM). Plasma RSNO
levels in healthy controls (6.0 ± 0.8 nM; n = 26) were significantly higher than
those of all patients (RP and SSc) (1.38 ± 0.4 nM; n = 61, P = 2.58
× 10−8). RP patients alone had lower levels than those of controls (2.12
± 0.6 nM; n = 16, P = 0.002). Even lower levels were found in SSc
patients (1.17 ± 0.5 nM; n = 45, P = 2.58 × 10−8).
Within the SSc subgroups, the RSNO levels were again significantly lower than
controls (lcSSc 1.31 ± 0.37 nM; n = 34, P = 10−6; dcSSc,
0.73 ± 0.41; n = 11; P = 0.00254) but not between each other (P =
0.34). There was no correlation between plasma RSNO and age or gender within any of
the patient groups.
Conclusions: Low concentrations of S-nitrosothiols measured in blood of
patients with RP and SSc indicate a profound disturbance in NO metabolism which may
play a key role in disease progress.
RESOLVIN D1 IN PREVENTIVE REGIMEN REDUCES PERITONEAL FIBROSIS IN MICE
1 Northwestern Scleroderma Program, Feinberg School of Medicine, Chicago,
USA
2 Department of Rheumatology, University Medical Centre Ljubljana,
Slovenia
3 University of Primorska, Faculty of Mathematics, Natural Science and
Information Technology, Koper, Slovenia
4 Center for Experimental Therapeutics and Reperfusion Injury, Department
of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, USA
Background: Peritoneal fibrosis following dialysis as well as
post-surgery is a major complication with no effective treatment. Progressive
fibrosis occurs in submesothelium, a thin layer of connective tissue underlying
single-layered mesothelial barrier. Resolvins are naturally occurring, omega-3 fatty
acid-derived, proresolution mediators exhibiting anti-inflammatory effects in
different mouse models. The roles and mechanisms of action of resolvins in fibrosis
are not well understood. We sought to investigate the actions of resolvin D1 (RvD1)
in the peritoneal fibrosis model.
Materials and methods: Peritoneal fibrosis was induced in C57BL/6J mice
with 0.1% chlorhexidine gluconate (CG) injections. RvD1 (100 or 300 ng) was used
daily for 3 weeks in preventive regimen, or given only during the last 2 weeks in
treatment regimen (n = 5/group). Peritoneum samples were harvested for analysis,
stained with Masson trichrome and hematoxylin/eosin and thickness measured at 10
sites per high power field (HPF; ×400) (30 measurements/mouse). Slides were
immunostained for F4/80 and α-smooth muscle actin. mRNA was analyzed by qPCR.
Results: 3 weeks of CG induced a dramatic, 6-fold thickening of
submesotelial layer. Thickeness was significantly reduced (median 40%) when mice
received RvD1 at 100ng/mouse in preventive regimen. When RvD1 was used in treatment
regimen the submesotelial layer thickness was not significantly reduced. In
CG-treated group collagen expression was increased (median) 20-32-fold and
fibronectin 17-fold. Lower dose of RvD1 in preventive regimen decreased expression
of these matrix genes 9-11 and 8-fold respectively. Both RvD1 doses in preventive
regimen significantly decreased the number of myofibroblasts accumulating within the
fibrotic mesothelial layer with CG treatment. Marked increase of macrophages
observed in CG-treated group was prevented by 50% with low dose RvD1 in both
preventive and treatment regimens (median 47 cells/HPF to 21-24 cells/HPF). RvD1 did
not selectively effect M1 or M2 polarization of macrophages or decreased expression
of mRNA of inflammatory markers SAA1/2, SAA3, TNFα in the lesional fibrotic
tissue.
Conclusions: RvD1 showed potent anti-fibrotic actions in preventive
regimen of a peritoneal fibrosis model, emphasizing the potential contribution of
omega-3 fatty acid DHA as RvD1 precursor in diet. These results support further
investigation of proresolving mediators in prevention and treatment of fibrosis.
TELOMERE LENGTH IN SYSTEMIC SCLEROSIS-ASSOCIATED PULMONARY FIBROSIS
1 Northwestern Scleroderma Program, Feinberg School of Medicine, Chicago,
USA
2 Department of Rheumatology, University Medical Centre Ljubljana,
Slovenia
3 University of Primorska, Faculty of Mathematics, Natural Science and
Information Technology, Koper, Slovenia
4 Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns
Hopkins University School of Medicine, Baltimore, USA
Background: Systemic sclerosis (SSc) is commonly associated with
pulmonary fibrosis. While SSc-associated pulmonary fibrosis shares clinical,
radiological and pathological features with idiopathic pulmonary fibrosis (IPF),
these two fibrosing lung diseases have distinct risk factors, histopathological
features, immunological alterations, temporal evolution, response to therapy and
outcomes. Recent studies have demonstrated mutations in telomerase and telomere
maintenance genes most commmonly mediate familial pulmonary fibrosis (FMF) by
causing telomere shortening. Here, we sought to assess telomere length in patients
with SSc-associated ILD.
Materials and methods: Telomere length was examined in 33 SSc patients.
Peripheral blood lymphocytes and granulocytes were analyzed by flow cytometry and
fluorescence in situ hybridization (FISH). Telomere length was expressed as the
difference from median value in age-matched healthy controls (delta telomere
length). Pulmonary function studies and chest CT imaging were analyzed. In three
patients with the shortest telomeres, the DNA sequence of FMF genes TERT, TR, DKC1,
TINF2, NAF1, RTEL1 was investigated.
Results: Median delta telomere length in granulocytes and lymphocytes
was similar in SSc patients to age-matched healthy controls. SSc patients with ILD
(n = 15) showed shorter telomeres in lymphocytes compared to those without ILD (n =
18) (median −0.420, range −2.79 to 0.06 vs. median −0.105, range −1.31 to 1.60; p =
0.07, Mann Whitney test). Moreover, topoisomerase-1 antibody (ATA)-positivity (n =
9) was associated with shorter lymphocyte telomeres compared to ATA-negative
patients (n = 24) (median −0.96, range −2.79 to 0.04 vs. median −0.16, range −1.20
to 1.60; p = 0.02). Delta telomere length in lymphocytes positively correlated with
age and age at disease onset. Three SSc patients with extremely short telomeres
(<-2 kb delta telomere length) were younger, had ILD, and were ATA-positive.
However, no mutations in genes related to telomere function were detected in this
group.
Conclusions: Telomere length is modestly reduced in lymphocytes in a
subset of SSc patients with early-age disease onset, ILD and ATA-positivity. Short
telomeres in SSc do not appear to be associated with mutations in telomere
maintenance genes. These findings highlight fundamental differences in idiopathic
compared to SSc-associated pulmonary fibrosis, which might be relevant for the
diagnosis, prognostication and management of patients suffering from these
conditions.
MESENCHYMAL STEM CELLS: PHENOTYPE MODULATION IN THE TREATMENT OF SCLERODERMA
BY CELL INJECTION
Lee R1, Reese C1, Makusij A1, Wolf
W1, Lopez GC1, Bonner M1, Silver
RM1, Hoffman S1,2, Tourkina E1,2
1 Division of Rheumatology and Immunology, Department of Medicine, Medical
University of South Carolina, Charleston, SC, USA
2 Regenerative Medicine and Cell Biology, Medical University of South
Carolina, Charleston, SC, USA
Introduction: Mesenchymal stem cells (MSCs) are non-hematopoietic
multipotent progenitor cells. The injection of MSCs has been proposed to be a
potential treatment for a variety of diseases including scleroderma (SSc). We
believe that this approach has value, but can be vastly improved by modulating the
function of MSCs. We propose to test the hypothesis that modifying the function of
MSCs with perturbants of CXCR4 and CCR5 function can be used to enhance the
beneficial effects of MSC injection.
Methods: MSCs (also called adipose derived stem cells, ADSCs) were
isolated from fat tissue. MSCs were cultured either in maintenance medium or
adipocyte induction medium for ten days with or without addition of
TGFβ, CCR5 inhibitor (maraviroc (MVC)), or CXCR4 inhibitor (AMD3100).
α-smooth muscle actin (ASMA), caveolin-1, and fatty acid binding protein 4 (FABP4)
expression were determined by Western blot and immunohistochemistry.
Results: MSCs from healthy subjects or saline treated mice contain
relatively high levels of caveolin-1 and low levels of the myofibroblast marker
ASMA, while the converse is true for MSCs from SSc patients or bleomycin treated
mice. A fibrogenic phenotype can also be induced in control MSCs by TGFβ treatment.
MSCs can differentiate into adipocytes when treated with Inducing Medium containing
troglitazone. While TGFβ promotes the fibrogenic differentiation of MSCs, it
inhibits their adipogenic differentiation. We find that the CCR5 antagonist MVC and
the CXCR4 antagonist AMD3100 are each capable of inhibiting fibrogenic
differentiation induced by TGFβ or of reversing the inhibition of adipogenic
differentiation induced by TGFβ.
Conclusions: MSCs from mice or humans with fibrotic disease exhibit
enhanced fibrogenic differentiation and inhibited adipogenic differentiation.
Modulation of the fate of MSCs by chemokine receptor antagonists has potential for
improving the treatment of SSc.
DIGITAL ARTERY VOLUME INDEX BY NON-CONTRAST MAGNETIC RESONANCE ANGIOGRAPHY OF
THE HAND AS QUANTITATIVE MEASURE OF NEOINTIMA PROLIFERATION IN RAYNAUD'S
PHENOMENON OF SYSTEMIC SCLEROSIS
Lettieri G1-3, Abignano G1,2,4, Eng S1,2,
Ridgway JP2, Evans R2, Buch M1,2, Emery
P1,2, O'Connor P2,5, Del Galdo
F1,2
1 Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of
Leeds, Leeds, UK
2 NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching
Hospitals NHS Trust, Leeds, UK
3 Department of Radiology, A.O.U. San Giovanni di Dio e Ruggi d'Aragona,
Salerno, Italy
4 Rheumatology Institute of Lucania (IReL), Rheumatology Department of
Lucania, San Carlo Hospital of Potenza and Madonna delle Grazie Hospital of Matera,
Potenza, Italy
5 Department of Radiology, Chapel Allerton Hospital, Leeds Teaching
Hospitals NHS Trust, Leeds, UK
Background: Neo-intima proliferation is a patognomonic feature of
Systemic Sclerosis (SSc) vasculopathy, responsible for the severity of Raynaud's
phenomenon and contributing to the major vascular manifestations in patients with
this condition.
Objectives: The aim of this study was to identify a surrogate outcome
measure of neo-intima proliferation in SSc using no-contrast time of flight (TOF)
magnetic resonance angiography (MRA) of digital arteries.
Methods: Twenty hands were scanned from 8 SSc patients (of which 4 with
current DU) and 10 healthy volunteers (HV). MRI scans were performed on a 3T
Magnetom Verio (Siemens Healthcare). A VIBE 3D T1 scan of 3 minutes and a 2D TOF
sequence of 8 minutes were performed using Time of Echo of 5 milliseconds (ms) and
repetition time (TR) ranging from 24 to 72 ms as needed. Digital arterial volume
index (DAVIX) was calculated as % of the ratio of digital arteries flow volume and
the respective finger volume. Cochin hand function questionnaire and capillary
pattern by nail fold video-capillaroscopy were assessed in patients and HV. DAVIX
values were analysed by Graph prism software by two-way-ANOVA or unpaired two-tailed
t-test as appropriate.
Results: Digital arteries were visualized at TR of 24 ms in 17/20 hands.
Three HV required increased TR. Seventeen native TOF images were post-processed to
obtain the volume of the digital arteries by OsiriX segmentation. DAVIX of HV was in
average 1.21 (±0.39) vs 0.37 (±0.18) of SSc patients (p<0.0001). These results
correlated with Cochin hand function score that was obviously worse in the SSc
patients vs HV (p<0.05). Within the SSc patients, fingers with DU had an average
DAVIX of 0.24 (±0.08) vs 0.40 (±0.18) of the fingers without DU (p = 0.02). DAVIX of
the different fingers in HV had no statistically significant difference.
Conclusions: Our study provides face and content validity of DAVIX as
non-invasive contrast free surrogate outcome measure of neo-intima proliferation in
SSc. Validation in large cohorts including demonstration of sensitivity to change
and predictive value for severe vasculopathy are warranted on large prospective
cohorts.
SKIN-RESIDENT EFFECTOR MEMORY CD8+CD28- T CELLS EXHIBIT A PRO-FIBROTIC
PHENOTYPE IN PATIENTS WITH SYSTEMIC SCLEROSIS
Gang Li1, Larregina AT1-3, Domsic RT4,
Stolz DB5, Medsger TA Jr.4, Lafyatis R4,
Fuschiotti P1,4
1 Department of Immunology, University of Pittsburgh School of Medicine,
Pittsburgh, USA
2 Department of Dermatology, University of Pittsburgh School of Medicine,
Pittsburgh, USA
3 McGowan Institute for Regenerative Medicine, University of Pittsburgh
School of Medicine, Pittsburgh, USA
4 Department of Medicine, Division of Rheumatology and Clinical
Immunology, University of Pittsburgh School of Medicine, Pittsburgh, USA
5 Department of Cell Biology, University of Pittsburgh School of Medicine,
Pittsburgh, USA
Background: Loss of CD28 expression by CD8+ T cells occurs with age and
during chronic inflammatory conditions. CD8+CD28- T cells are a heterogeneous cell
subpopulation whose function ranges from immunosuppressive to effector. Here we
analyzed the role of CD8+CD28- T cells in the pathogenesis of systemic sclerosis
(SSc), a connective tissue disorder characterized by autoimmunity, vasculopathy and
extensive cutaneous and visceral fibrosis.
Methods: We purified CD8+ T cells from the blood and lesional skin of
patients with SSc. Flow cytometry was used to determine cell surface expression of
skin-homing receptors, markers of residency in the skin and phenotype. Cytokine
production was determined by intracellular cytokine staining. Co-culture experiments
were performed to determine the ability of SSc CD8+ T cells to induce a pro-fibrotic
phenotype and tissue damage in dermal fibroblasts.
Results: The frequency of CD8+CD28- T cells is increased in the blood
and affected skin of SSc patients, independent of patient age, and correlates with
the extent of skin fibrosis. The majority of skin-tropic CD8+CD28- T cells are
resident in the skin lesions of patients in the early stage of the disease, exhibit
an effector memory phenotype and present a strong cytolytic activity ex
vivo. Skin-resident and circulating SSc CD8+CD28- T cells produce high
levels of the pro-fibrotic cytokine IL-13, which induces collagen production by
normal and SSc dermal fibroblasts.
Conclusions: Our findings indicate that CD8+CD28- T cells represent a
pathogenic T-cell subset in SSc and likely play a critical role in the early stage
of SSc skin disease.
IDENTIFICATION OF PIRIN AS A NOVEL THERAPEUTIC TARGET OF CCG-222740 IN
FIBROSIS
Lisabeth EM1, Kahl D2, Gopallawa I1, Haynes
SE3, Misek SA1, Khanna D4, Fox
DA4, Martin BR3, Larsen SD2,5, Neubig
RR1
1 Department of Pharmacology & Toxicology, Michigan State University,
East Lansing, MI, USA
2 Department of Medicinal Chemistry, University of Michigan, Ann Arbor,
MI, USA
3 Department of Chemistry, University of Michigan, Ann Arbor, MI, USA
4 Department of Internal Medicine, Division of Rheumatology, University of
Michigan Medical Center, Ann Arbor, MI, USA
5 Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of
Michigan, Ann Arbor, MI, USA
The CCG-203971 series of compounds was identified through a phenotypic screen to
reduce gene transcription induced by the Rho/MRTF/SRF pathway. They also inhibit
fibrosis in several in vitro and in vivo models; they strongly reduce TGF-b mediated
expression of a-SMA in vitro and reduce skin thickness and collagen production in a
bleomycin skin fibrosis model in vivo. However, the molecular target(s) of
CCG-203971 and its optimized analog, CCG-222740, have not yet been identified.
Therefore, we undertook a mass spectrometry proteomic approach using an immobilized
CCG-222740 probe to identify molecular targets. These results identified an
intranuclear iron-binding protein, pirin, as a potential target of CCG-222740. Pirin
is a highly conserved and ubiquitously expressed nuclear protein that interacts with
NF-I/CTF1 and NF-kB transcription factors. Inhibition of pirin has been shown to be
beneficial in melanoma and ovarian cancer models; however, pirin has not yet been
implicated in fibrosis. CCG-222740 binds to purified pirin in vitro and reduction of
pirin using siRNA reduces TGF-b induced ACTA2 expression in primary dermal
fibroblasts, a hallmark of the fibroblast to myofibroblast transition. In addition,
a bioinformatics analysis of genes regulated by pirin and by CCG-1423 (a early
analog of CCG-222740) shows significant overlap. Overall, these studies suggest that
pirin may be a target of our series of compounds and a novel potential therapeutic
target in fibrosis.
Acknowledgments: Supported by NIH R01 GM115459-02 and R01 AR0660
49-03.
INTRINSIC SUBSETS ARE CONSERVED ACROSS MULTIPLE ORGANS IN SYSTEMIC
SCLEROSIS
1 Department of Molecular and Systems Biology, Geisel School of Medicine
at Dartmouth, Hanover, NH, USA
2 Program in Quantitative Biomedical Science, Geisel School of Medicine at
Dartmouth, Hanover, NH, USA
3 Stanford University School of Medicine, Palo Alto, CA, USA
4 Division of Rheumatology, University of California, San Francisco,
USA
5 Pulmonary Division, Department of Medicine, University of California,
San Francisco, USA
*Mehta BK and Franks JM contributed equally to this work.
Background: While skin fibrosis is a hallmark of systemic sclerosis
(SSc), internal organ involvement is the primary cause of morbidity and mortality.
Pulmonary Arterial Hypertension (PAH), Interstitial Lung Disease (ILD), and
gastrointestinal (GI) dysfunction are common in patients. Here we tested the
hypothesis, generated from a meta-analysis of ten different SSc datasets, that any
single patient with SSc would have the same deregulated molecular signatures across
multiple organ systems, consistent with the systemic nature of the disease.
Methods: RNA sequencing (RNA-seq) was performed on at least four organ
biospecimens (skin, esophagus, fundus, duodenum, and/or blood) from 14 patients who
met 2013 ACR/EULAR criteria for SSc. RNA was sequenced by 75bp paired-end RNA-seq at
>80 million reads per sample and aligned to the reference genome (hg19). Each
sample was assigned to an intrinsic subset (inflammatory, proliferative, or
normal-like) using Support Vector Machine (SVM) classification and unsupervised
hierarchical clustering with profiles of normalized Reads Per Kilobase of transcript
per Million mapped reads (RPKM) values.
Results: Hierarchical clustering of the RPKM data showed that samples
from a single tissue type cluster tightly together. This clustering is driven by
each organ having distinct gene expression profiles that were more similar within
that organ rather than between organs. Analysis of skin and esophagus recapitulated
the intrinsic subsets previously observed in these organs. Furthermore, for the
first time, the inflammatory, proliferative, and normal-like intrinsic subsets were
discovered in the fundus and duodenum. The blood of patients with SSc also displayed
gene expression signatures of the inflammatory, proliferative, and normal-like
intrinsic subsets. Importantly, all five organs assessed were represented in each
intrinsic subset. The majority of patients (9/14) showed >60% concordance of
intrinsic subset assignment across the multiple organs analyzed. Moreover, tissues
from the gastrointestinal tract tended to belong to the same subset (5/14 with 100%
concordance).
Discussion: We have performed the first molecular analysis of
biospecimens from multiple organs within an individual patient with SSc. Our data
suggest that SSc molecular subsets are reproducible in different tissues and that
dysregulated molecular pathways are conserved across organ systems within the same
patient.
INTERFERON GAMMA (IFN-γ) AND TUMOR NECROSIS FACTOR-α (TNF-α) SUBPOPULATIONS IN
SKIN HOMING T CELLS OF LOCALIZED SCLERODERMA
1 Pediatric Rheumatology, University of Pittsburgh Medical Centre,
Pittsburgh, PA, USA
2 Department of Pediatrics, Stanford University, Stanford, CA, USA
Background: Localized scleroderma (LS) has both inflammatory and
fibrotic components contributing to its effect on the skin and underlying tissue.
The extent and duration of inflammation (active disease) is thought to be the major
contributor of long-term disease damage and disability in LS. Current commercially
available serological markers of disease activity have limited utility in LS.
Therefore, identifying new circulating biologic markers reflecting active disease
status would assist clinicians when to initiate or discontinue therapy in unclear
cases. The purpose of this study is to investigate cellular populations of skin
homing cells in peripheral blood that may directly contribute to skin inflammation
and reflect activity at lesion sites.
Methods: PBMCs were isolated from 10 pediatric LS subjects with paired
active/inactive blood specimens. Flow cytometry was performed to characterize T
cells using antibodies for skin homing (CCR10 and CLA), skin inflammation (IFN-γ,
TNF-α and IL-13) and skin homing-adhesion markers (CD62L). Cell populations were
compared between LS active and inactive disease states using paired Mann-Whitney U
tests. Sub-analysis was performed to evaluate impact of disease duration on these
phenotypes.
Results: General T cell proportions were similar between active and
inactive disease states, trending toward increased CD4+ (TH) and CD8+
(TC) in active LS. Skin homing markers provided with more differences
with trend towards significance of CCR10+ TH populations and
significantly elevated TC CCR10+ IFN-γ+ and TNF-α+ subsets (p = 0.02 and
0.03, respectively)in the active disease state. Sub-analysis into short (<2
years), intermediate (2-4 years) and long (>4 years) disease duration showed a
trend of CCR10+ and CLA+ cells to be elevated in active disease, especially in the
TC subset, which remained persistent regardless of timing from
disease onset.
Conclusions: This is one of the first dedicated studies to examine the
circulating immunophenotype in pediatric-onset localized scleroderma, with emphasis
on examining T skin homing cell subtypes in disease activity. These findings support
increased skin homing (CCR10+) with cytokine producing subpopulations (IFN-γ+ and
TNF-α+) in active disease for both TH and TC cells. This
indicates circulating blood cellular and cytokine phenotypes targeting the skin that
could serve as biomarkers provide background for disease propagation.
MORBIDITY AND MORTALITY OF SCLERODERMA IN AFRICAN AMERICANS
Moore DF, Kramer EG, Eltaraboulsi RR, Steen VD
Department of Medicine, MedStar Georgetown University Hospital, Washington, DC,
USA
Background: Retrospective cohorts have demonstrated that African
Americans (AAs) are more likely to have diffuse skin disease, end-organ dysfunction,
and higher mortality than non-AAs. A prior study concluded that this excess risk of
disease burden and mortality was not fully explained by differences in rates of
diffuse disease and autoantibody status.
Methods: A single-center, retrospective study comparing AA and non-AA
patients with scleroderma was performed. Patients were evaluated by the senior
author between 2008 and 2016. AA and non-AAs were matched based on sex, age at first
visit, date of first visit, disease duration at first visit, and limited vs. diffuse
cutaneous disease. Demographic, serologic, and clinical features were compared.
Mortality risks were assessed in an unadjusted manner and by a Cox proportional
hazards model with covariates of race, median household income by ZIP code, marital
status, education level, employment status, and insurance type.
Results: AAs comprised 203 of the 402 patients analyzed. The groups were
statistically similar regarding all factors by which they were matched. AAs were
less likely to have inflammatory joints, tendon rubs, telangiectasias, or calcinosis
(all p<0.001). However, they had significantly reduced FVC (% predicted, 68.4 vs.
84.1, p<0.001) and DLCO (% predicted, 45.6 vs. 63.7, p<0.001). Fibrosis per CT
was more severe in AAs (p = 0.002), and more AAs had any type of pulmonary
hypertension (20.7% vs. 12.6%, p = 0.029). Autoantibody profile statistically
differed among the two groups (p<0.001). This difference was driven by the higher
prevalence of isolated nucleolar ANA and anti-U1RNP in AAs and anti-centromere and
anti-RNA polymerase III antibodies in non-AAs. Death during follow-up was 21.2% in
AAs vs. 11.1% in non-AAs (p = 0.006). AA status demonstrated an unadjusted hazard
ratio for death during follow-up of 2.063 (p = 0.008), but when adjusted for the
aforementioned socioeconomic covariates, the hazard ratio of AA status declined to
1.170 (95% CI 0.448 – 3.054, p = 0.749).
Conclusions: African American patients with scleroderma have more severe
pulmonary disease and a higher unadjusted risk of mortality than matched non-African
Americans. Adjusting for available socioeconomic factors, African American race did
not have a significant effect on mortality.
DEVELOPMENT OF A NOVEL EPITOPE-BASED DIAGNOSTIC ASSAY FOR SYSTEMIC
SCLEROSIS
1 Department of Molecular and Clinical Sciences, Università Politecnica
delle Marche, Ancona, Italy
2 School of Biosciences and Veterinary Medicine, University of Camerino,
Camerino, Italy
3 Department of Molecular Medicine, Federico II University, Naples,
Italy
4 Department of Medical Sciences, University of Turin, Turin, Italy
Purpose: To develop an assay for the classification of systemic
sclerosis (SSc) clinical subsets, based on the epitopes recognized by different
autoantibodies.
Materials and methods: 25 SSc (12 limited, 13 diffuse) and 25 healthy
control (HC) serum samples were tested by three subsequent arrays:
the large human PDGF receptor alpha (PDGFRα) conformational peptide library
formerly used for epitope mapping of monoclonal anti-PDGFRα antibodies
a novel library containing the top 20 PDGFRα conformational binders and 60
conformational and linear peptides of a cognate protein forming a complex
with PDGFRα
a novel library containing the top cognate protein peptide binders and 15
chimeric PDGFRα/cognate protein peptides chosen among the best binders.
Statistical analysis was performed by Wilcoxon-Mann-Whitney test.
Serological and clinical data were correlated.
Results: Library a) identified an immunodominant peptide discriminating
SSc from HC serum samples. Library b) confirmed this finding, highlighting also one
immunodominant epitope from the cognate protein, and identified two cohorts of SSc
samples (reactive vs nonreactive, the latter undistinguishable from HC), each
composed of limited and diffuse SSc subsets. Library c) identified the chimeric
epitope bound by the reactive SSc serum samples, taken from patients with active,
progressive disease regardless of limited vs diffuse subsets, whereas the
nonreactive SSc samples were taken from subjects with less active, non progressive
disease.
Conclusions: We developed a conformational epitope-based assay detecting
SSc-specific, agonistic serum autoantibodies. This novel array may identify SSc
patients with active disease, regardless of the canonical ‘limited/diffuse’
classification. We propose this assay for the prospective screening of large cohorts
of patients affected by, or suspected for, SSc, to validate it as a tool for disease
activity assessment and/or early diagnosis.
GENOME-WIDE DNA METHYLATION PATTERN IN SYSTEMIC SCLEROSIS MICROVASCULAR
ENDOTHELIAL CELLS (SSC-MVECS): IDENTIFICATION OF EPIGENETICALLY AFFECTED KEY
GENES AND PATHWAYS IN SSC-MVECS
1 Department of Internal Medicine, University of Toledo, Toledo, OH,
USA
2 Division of Rheumatology, University of Toledo, Toledo, OH, USA
Background: The etiology of systemic sclerosis (SSc) is not clear, but
there is evidence suggesting a critical role for epigenetic alterations in disease
pathogenesis and clinical expression. We sought in this study to characterize the
genome wide DNA methylation signature in SSc Microvascular Endothelial Cells
(MVECs).
Methods: We performed a genome-wide DNA methylation study in MVECs
derived from 7 diffuse cutaneous SSc (dSSc) patients compared to 7 age, sex, and
ethnicity-matched healthy controls. Cytosine methylation was quantified across the
genome. We divided samples from patients and controls in two groups of matched SSc
subjects and controls. Differentially methylated CpG sites between patients and
controls were identified by fold difference in methylation level ≥1.2, and false
discovery rate (FDR) adjusted P value <0.01. Moreover, quantitative real-time
RT-PCR was performed to assess the correlation between DNA methylation and gene
expression levels in selected genes.
Results: We identified 71,353 differentially methylated CpG sites in
SSc-MVECs using Infinium MethylationEPIC microarray in the first group (0.081% of
representative probes), and 33,170 CpG sites in the second group using
HumanMethylation 450 microarray (0.073% of representative probes) in dcSSc-MVEC.
Among the two groups of subjects, we identified differential methylation of 2,455
CpG sites, representing 1,301 genes. Most of the differentially methylated CpG sites
were hypermethylated (1,625 CpG), corresponding to 910 genes. There were 830
hypomethylated CpG sites, representing 485 genes. Common Hypermethylated genes in
SSc MVECs include NOS1, which encodes for nitric oxide synthase −1,
DNMT3A, DNMT3B, HDAC4 and ANGPT2. We also
identified hypomethylation of IL17RA, CTNNA3, ICAM2 and
SDK1in SSc MVECs. Furthermore, we demonstrate significant
inverse correlation between DNA methylation status and gene expression in the
majority of genes evaluated.
Gene ontology analysis of hypermethylated genes demonstrated enrichment of genes
involved in homophilic cell adhesion via plasma membrane adhesion molecules (P =
2.10E-07) and angiogenesis (P = 0.0006). Pathway analysis of hypomethylated genes
includes genes involved in Wnt signaling pathway (P = 0.001), vascular smooth muscle
contraction (P = 0.014) and adherens junctions (P 0.013).
Conclusions: Our data suggest the presence of significant genome-wide
DNA methylation aberrancies in SSc-MVEC, and identify novel affected genes and
pathways in SSc MVECs.
OPTIMIZATION OF GENE TRANSCRIPTION INHIBITORS AS NOVEL ORAL ANTI-FIBROTIC
AGENTS
1 Department of Pharmacology & Toxicology, Michigan State University,
East Lansing, MI, USA
2 Department of Medicinal Chemistry, University of Michigan, Ann Arbor,
MI, USA
3 Department of Internal Medicine, Division of Rheumatology, University of
Michigan Medical Center, Ann Arbor, MI, USA
4 Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of
Michigan, Ann Arbor, MI, USA
Tissue fibrosis contributes to a wide array of chronic diseases such as scleroderma
or systemic sclerosis; existing therapies are only marginally effective. Mechanisms
underlying later stages of fibrosis are conserved across many organs and tissues.
The conversion of quiescent fibroblasts to the proliferative, contractile, and
synthetic myofibroblast phenotype represents a key step in fibrosis. There is
abundant evidence linking myofibroblast conversion to transcription mechanisms,
including Rho GTPase and actin-dependent gene-transcription mechanism mediated by
serum response factor (SRF) and its transcriptional co-activator myocardin-related
transcription factor (MRTF-A, a.k.a. MKL1). Also, many key fibrosis-related genes
(CTGF, ACTA2 - alpha smooth muscle actin, MYL9, and several collagen genes) are
direct targets for MRTF/SRF-regulated gene transcription. We have described
compounds that inhibit transcription induced by the MRTF-regulated gene
transcription pathway (e.g. CCG-203971). They have been shown to reduce fibrosis in
multiple in vivo models. Key limitations of this compound are low potency and rapid
metabolism that necessitate high doses for in vivo efficacy. Using an SRE-Luciferase
reporter assay and modulation of native gene expression in dermal fibroblasts
derived from normal individuals and from affected skin from individuals with diffuse
systemic sclerosis (dSSc), we have optimized this compound series to reduce
metabolism in liver microsomes while maintaining good potency and activity. New
analogs (CCG-222740 and CCG-257081) have improved potency and/or pharmacokinetics
which should facilitate in vivo use and potential clinical translation.
Acknowledgments: Supported by R01 AR066049-03 (to SDL) and NIH R01
GM115459-02 (to RRn).
DYREGULATED SIRTUIN-1 IN SYSTEMIC SCLEROSIS
O'Reilly S, Henderson J, Ford D
Faculty of Health and Life Sciences, Northumbria University, Ellison Place, Newcastle
Upon Tyne, UK
Background: Systemic sclerosis is an idiopathic autoimmune disease in
which there is now an acceptance that epigenetic modifications are clearly playing a
role. Multiple epigenetic modifications exist and include non coding RNAs, including
microRNAs, methylation of DNA and histone modifications including acetylation.
Acetylation of histones alters transcription of DNA and is regulated by histone
acetylases enzymes that add the acetyl group. Sirtuin-1 is a histone deactylase that
removes the acetyl group from histone tails and used NAD as a co factor. We
investigated the role of Sirt-1 in fibrosis.
Methods: Using systemic sclerosis samples we observed much reduced
levels of Sirt-1 compared to controls. Using dermal fibroblasts we used the Sirt-1
activator Resveratrol in vitro and to confirm activation of Sirt-1 we use acetylated
P53 protein as a proxy readout system compared to the total P53 levels. We could
confirm activation of Sirt-1 after Resveratrol stimulation that was co-incident with
a large reduction in collagen protein levels suggesting Sirt-1 regulates collagen.
Mechanistically this is due to deacetylation of Activator Protein-1 (AP-1) thus
altering AP-1 binding to appropriate promoters. Incubation of the profibrotic
cytokine TGF-beta1 did not result in reduction of Sirt1 suggesting its
downregulation is not due to TGF-beta1 in SSc. However, the acetyltransferase
important in promoting acetylation was elevated after TGF-beta1 incubation.
Conclusions: Acetylation of AP-1 is important in driving the deposition
of collagen in SSc. Reduced Sirt1 is important as this leads to increased
acetylation of AP-1 and this is not regulated directly by TGF-beta-1, however,
TGF-beta1 does regulate P300 acetyltransferase activity. Resveratrol is a key
inducer of Sirt1 and we see this as a negative regulator of collagen and is a safe
and tolerable nutraceutical.
1 Department of Medicine, Divisions of Rheumatology and Immunology,
Medical University of South Carolina, Charleston, SC, USA
2 Pulmonary and Critical Care, Medical University of South Carolina,
Charleston, SC, USA
3 Department of Cardiology, Medical University of South Carolina,
Charleston, SC, USA
It is controversial whether the role of cells of the hematopoietic lineage in
fibrosis is solely due to monocytes differentiating into macrophages that activate
resident fibroblasts, or whether monocytes also differentiate into fibroblasts. We
showed that in the fibrotic lung tissue of bleomycin-treated mice, there are
CD45+/ColI+ cells (fibrocytes) present in greater numbers than in control tissue.
Here we confirm by transplantation of EGFP+ bone marrow that both CD45+/ColI+ and
CD45+/ColI- cells are EGFP+ while few CD45- cells are EGFP+. We further confirm by
immunohistochemistry that many CD45+/ ColI+ fibroblasts are present in fibrotic lung
tissue but not in control tissue. Next we compared macrophage markers present on
CD45+/ColI+ and CD45+/ ColI- cells. Although CD11b, CD16, CD68, and CD206 were
present at similar levels on CD45+/ColI- cells from control and fibrotic mouse lung,
they were present at much higher levels on CD45+/ColI+ cells from fibrotic lung
compared to control lung. To trace CD45+/ColI+ cells, fibroblasts were isolated from
lung tissue, then analyzed by flow cytometry. To ensure that fibroblasts retain
their in vivo phenotype, we studied fibroblasts that were not passaged. CD45, CD11b,
CD16, CD68, and CD206 levels were high in cultures from fibrotic lung tissue but not
control tissue. These results with fibroblasts are similar to those obtained with
fibrocytes, suggesting that fibrocytes are indeed fibroblasts or fibroblast
precursors. While for many it is dogma that fibroblasts are CD45-, our results
reveal a flaw in this dogma. We find that fibroblasts are detected as CD45+ (and
CXCR4+ and CCR5+) by flow cytometry only after fixation/permeabilization, not when
they are live. To demonstrate that CD45+/ ColI+ fibroblasts synthesize ColI rather
than take it up from the environment, we analyzed mice expressing EGFP under the
control of the ColIα1 promoter. Indeed, CD45+ fibroblasts were EGFP+ indicating that
they express ColI. While these studies provide convincing evidence that fibroblasts
can be derived from CD45+ cells and express CD45, a final proof will require lineage
tracing using Cre technology.
THE BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTOR SCLERAXIS (SCX) IS
OVEREXPRESSED IN SYSTEMIC SCLEROSIS AND LUNG FIBROSING DISEASES
1 Department of Immunology and Rheumatology, Instituto Nacional de
Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
2 Laboratory of Immunobiology and Genetics, Instituto Nacional de
Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
3 Department of Pulmonary Fibrosis, Instituto Nacional de Enfermedades
Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
4 National Autonomous University of Mexico, Mexico City, Mexico
Background: The transcription factor scleraxis (Scx) is required for the
expression of several extracellular matrix genes and is a critical regulator of
fibroblast phenotype and fibroblast to myofibroblast transformation in several
tissues like heart and tendons. Its participation in the pathogenesis of lung
fibrosis and systemic sclerosis (SSc) has not been studied.
Objectives: To explore if Scx is involved in the pathogenesis of
idiopathic pulmonary fibrosis (IPF) and systemic sclerosis we evaluated the serum
levels of scleraxis in SSc patients and healthy controls and correlated scleraxis
levels with SSc clinical features.
Methods: We included 123 SSc patients, 18 with IPF and 56 healthy
controls. We performed clinical evaluation, presence and severity of internal organ
involvement using Medsger severity scale, SSc-specific antibodies and treatment.
Levels of Scx were measured in serum by an ELISA assay. Clinical and serological
variables were collected for SSc and IPF patients. Analyses were performed using
SPSS v.22 software; Student's t test was used for numerical variables and Chi square
test was used for categorical variables; multivariable analyses were performed,
adjusting for confusing variables.
Results: Differences in age and gender among groups were not detected.
We found significantly higher levels of Scx in both, SSc (0.6992 ± 0.25 ng/mL,
p<0.001) and IPF patients (0.8932 ± 0.40 ng/mL, p<0.001) when compared to
healthy controls (0.4862 ± 0.14 ng/mL). SSc patients with calcinosis had lower
scleraxis levels (0.6289 ± 0.0931 vs. 0.7218 ± 0.29, p = 0.008). Scleraxis levels
were similar in dcSSc (0.699) than in lcSSc (0.698) patients, as well as in SSc
patients and SSc subsets when analyzed by presence and severity of internal organ
involvement.
Conclusions: Patients with SSc and with IPF exhibit increased serum
levels of Scx. This molecule may be an indicator of the increased fibrotic pathway
activation in these diseases. Further functional analysis should follow.
CIRCULATING MICROPARTICLE SUBSETS ARE ASSOCIATED WITH EXTENDED FIBROTIC
PHENOTYPE IN SYSTEMIC SCLEROSIS
1 Division of Rheumatology, Department of Internal Medicine, Keio
University School of Medicine, Tokyo, Japan
2 Department of Allergy and Rheumatology, Nippon Medical School Graduate
School of Medicine, Tokyo, Japan
Background: Microparticle (MP) is a small membrane vesicle, released
from various kinds of cells, which contains various growth factors and cytokines.
Recent studies reported that MP as a potentially valuable maker for inflammation or
endothelial damage. Although, association between MP subsets and systemic sclerosis
(SSc) remains still unclear.
Objectives: To elucidate the association between circulating
microparticle subsets and clinical characteristic of SSc.
Methods: Thirty-six patients with SSc and 13 healthy controls (HC) were
enrolled. Platelet-rich plasma containing MPs was isolated from whole blood using
gradient centrifugation and the number of each MP subset was analyzed by flow
cytometer. MP was defined as a particle smaller than 1.0 μm diameter using
Megamix®, and each MP subset (Platelet-derived MP:PltMP, Endothelial
cell-derived MP:EndoMP, and Monocyte-derived MP:MoMP) was defined based on the
expression of cell type-specific surface markers. Clinical data were retrospectively
collected, and the correlation between the number of each MP subset was
analyzed.
Results: Mean age of patients with SSc was 60.9 ± 1.8 years, 94% was
female, and mean disease duration was 11.2 ± 9.5 years. The proportion of diffuse
cutaneous SSc (dcSSc) was 17%. The number of total MP was significantly higher in
SSc than those in HC (3072 ± 188 vs 1780 ± 452, p<0.05). Notably, the numbers of
all MP subsets were significantly higher in patients with dcSSc compared to lcSSc
(p<0.01). In addition, the numbers of PltMP and EndoMP were higher in patients
with interstitial lung disease than those without (p<0.05). Moreover, all MP
subsets were inversely correlated with the percentage of predicted forced vital
capacity (p<0.05, p<0.01, and p<0.01, respectively).
Conclusions: Our results suggest that MP subsets are associated with
extended fibrotic phenotype in SSc, and might be novel biomarkers reflecting extent
of remodeling.
ANALYSIS OF ECM CROSS-LINKING IN DERMAL FIBROBLAST CULTURES
Semkova MS1,2, Ong VH2, Tabor AB3, Hsuan
JJ1
1 Institute for Liver and Digestive Health, Division of Medicine,
University College London, London, UK
2 Centre for Rheumatology and Connective Tissue Diseases, Division of
Medicine, University College London, London, UK
3 Department of Chemistry, University College London, London, UK
One common phenomenon observed for the majority of SSc patients is the abnormalities
in ECM metabolism that trigger accumulation of ECM proteins with resistance to ECM
remodelling that leads to skin fibrosis. Evidence suggests that increased ECM
cross-linking is a key mechanism of fibrotic progression and targeting this
phenomenon may help resolve fibrosis.
Targeting the cross-linking formation by the enzyme lysyl oxidase-like-2 (LOXL2) has
not been successful in recent Phase 3 trials in lung and liver fibrosis, and
pro-resolution strategies in advanced disease may require degradation of
cross-links.
The aim of this project is to characterise cross-linking changes in the ECM during
disease progression and to investigate the contribution of these modifications to
the resistance to ECM remodelling by matrix metalloproteinases (MMPs).
The initial objectives were to:
Synthesize peptides conjugated by naturally occurring cross-links that will
be further used to develop a strategy to analyse cross-linked peptides using
mass spectrometry (MS).
Isolate a cell-free ECM fraction from control and SSc patient dermal
fibroblast cultures, from frozen skin tissue samples from SSc patient and
murine SSc model.
Identify and quantify the ECM proteins in these preparations.
Due to the less complex matrisome produced in cell cultures in comparison with skin
tissue, albeit in lower quantity, analysis of cross-linking differences between
control fibroblasts and fibrotic myofibroblasts provides important preliminary
information about cross-linking changes in SSc. We present preliminary results in
the synthesis and MS based analysis of cross-linked peptides together with methods
employed to extract and isolate cross-linked peptides from dermal fibroblast cell
cultures. A combination of these techniques is being developed to investigate
cross-linking differences between control fibroblast and fibrotic myofibroblast cell
cultures and skin tissue samples.
THE ATM KINASE AND PTEN, DRIVE MYOFIBROBLASTS DIFFERENTIATION BY ACTIVATING
THE TGF β AUTOCRINE LOOP
1 Centre for Rheumatology and Connective Tissue Diseases, UCL Medical
School (Royal Free Campus), London, UK
2 Interstitial Lung Disease Unit, NHLI, Royal Brompton Hospital/Imperial
College, London, UK
Background: Pulmonary fibrosis is a major cause of mortality in
scleroderma (SSc) and Idiopathic Pulmonary Fibrosis (IPF). Fibrosis is driven by
inappropriate myofibroblast differentiation and persistence. Understanding this
process, is vital for developing an effective treatment. Angiotensin II, is
implicated in fibroblast activation in the heart and kidney, through interactions
with growth factors (e.g. EGF and TGFβ). We examined the role of Angiotensin II in
myofibroblast activation in the lung.
Methods: Lung fibroblasts were isolated from SSc, IPF, or control
patient lungs (6 each). Fibroblasts were also cultured from PTEN null and wild-type
mice. Protein expression after angiotensin II treatment (AngII) was investigated by
western blotting. Myofibroblast differentiation and function was assayed through the
contraction of 3D collagen gels and scratch migration assays. The signalling
pathways involved were dissected using specific inhibitors: PI3-kinase/AKT
(wortmannin, LY294002), TGFβ (1d11 neutralizing antibody, SB431542 ALK5 inhibitor)
Ataxia-Telangiectasia Mutated (ATM - Ku55933), AngII (Losartan).
Results: SSc and IPF lung fibroblasts showed increased AKT
phosphorylation and suppressed PTEN expression (p<0.05). Their phenotype was more
myofibroblast-like, with higher αSMA expression (P<0.05), increased collagen gel
contraction (control; 207 ± 14 vs SSc; 93 ± 15 vs IPF 91 ± 21, p<0.05), and
enhanced migratory capacity (P<0.05). PTEN-null fibroblasts showed a similar
phenotype. AngII treatment activated AKT, suppressed PTEN and induced myofibroblast
differentiation in normal lung fibroblasts. In both AngII-treated and PTEN lung
fibroblasts AKT activation required the ATM kinase. Inhibition of AKT either with
PI3K or ATM inhibitor abrogated these effects. The increased expression of
Myofibroblast-related genes after AngII treatment, was also blocked by inhibition of
TGFβ with a neutralizing antibody or an ALK5 inhibitor. AKT phosphorylation on the
other hand was only partially blocked was partially blocked by TGFβ inhibition.
Conclusions: Our data demonstrate for the first time that AngII signals
via the ATM kinase, which together with PTEN suppression are essential for the
activation of AKT by AngII. AngII promotes myofibroblast differentiation, by
stimulating the fibroblast TGFβ autocrine loop through AKT. Our data shows that
activation of AKT through ATM and PTEN, may serve as the molecular link between
pulmonary hypertension and lung fibrosis in fibrotic diseases.
SAFETY AND SUITABILITY OF DABIGATRAN ETEXILATE TO INHIBIT THROMBIN IN
SCLERODERMA-ASSOCIATED INTERSTITIAL LUNG DISEASE: A PRELIMINARY REPORT
1 Divisions of Rheumatology, Department of Medicine, Medical University of
South Carolina, Charleston, SC, USA
2 Department of Public Health Sciences, Medical University of South
Carolina, Charleston, SC, USA
3 Divisions of Pulmonology, Department of Medicine, Medical University of
South Carolina, Charleston, SC, USA
4 Department of Pulmonary Medicine, Cleveland Clinic, Cleveland, OH,
USA
Background: Studies from our laboratory and others implicate thrombin in
the pathogenesis of SSc-ILD. Thrombin activity is high in SSc-ILD and can transform
normal lung fibroblasts to a myofibroblast phenotype with features of high
contractility and collagen synthesis. Dabigatran etexilate (Pradaxa®) is
a selective thrombin inhibitor approved for prevention of thromboembolic
complications in patients with atrial fibrillation.
Methods: Based on strong preclinical in vitro and animal data
demonstrating that thrombin inhibition by dabigatran etexilate ameliorates lung
fibrosis, we are conducting a single-site, open-label study to establish the safety
of dabigatran etexilate, 75 mg twice daily administered for 6 months, to 15 patients
with SSc-ILD (ClinicalTrials.gov Identifier NCT02426229). We are also conducting
exploratory studies of the potential effects of dabigatran etexilate on the skin and
lungs, including effects on the biological activity of thrombin in plasma and
bronchoalveolar lavage fluid, as well as on the biologic behavior of SSc skin and
lung fibroblasts.
Results: To date, 12 SSc-ILD patients have been enrolled, and 8 have
completed treatment. Baseline characteristics are as follows: 10 females and 2
males; 5 limited and 7 diffuse cutaneous SSc; 5 African Americans and 7 Caucasians;
age 47 + 9.8 years (mean + SD); disease duration 4.5 + 3.5 years; mRSS 14.8 + 10.7;
SHAQ 1.4 + 0.6; UCLA SCTC GIT score 0.87 + 0.61; Baseline Dyspnea Index 12 + 8.1;
FVC% predicted 69.8 + 16.4; DLCO% predicted 54.6 + 13.5. Overall, dabigatran
etexilate has been well tolerated. To date, there have been no serious adverse
events. Mild adverse events reported include: headache (n = 1), fatigue (n = 1),
epistaxis (n = 2), and menorrhagia (n = 1). Monthly laboratory monitoring has shown
no clinically significant change in CBC, CMP, PT/INR or PTT, but has shown the
expected prolongation of thrombin clotting time (TCT). Treatment was interrupted in
one subject for an elective surgical procedure that was performed under normal
coagulation parameters and without bleeding complications.
Conclusions: If the safety and tolerability of dabigatran etexilate are
confirmed, we will propose a larger, double-blind RCT to establish the safety and
determine the efficacy of dabigatran etexilate in patients with SSc-ILD.
Acknowledgements: Funded by NIAMS/NIH R21 AR065089 and SC
Smart-State®.
EFFECT OF OSM AND IL-6 ON ENDOMT IN HUMAN DERMAL MICROVASCULAR CELLS
1 Arthritis Centre, Boston University, Boston, USA
2 Immuno-Inflammation Therapeutic Area Unit, GSK Medicines Research
Centre, Stevenage, UK
Background: Oncostatin-M (OSM) and interleukin-6 (IL-6) are members of
the IL-6 superfamily and signal via the common signalling component, glycoprotein
130 (gp130). IL-6 can signal via gp130 with the IL-6 receptor (IL-6R) or by
trans signalling with soluble IL-6R (sIL6-R). Endothelial cells
(ECs) express both gp130 and OSM receptors, however it is unclear whether ECs
express IL-6R. Endothelial to mesenchymal transition (EndoMT) is the phenotypic
transition of ECs into mesenchymal cells where ECs lose their specific EC markers,
detach from the endothelial layer and initiate the expression of mesenchymal cell
products. The role of OSM and IL-6 in EndoMT has not yet been fully elucidated.
Objectives: To determine the effect of OSM and IL-6/sIL-6R on
microvascular EC migration, proliferation and EndoMT.
Methods: Human dermal microvascular ECs (HDMECs) were treated with human
recombinant proteins OSM (1-100 ng/mL), IL-6 (10-100 ng/mL) and sIL-6R (10-100
ng/mL). Cell migration and proliferation were measured with Live-Cell imaging system
and analysed using a two-way ANOVA. Collagen type I protein was measured by western
blot analysis. Changes in VE-Cadherin and F-actin expression were examined by
immunofluorescence. Gene expression was measured using quantitative RT-PCR analysis
and analysed by Student's paired T-Test.
Results: OSM and IL-6, with or without IL-6R, significantly increased
(P<0.001, n = 3 donors) HDMEC migration and proliferation and secretion of
extracellular matrix (ECM) protein Collagen I compared to the control group. OSM,
but not IL-6 with or without sIL-6R, reduced expression of EC marker VE-Cadherin and
increased expression of elongated F-actin stress fibres (n = 2 donors). OSM
significantly increased (P<0.05, n = 3 donors) expression of EndoMT genes
SNAIL1, SNAIL2, SNAIL3 and TWIST and ECM genes
MMP1, MMP2 TIMP1 and TIMP2 compared to the
control. Many of the gene changes in response to OSM were further augmented by
co-stimulation with sIL-6R. IL-6 with or without sIL-6R only significantly affected
(P<0.05, n = 3 donors) EndoMT genes SNAIL1 and
SNAIL3 and ECM gene TIMP1.
Conclusions: OSM induced a stronger EndoMT phenotype in HDMECs in
comparison to IL-6, suggesting that OSM is capable of initiating EndoMT activity in
microvascular cells. OSM may contribute to dermal fibrosis in systemic sclerosis by
inducing microvascular cell activation and EndoMT.
DYSREGULATION OF AUTOREACTIVE IL-6-PRODUCING TRANSITIONAL B LYMPHOCYTES IN
SYSTEMIC SCLEROSIS
Taher ET1, Ong VH2, Hillion S1, Simon
Q1, Denton CP2, Pers JO1, Abraham
DJ2, Mageed RA1
1 Centre for Experimental Medicine and Rheumatology, William Harvey
Research Institute, Queen Mary University of London, London, UK
2 Centre for Rheumatology and Connective Tissue Diseases, UCL Medical
School Royal Free Campus, London, UK
A key feature of autoimmunity in scleroderma (SSc) is high levels of auto-antibodies.
The mechanisms that underpins the generation of pathogenic autoantibody-producing B
lymphocytes and their role in SSc remains unclear. Defective tolerance and
deregulation of autoreactive B cell responses are potential causes of B
cell-mediated pathology.
We aim to elucidate the potential mechanisms that contribute to the pathogenic B cell
generation relevant to SSc disease process and to assess the altered distribution
and function of B cells.
Multi-colour flow cytometry analysis was used to determine B cell subset
distribution, cytokine production and tolerance induction in SSc patients and
healthy controls. Cytokine production following stimulation of the cells ex
vivo was carried out by multiplex analysis.
The cohort consisted of 80 patients; 84% of whom were females. 46% had diffuse
cutaneous SSc (dcSSc) with mean age 49.2 ± 12 years and disease duration 7.6 ± 7
years. 22.5% harboured anti-Scl70 antibodies and all had interstitial lung disease.
More transitional B cells (CD24hiCD38hi)(Mean ± SEM) (19.85 ±
1.87 cells/μL, 13.6 ± 0.93% of total B cells) were detected in SSc patients compared
with healthy controls (HC)(7.04.5 ± 1.09 cells/μL, 6.81 ± 0.62% of total B cells;
p<0.001).
There was differential expression of transitional subsets within
CD24hiCD38hi B cells based on the expression of CD27, IgM,
IgD and CD32 markers. The number and frequency of IL-6+ transitional B cells were
2.8 ± 0.7 cells/μL (14.0 ± 3.1% of transitional B-cells) in SSc compared with 0.51 ±
0.13 cells/μL (6.7 ± 1.0% of transitional B-cells) (p<0.01 and <0.05,
respectively) in the HCs. When stimulated with TLR9 and CD40, B cells in SSc
produced significantly less IL-10 (82.0 ± 36.0pg/mL and 44.2 ± 18.0pg/mL,
respectively) than B cells from the HCs (190.3 ± 35.7pg/mL and 163.4 ± 37.9pg/mL,
respectively) (p<0.05). B cell receptor-mediated apoptosis in transitional B cell
subsets showed reduction in apoptosis in all subsets in SSc patients (13.6 ± 4.1)
compared with the HCs (47.1 ± 12.9) (p<0.05). ELIspot analysis
of IgM anti-Scl-70-producing transitional B cells revealed that the subset medium
expression levels of IgM, IgD, CD10 and CD32 with high CD21 has maximal B cells with
IgM autoantibodies bound to Scl70 antigen.
This study provides new insights into mechanisms underlying defective B lymphocyte
responses with specific defects in regulation of B lymphocyte tolerance, development
and functions in SSc.
PATIENTS WITH EARLIER AND MILDER DISEASE WITH INTERSTITIAL LUNG INVOLVEMENT IN
SYSTEMIC SCLEROSIS HAVE BETTER RESPONSE TO IMMUNOSUPPRESSIVE TREATMENT
1 Division of Rheumatology, Department of Internal Medicine, Keio
University, Tokyo, Japan
2 Department of Allergy and Rheumatology, Nippon Medical School Graduate
School of Medicine, Tokyo, Japan
3 Department of Radiology, Keio University School of Medicine, Tokyo,
Japan
Background: Interstitial lung disease (ILD) is an important organ
involvement in patients with systemic sclerosis (SSc), which determines their
prognosis. It was reported that extent of diseased area of ILD determine by CT scan
was associated with mortality. However, whether the population with severe disease
is truly a good candidate for the treatment was still unclear.
Objectives: To determine whether we can decide a target for the
treatment based on the extent of diseased area of ILD in SSc.
Methods: SSc-ILD patients treated with cyclophosphamide during 2008 to
2017 were stratified into extensive or limited lung disease (ED or LD) based on the
extent of ILD using HRCT following a previous report. (AJRCCM. 2008). Response to
the treatment determined by the change of% FVC, and baseline clinical parameters
were compared between two groups.
Results: Twenty-two patients were involved. Mean age was 54.1 ± 14.4
years, female was 86%, and diffuse cutaneous SSc were 55%. Disease duration from
onset of SSc to initiation of the treatment was 37 ± 66 months. Twelve were
stratified as ED and 10 as LD. Treatment response at one year was better in patients
with LD compared to ED (9.98 ± 6.49 vs 2.84 ± 7.68% improvement, p<0.05).
Baseline% FVC was higher and disease duration was shorter in LD (p<0.05). Other
clinical parameters including age, sex, smoking, autoantibody profiles and serum
KL-6 levels were comparable between two groups.
Conclusions: SSc patients with LD, who had earlier and milder disease,
showed better short-term response to the treatment compared to those with ED,
suggesting that earlier intervention could lead to better prognosis in patients with
SSc.
TH2 CYTOKINE IL-31 IS ELEVATED IN SYSTEMIC SCLEROSIS AND PROMOTES MULTIPLE
PATHOGENIC MECHANISMS
Taki Z1, Zafar S1, Rosario H1, Black
S1, Etomi O1, Abdi Ahmed B1, Tam
A1, Lopez H2, Scolamiero L1, Hart
A1, Arumala N1, Renzoni E1, Wells
A3, Renzoni E3, Venturini C1, Mohamed
A1, Mazumder N1, Shiwen X1, Xing
F1, Abraham D1, Denton C1, Stratton
R1
1 Centre for Rheumatology and Connective Tissue Disease, Royal Free
Hospital Campus, University College Medical School, London, UK
2 MuriGenics, Inc., Vallejo, CA, USA
3 Imperial College London, Royal Brompton Campus, London, UK
Objectives: In systemic sclerosis (SSc) Th2 cytokines provide a possible
mechanistic link between autoimmunity and the fibrotic pathology seen. IL31 is a
recently described Th2 cytokine which signals via the oncostatin M receptor/IL-31RA
dimer. We sought to determine whether IL-31 is elevated in SSc plasma or blister
fluid (BF), and test whether the IL-31RA is expressed by cells relevant to the
disease process. Responses of target cells to IL-31 were evaluated.
Methods: IL-31 was measured by ELISA of plasma (SSc n = 43, HC n = 27)
and BF (SSc n = 44, HC n = 22). Itch severity was determined by the 5D pruritus
scale. IL31RA mRNA was assayed by qPCR of SSc and HC fibroblasts, epidermal tissue
extracts, and fat derived MSCs. Responses of target cells to IL-31 were measured by
next generation sequencing (NGS), migration assays, qPCR and Western blotting.
Results: Plasma IL-31 was elevated in SSc patients (1370 ± 567 SSc vs
196 ± 47 pg/ml HC, p<0.023). Both BF and plasma IL-31 levels were higher in
diffuse cutaneous subset (plasma IL-31 2193 ± 1025 dcSSc vs 486 ± 236 lcSSc,
p<0.05, BF 565 ± 181 dcSSc vs 173 ± 123 lcSSc, p = NS), but were not correlated
with itch severity or disease duration. IL-31RA was increased in SSc cells and
tissues (dermal fibroblasts 1.00 ± 0.067 vs 0.34 ± 0.017 (p<0.035), lung
fibroblasts 0.97 ± 0.15 vs 0.32 ± 0.064 (p<0.05), epidermal tissue 18.4 ± 2.09 vs
8.2 ± 2.47, (p NS), MSCs (relative copy number mean ± SEM)). Adding recombinant
IL-31 to dermal fibroblasts induced NGS patterns associated with Wnt, PI3K, TGFβ
signalling, angiogenic suppression, and specifically induced IL-33 and CXCL5.
Overlap with TGFβ induced genes was modest. Collagen I and CTGF mRNA, induced as
expected by TGFβ, were not altered by IL-31 treatment, but collagen I protein
release was induced. In MSCs, IL-31 enhanced migration and osteogenic
differentiation.
Discussion: IL-31 is increased in SSc where multiple disease-associated
cells express IL-31RA. Absolute levels of IL-31 greatly exceed those of other
inflammatory factors studied previously in plasma or BF. IL-31 induces phenotypic
and gene expression changes in target cells consistent with a pathogenic role
distinct from and additive to TGFβ.
CONNECTIVE TISSUE GROWTH FACTOR GENE DELETION ATTENUATES LUNG FIBROSIS IN A
BLEOMYCIN MOUSE MODEL OF SYSTEMIC SCLEROSIS
Tam A1, Jones H1, Trinder SL1, Khan
K1, Good RBW1, Ramanayake W1, Denton
CP1, Holmes AM1, Norman JT2, Abraham
DJ1
1 Division of Medicine, Centre for Rheumatology and Connective Tissue
Disease, University College London, London, UK
2 Centre for Nephrology, Division of Medicine, University College London,
London, UK
Background: Systemic sclerosis (SSc) is an autoimmune disease
characterized by inflammation, vasculopathy and fibrosis. Interstitial lung disease
is common in SSc and a major cause of death as there is currently no treatment to
reverse lung fibrosis. Connective tissue growth factor (CTGF, CCN2) is a member of
the CCN family of matricellular proteins and its expression is upregulated in SSc
and correlates with severity of fibrosis. CCN2 contributes to fibrosis by regulating
inflammatory mediators, myofibroblast differentiation and overexpression of
collagen. CCN2-overexpression mice display a fibrotic phenotype while CCN2-deficient
mice are resistant to bleomycin-induced skin fibrosis. Based on the importance of
CCN2 in fibrosis, we are interested in whether CCN2 deletion is able to attenuate
the development of lung fibrosis.
Methods: Conditional CCN2 knockout mice were generated by crossing
CCN2flox/floxmice and ROSA-CreERT2mice. Deletion of CCN2
exon-2 was induced by IP injection of tamoxifen. Bleomycin or saline were
administered via oropharyngeal instillation and mice were sacrificed after 21 days
(4-mice/group; unpaired t-test).
Results: Micro-CT 3D-imaging of whole lungs and H&E staining of
paraffin embedded lung sections showed the presence of florid fibrosis in
bleomycin-treated mice compared to saline-treated mice. Using the modified Ashcroft
Scale, lung sections from bleomycin-treated wild-type mice showed a higher fibrotic
index compared with those from saline-treated wild-type mice (p<0.05). A lower
fibrotic index was observed in the lungs of bleomycin-treated CCN2 KO mice compared
to bleomycin-treated wild-type mice (grade 4 vs 5, p = 0.0277). Picrosirius red
staining showed that bleomycin induced less collagen expression in the lungs of CTGF
KO compared to wild-type mice.
Conclusions: Knocking out CCN2 in mice reduced bleomycin-induced lung
fibrosis. These results suggest that CCN2 mediates bleomycin-induced lung fibrosis,
and that therapeutic strategies targeting CCN2 may be an effective means of treating
lung fibrosis in SSc.
TOWARDS A TRANSCRIPTOMIC MAP OF RHEUMATIC DISEASE
Taroni JN1,2, Greene CS1-3
1 Department of Systems Pharmacology and Translational Therapeutics,
University of Pennsylvania, Philadelphia, PA, USA
2 Institute for Translational Medicine and Therapeutics, University of
Pennsylvania, Philadelphia, PA, USA
3 Institute for Biomedical Informatics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
Systemic sclerosis is a rare, heterogeneous rheumatic disease characterized by
fibrosis, vascular abnormalities, and multi-organ involvement. We have previously
demonstrated that insight into disease pathogenesis or treatment effects can be
extracted from data that are not from direct assays of SSc. To that end, we are
building a multi-tissue pan-rheumatic gene expression compendium from all publicly
available expression data that assay rheumatic and autoimmune diseases. This
compendium is being constructed with precision rheumatology in mind.
We present validated strategies for combining datasets from multiple platforms and
technologies. We find that several normalization methods, including quantile
normalization and Training Distribution Matching, allow for the effective
combination of microarray and RNA-seq data into a training set for machine learning
(ML) applications. Furthermore, in this study, we specifically utilize single-sample
(microarray) normalization methods designed for personalized medicine workflows. We
find that the SCAN method (Piccolo, et al. 2012.) performs as well or better than
commonly used multi-array normalization methods.
We estimate that the final compendium will contain over twelve thousand samples,
which is greater than ten times the number of assays analyzed in any single,
publicly available study of rheumatic disease. By analyzing a resource of this
magnitude, we will address the following: 1) the identification molecular mechanisms
shared between diseases, including immune cell phenotypes and 2) the development of
an analysis framework for genome-scale data from rare rheumatic diseases, such as
SSc and vasculitides, that utilizes information from more common conditions.
We will employ unsupervised ML techniques to identify robust expression patterns
suitable for shared molecular mechanism detection and biomarker development. A
distinct advantage of using unsupervised methods is that we do not need to design
contrasts between cases and controls a priori and therefore, they
are well-suited to capture the heterogeneity of SSc and other rheumatic disorders.
We expect that shared pathways and immune cell-specific phenotypes can be learned
from heterogeneous multi-tissue and multi-disease data, leading to rational targets
for therapeutic intervention (e.g., through drug repositioning) and patterns that
can be used to guide clinical assay creation (e.g., for a gene set associated with
rituximab response).
THERAPEUTIC TARGETING OF TAZ AND YAP BY DIMETHYL FUMARATE IN SYSTEMIC
SCLEROSIS FIBROSIS
1 Arthritis Center, Boston University School of Medicine, Boston, MA,
USA
2 Department of Biomedical Engineering, Boston University, Boston, MA,
USA
3 Department of Biochemistry, Boston University School of Medicine,
Boston, MA, USA
Systemic sclerosis (scleroderma, SSc) is a devastating multi-organ fibrotic disease
with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF,
Tecfidera®) have shown therapeutic effects in several disease models,
prompting us to determine whether DMF is effective as a treatment for SSc dermal
fibrosis. We found that DMF blocks the pro-fibrotic effects of TGFβ in healthy- and
SSc patient-derived skin fibroblasts. Interestingly, the canonical pathway of
Smad2/3 in skin fibroblasts was not affected by DMF. On the other hand, DMF
decreased TAZ and, to a lesser degree, YAP protein levels, but it did not affect
TAZ/YAP mRNA levels. Depletion of TAZ/YAP in dermal fibroblasts recapitulated the
anti-fibrotic effects of DMF. We further showed that DMF significantly diminished
nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface.
Mechanistically, we found that TGFb upregulated TAZ, but not YAP protein levels via
Akt1-mediated inhibitory phosphorylation of GSK3b and a subsequent inhibition of
bTRCP-mediated proteasomal degradation of TAZ, as well as a corresponding increase
of TAZ/YAP transcriptional targets. DMF treatment abrogated these TGFb effects. We
further showed that blockade of PI3K (LY294002) or Akt1 (MK-2206) reduced nuclear
localization of TAZ. These findings suggest that DMF differentially affects TAZ and
YAP activity. Whereas DMF affects TAZ protein levels and nuclear localization, it
primarily regulates YAP nuclear localization. Importantly, DMF prevented
bleomycin-induced skin fibrosis in mice. We also confirmed the increased presence
and the nuclear localization of fibroblast TAZ/YAP in skin biopsies from patients
with SSc. Together, our work demonstrates a novel mechanism of the anti-fibrotic
effect of DMF via inhibition of Akt1/GSK3b/TAZ/YAP signaling and confirms a critical
role of TAZ/YAP in mediating the pro-fibrotic responses in dermal fibroblasts. This
study supports the use of DMF as a treatment for SSc dermal fibrosis.
REGULATION OF FIBROBLAST PHENOTYPE, FUNCTIONALITY AND MATRIX PRODUCTION BY THE
NF-κB SUBUNIT C-REL IN FIBROSIS
Worrell JC1, Leslie J1, Smith G1, O'Reilly
S2, Zaki M1, van Laar JM1, Mann
DA1, Oakley F1
1 Newcastle Fibrosis Research Group, Institute of Cellular Medicine,
Newcastle University, Newcastle upon Tyne, UK
2 Department of Health and Life Sciences, Northumbria University,
Newcastle upon Tyne, UK
Background: NF-kappaB (NF-κB) regulates over 200 genes controlling
various biological processes including inflammation, cell proliferation and
survival. NF-κB signalling is important for normal skin physiology, and
dysregulation of this pathway is implicated in disease. Systemic sclerosis (SSc) is
a rare but debilitating disease characterised by fibrosis that occurs in the skin,
lungs and blood vessels. Mice that lack the NF-κB subunit c-Rel are protected from
developing skin fibrosis. c-Rel expression is elevated in the skin of scleroderma
patients with abnormal cellular localization. This study aims to ascertain if c-Rel
has pro-inflammatory or pro-fibrogenic activities in the context of in vivo models
of fibrosis and systemic sclerosis.
Materials and methods: Differential expression analysis of publically
available microarray data comparing normal and SSc skin (N = 78 diffuse, N = 16
lesional and N = 30 control) was performed. Chromatin immunoprecipitation (ChIP) was
used to investigate cRel binding to promoters in normal and SSc fibroblasts. c-Rel
knockdown (siRNA) was performed on primary human dermal fibroblasts. RNA sequencing
and pathway analysis was performed on unpassaged lung and dermal fibroblasts from
Wild type and cRel-/-mice. Functional assays were performed examining
fibroblast phenotype and metabolism. Mice were given bleomycin either subcutaneously
or via intra-tracheal administration chronically and histological
analysis carried out.
Results: A unique NF-κB gene signature was identified in diffuse
scleroderma skin versus control. c-Rel directly binds the COL1A1 gene promoter and
this interaction was enhanced in myofibroblasts originating from SSc patients. c-Rel
knockdown in primary human dermal fibroblasts significantly decreased gene
expression of TIMP-1, COL12A1, COL8A1 with decreased TGFB1 production. c-Rel
expression was significantly elevated in mouse lung and skin in response to chronic
injury induced by bleomycin; increased staining was observed in fibroblasts and
immune cells. RNA sequencing revealed significant downregulation of genes involved
in the production of extracellular matrix. Common pathways identified by KEGG
analysis included ECM-receptor interaction and focal adhesion. c-Rel-/-
fibroblasts displayed altered functional capabilities compared to WT
fibroblasts.
Conclusions: c-Rel is required for the excessive production of collagen
by dermal myofibroblasts. Targeting c-Rel in fibroblasts from patients with
scleroderma may suppress their fibrogenic activities leading to novel strategies to
treat the disease.
DO SERUM BICARBONATE LEVELS CORRELATE WITH RENAL DECLINE IN SYSTEMIC
SCLEROSIS?
Wright J1, Raphael KL2, Frech TM3
1 Department of Internal Medicine, Resident Physician, University of Utah,
Salt Lake City, Utah, USA
2 Department of Internal Medicine, Division of Nephrology, University of
Utah and Veterans Affair Medical Center, Salt Lake City, Utah, USA
3 Department of Internal Medicine, Division of Rheumatology, University of
Utah and Veterans Affair Medical Center, Salt Lake City, Utah, USA
Background: End-stage organ manifestations that affect serum bicarbonate
levels in systemic sclerosis (SSc) include interstitial lung disease (ILD),
pulmonary arterial hypertension (PAH), and scleroderma renal crisis (SRC).
Retrospective analyses have indicated that low serum bicarbonate predicts death in
non-SSc chronic kidney disease (CKD) and that base substitution might reduce the
decline of kidney function. The objective of this study was to examine serum
bicarbonate levels in a cohort of SSc patients and to assess the association of
serial low serum bicarbonate (<20 mmol/L) with the development of renal
dysfunction.
Methods: Patients were recruited and consented for participation from
the University of Utah SSc Clinic. Clinical characteristics were recorded at the
time of enrollment. We assessed serial serum bicarbonate levels and estimated
glomerular filtration rate (eGFR) and determined the percent who developed incident
low eGFR based on bicarbonate concentration.
Results: In this SSc cohort, 159 had at least two serum bicarbonate with
concurrent creatinine levels. Of these 159 patients, 19 patients (18%) had two or
more serial low bicarbonate levels >6 months a part. They were mostly female (n =
17) with limited cutaneous involvement (n = 13). All were ANA positive with
additional autoantibodies including U1RNP (n = 3), U3RNP (n = 2), Th/To (n = 1),
centromere (n = 7), SCL70 (n = 1), and RNApol3 (n = 6). In this serial low
bicarbonate group, 8 patients had PAH and 6 patients had ILD. Of the 19 patients
with serial low bicarbonate levels, 8 patients (42%) developed renal insufficiency.
Only one of those 8 patients met criteria for SRC and only the U1RNP autoantibody
patients (n = 3) lacked PAH and/or ILD. None of the patients were deceased.
Conclusions: In a single center SSc cohort, low serial serum bicarbonate
was associated with the development of renal insufficiency in almost half of the
patients with this clinical finding. The majority of patients with low serial
bicarbonate levels had the presence of lung disease, which highlights the importance
of obtaining an arterial blood gas for better characterization of the acid-base
state. Rheumatologists should be aware of the association of low serum bicarbonate
levels with incident CKD in order to prescribe replacement if indicated.
PLASMA LEVELS OF MICRO-RNA IN PULMONARY ARTERIAL HYPERTENSION ASSOCIATED WITH
ANTI-CENTROMER POSITIVE SYSTEMIC SCLEROSIS
1 Department of Clinical Sciences Lund, Rheumatology, Lund University and
Skåne University Hospital, Lund, Sweden
2 Department of Autoimmunology & Biomarkers, Statens Serum Institut,
Copenhagen, Denmark
3 Department of Clinical Sciences Lund, Cardiology, Lund University and
the Section for Heart failure and Valvular Disease, VO Heart and Lung Medicine,
Skåne University Hospital, Lund, Sweden
4 Department of Clinical Biochemistry and Pharmacology, Odense University
Hospital, University of Southern Denmark, Odense, Denmark
5 Institute of Clinical Research, Clinical Biochemistry, University of
Southern Denmark, Odense, Denmark
Objective: To evaluate plasma levels of micro-RNAs (miRNA) in patients
with anti-centromere positive limited cutanous systemic sclerosis (lcSSc) that have
developed pulmonary arterial hypertension (APAH-SSc) without significant pulmonary
fibrosis compared to those patients with anti-centromere positive lcSSc that have
not developed APAH-SSc despite similiar disease phenotype and disease duration.
Material and methods: At the longitudinal systemic sclerosis cohort in
Lund 27 female patients with anti-centromere positive lcSSc had developed APAH-SSc
without significant pulmonary fibrosis over a time period of 32 years. Sixty-six
lcSSc patients without APAH-SSc and without significant pulmonary fibrosis were
matched for antibody status, gender and disease duration. Using quantitative RT-PCR
the amount of 44 mature miRNA in plasma was determined. MiRNA expression levels were
correlated with clinical data relevant for APAH-SSc.
Results: The lcSSc patients with APAH-SSc had median age of 70 years and
were 4.5 years older than the lcSSc patients without APAH-SSc (p<0.05) despite
our attempt to match the groups also for age. Both groups had median disease
durations of 11 years from non-Raynaud symptom onset. Let-7a-5p, Let-7c-5p,
Let-7d-5p, miR-17-5p, miR-20a-5p, miR-140-5p, miR-191-5p, miR-199a/b-3p, miR-203a-3p
and miR-223a-3p levels were lower in plasma of patients with APAH-SSc compared to
the lcSSc patients without APAH-SSc. miR-21-5p and miR-486-3p displayed higher
plasma levels in patients with APAH-SSc compared to the lcSSc patients without
APAH-SSc. Six of these miRNAs remained as predictors for APAH-SSc after adjustment
for age (p<0.05 for let-7d-5p, miR-17-5p, miR-140-5p, miR-191-5p;
p<0.01 for miR-20a-5p and miR-203a-3p). The plasma levels of
Let-7a-5p, miR-20a-5p and miR-203a-3p showed inverse correlations with the estimated
pulmonary arterial pressure obtained by heart echography (r = −0.31, p<0.01 for
miR-20a-5p; r = −0.38, p<0.001 for Let-7a-5p; and
r = −0.41, p<0.001 for miR-203a-3p) and with NT-pro-brain
natriuretic peptide (r = −0.32, p<0.01 for Let-7a-5p; r = −0.41,
p<0.001 for miR-20a-5p; and r = −0.44, p<0.001 for
miR-203a-3p), a measure for strain on the heart musculature.
Conclusion: Our study links expression levels of specific circulating
plasma miRNAs to APAH-SSc in anti-centromer positive female patients with lcSSc.
VALIDATION OF DETECT ALGORITHM IN PATIENTS WITH SYSTEMIC SCLEROSIS –
VALIDATION IN A COHORT OF JAPANESE SINGLE CENTER
1 Division of Rheumatology, Department of Internal Medicine, Keio
University School of Medicine, Tokyo, Japan
2 Department of Allergy and Rheumatology, Nippon Medical School Graduate
School of Medicine, Tokyo, Japan
3 International University of Health and Welfare, Mita Hospital, Tokyo,
Japan
Background: Pulmonary arterial hypertension (PAH) is one of leading
causes of death in patients with SSc. Annual screening is recommended to improve
their prognosis. DETECT algorithm is reported as a good tool to identify candidates
who need right heart catheterization (RHC), with high sensitivity. However, its
usefulness has not been validated in Japanese cohorts.
Objectives: To validate the effectiveness of DETECT algorithm in
Japanese patients with SSc.
Methods: Patients with SSc who visited Keio University Hospital between
2005 and 2016 were included in the study. Patients over 18 years old, with disease
duration more than 3 years, and DLCO predicted less than 60% were selected and
clinical information was retrospectively collected. The sensitivity and specificity
were calculated in a cohort of PAH patients plus non-PAH patients, in whom RHC data
were available. Validation with patients having data minimally-required for
evaluation by algorithm were also examined.
Results: Three hundred four patients were visited our hospital during
from 2005 to 2016. Patients who fulfilled criteria and had data minimally-required
for algorithm were 126 cases. 50 were examined RHC, 26 (21%) were diagnosed as
pulmonary hypertension and 21 (15%) were PAH. When a cohort of SSc patients who
examined RHC was applied to DETECT algorithm, missed diagnosis of PAH was 0%.
Sensitivity/specificity for detecting PAH patients were 100%/42%, respectively.
Evaluation of 126 patients with data minimally-required for algorithm was also
examined. Referral rate to RHC evaluation was 43%, missed diagnosis of PAH was 0%,
and sensitivity/specificity were 100%/72%, respectively. However, there was a
patient who was initially excluded at Step 1, but developed PAH one year later.
Conclusion: The DETECT algorithm was reassured as a good tool for the
screening of PAH in SSc patients. However, unnecessity of RHC referral judged by
this algorithm does not guarantee the patient to be free from future development of
PAH.
