Synergistic inhibition of CitH3 and S100A8/A9: A novel therapeutic strategy for mitigating sepsis-induced inflammation and lung injury

Abstract

Objective:

Sepsis is a life-threatening condition with high global morbidity and mortality. Citrullinated histone H3 (CitH3) has gained recognition as a significant biomarker for early sepsis diagnosis and management. This study aims to investigate the therapeutic potential of targeting both CitH3 and S100A8/A9 to reduce sepsis-induced inflammation and organ damage.

Methods:

Using a novel CitH3 antibody distinct from commercial options, we analyzed serum samples from LPS-treated mice through a co-immunoprecipitation assay followed by LC-MS/MS proteomic analysis to explore the interaction between CitH3 and S100A8/A9 proteins in peripheral blood. Additionally, in a Pseudomonas aeruginosa (PA)-induced lung injury model, we assessed CitH3 and S100A8/A9 levels in bronchoalveolar lavage fluid (BALF), alveolar samples, and neutrophils to determine their influence on neutrophil activation and inflammatory responses.

Results:

Our study revealed, for the first time, that CitH3 and S100A8/A9 synergistically promoted neutrophil activation, inflammatory responses, and NETosis, which exacerbated lung injury in sepsis. Dual targeting of CitH3 and S100A8/A9 significantly reduced neutrophil recruitment, NETosis, and inflammation in the PA-induced lung injury model. This therapeutic approach improved lung injury and survival rates, accompanied by a shift in cytokine profiles, with reductions in pro-inflammatory cytokines and increases in anti-inflammatory cytokines.

Conclusion:

These findings underscore the potential of dual targeting CitH3 and S100A8/A9 as a novel therapeutic approach for sepsis. This combined intervention shows promising effects in reducing inflammation and enhancing survival, offering a groundbreaking strategy for sepsis diagnosis and treatment.

Keywords

sepsis CitH3 S100A8/A9 hCitH3-mAb NETosis survival

Introduction

Sepsis remains a critical global health concern, characterized by a dysregulated host response to infection leading to life-threatening organ dysfunction.¹ Despite advances in medical care, sepsis remains a leading cause of morbidity and mortality worldwide.² The complexity of sepsis pathophysiology, encompassing an intricate interplay between the host immune system and microbial factors, poses significant challenges in its diagnosis and management.³

In this context, the identification of reliable biological indicators and therapeutic targets is imperative for advancing sepsis diagnosis and treatment. CitH3 has emerged as a promising blood marker with diagnostic and therapeutic implications in sepsis.⁴ Additionally, S100A8/A9, integral members of the S100 protein family, calcium-binding proteins predominantly found in myeloid cell cytoplasm, have surfaced as potential indicators of sepsis and related organ injury.^5,6 During acute lung injury, neutrophils are the first cells recruited to the site of inflammation, where they deploy a potent antimicrobial arsenal that includes oxidants, proteinases, and cationic peptides. However, under pathological conditions, the unregulated release of these microbicidal compounds into the extracellular space can paradoxically damage host tissues.^7–9 NETosis, the process of neutrophil extracellular trap (NET) formation, involves creating fibrous structures containing neutrophil proteins and DNA.¹⁰ While NETs aid in pathogen clearance as part of innate immunity, they also contribute to inflammatory damage in both sterile and infectious contexts. Interventions aimed at reducing NET formation, such as DNase I treatment, have shown potential in mitigating lung injury and improving outcomes in various inflammatory lung conditions.^11,12 Additionally, CitH3, a hallmark of NETosis, signifies neutrophil activation and plays a crucial role in the innate immune response during early-stage infections.¹³ S100A8/A9, during NETosis, translocates to the nucleus, regulating chromatin decondensation—an essential step in NETs formation.^7,9,14 Their presence within NETs not only amplifies antimicrobial potential but also potently intensifies the inflammatory response.

Both CitH3 and S100A8/A9 have emerged as promising clinical markers in inflammatory and infectious conditions, highlighting their significance as potential indicators and therapeutic targets, particularly in sepsis. Despite advancements in understanding their roles, the precise mechanisms and interactions of these markers during sepsis, especially their involvement in NETosis, remain largely unexplored. Our study reveals, for the first time, the association of CitH3 and S100A8/A9 in peripheral blood, lung tissue, and neutrophils during sepsis. Additionally, our findings demonstrate that a combination treatment targeting CitH3 and S100A8/A9 significantly improves the survival rate of mice with sepsis, offering a novel approach for therapeutic intervention.

Materials and methods

Animals

Male C57BL/6J mice aged between 8 and 10 weeks were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) for this investigation. These mice were accommodated in a specific pathogen-free facility under controlled environmental conditions, maintaining a temperature of 23 ± 1.5°C and relative humidity of 70% ± 20%. All experimental procedures and animal care practices adhered to the guidelines established by the University of Michigan Institutional Animal Care and Use Committee, ensuring compliance with approved protocols. The animal ethics approval number is PRO00011567.

Mouse neutrophil isolation

Mouse neutrophils were isolated by harvesting bone marrow cells from femurs and tibias, followed by centrifugation at 427 × g for 7 min at 4°C in RPMI. After lysing red blood cells on ice for 10 min and repeating the centrifugation, cells were resuspended in 1–3 mL of sterile PBS. Histopaque 1119 (3 mL) was layered over with 3 mL of Histopaque 1077 in a 15-mL conical centrifuge tube. The bone marrow cell suspension was overlaid on top, and caution was taken during centrifugation at 872 × g for 30 min at room temperature. Neutrophils were collected at the interface of the Histopaque 1119 and 1077 layers, followed by two washes with RPMI 1640. Mouse neutrophils were re-suspended in RPMI 1640 medium with 10% FBS, the cells were challenged by PMA (500 nM) followed by another 3 h incubation after incubation for 30 min at 37°C.

Serum preparation of LPS-induced endotoxic mouse model

The LPS-induced endotoxic shock model was established by intraperitoneal (i.p.) injection of mice with lipopolysaccharide (LPS) at a dosage of 25 mg/kg, while a control group received an equivalent volume of normal saline i.p. Serum samples were meticulously collected at 18 h post-LPS injection. Subsequently, to enhance the specificity of the subsequent LC-MS/MS analysis, the collected serum underwent IgG depletion treatment (R&D, MIDR002-020). This step aimed to selectively remove high-abundance immunoglobulins, allowing for increased sensitivity in detecting low-abundance proteins. The treated serum was then utilized for co-immunoprecipitation, a technique employed to isolate and enrich specific proteins of interest, particularly CitH3 related proteins in this context. Following the co-immunoprecipitation step, the enriched proteins were subjected to comprehensive LC-MS/MS analysis, enabling the identification and quantification of alterations in the protein profile associated with CitH3 in the LPS-induced endotoxic shock model.

Liquid chromatographic and mass spectrometric conditions (LC-MS/MS)

A Shimadzu Nexera LC-30AD LC-MS/MS system, coupled with a Shimadzu LC-MS 8060 using APCI in positive mode, was employed for bioanalysis. Separation utilized a Phenomenex Synergi Fusion C18 column with a gradient of 0.1% formic acid in water (A) and methanol (B). The total run time was 12 min, with a flow rate of 0.25 mL/min and an injection volume of 10 µL. The LC-MS/MS operated in positive APCI mode with MRM, using 118.15 > 91.1 m/z for indole and 124.15 > 96.1 m/z for indole d7. The Shimadzu Labsolutions software (Version 5.99) was used for auto-optimization of compound-dependent LC-MS/MS parameters. Indole (1 µg/mL) and indole-d7 IS (1 µg/mL) in methanol were utilized for method development. Source settings included nebulizer gas at 3.0 L/min, heating gas at 10 L/min, drying gas at 5 L/min, interface temperature at 300°C, a desolvation line temperature at 200°C, and heat block temperature at 200°C. MRM transitions, optimum LC-MS/MS parameters, and data acquisition/quantitation were managed using Shimadzu Labsolutions LCMS software.

Pulmonary infection model

The bacterial strain PA 19660 obtained from ATCC (Manassas, Virginia, USA) was prepared in phosphate-buffered saline (PBS) at a concentration of 8.25 × 10⁷ colony-forming units (CFU)/mL. To establish the PA pneumonia-induced sepsis model, 8–10-week-old mice underwent a standardized procedure.¹⁵ An anesthetic combination of ketamine (100 mg/kg body weight) and xylazine (20 mg/kg) was administered, after which mice were positioned vertically for nasal inoculation with a total of 30 μL of the PA solution (15 μL in each nostril), resulting in a final bacterial load of 2.5 × 10⁶ CFU. Sham control mice received sterile PBS inoculation. For non-survival studies, mice were humanely euthanized using CO₂ at 24 h post-inoculation, with serum, bronchoalveolar lavage fluid (BALF), and organs collected and stored at −80°C for subsequent analysis. In survival studies, groups of 15 mice were monitored for 10 days, with moribund animals euthanized using CO₂. The observational endpoint also determined euthanization. This model provided a platform to explore the progression of PA pneumonia-induced sepsis, offering valuable insights into the effects of S100A8/A9 inhibitor paquinimod (MCE, HY-100442) and humanized CitH3 antibody (hCitH3-mAb) on host defense mechanisms and bacterial spread during PA-induced sepsis.

Co-immunoprecipitation

For serum samples

The depletion of mouse serum IgG was carried out using mouse serum protein immunodepletion resin (R&D, MIDR002-020) as per the manufacturer’s protocol. The serum was diluted to 500 μL with IP Lysis/Wash Buffer and allowed to form the immune complex with our novel CitH3 antibody (hCitH3-mAb) overnight at 4°C. Pierce Protein A/G Magnetic Beads (25 µL, 0.25 mg) were prepared by washing them twice with IP Lysis/Wash Buffer in a 1.5 mL microcentrifuge tube. Following the formation of the immune complex, it was added to the pre-washed magnetic beads and incubated for 1 h at room temperature with mixing. Subsequently, the beads were collected using a magnetic stand, and the unbound sample was preserved for analysis. A double wash with IP Lysis/Wash Buffer, succeeded by a rinse with ultra-pure water, was conducted. For elution, 100 µL of Elution Buffer was added to the tube, followed by a 10-min incubation at room temperature with mixing. The supernatant containing the target antigen was separated magnetically. To neutralize the low pH, Neutralization Buffer was added at a ratio of 10 µL per 100 µL of eluate. All steps were performed in accordance with the provided kit protocol (Thermo Fisher Scientific, 88084).

For non-serum samples

The immune complex formation step with hCitH3-mAb was carried out similarly as described above, without using mouse serum protein immunodepletion resin. Pierce Protein A/G Magnetic Beads and subsequent steps were used as described above, starting from the washing step. All procedures were conducted in accordance with the provided kit protocol (Thermo Fisher Scientific, 88084).

Western blot

The eluted proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes for immunoblotting. For minimizing non-specific interactions, the membranes were blocked for 1 h at room temperature using a solution of 5% skimmed milk in Phosphate-buffered saline (PBS) supplemented with 0.1% Tween 20. Primary antibodies (S100A8/A9, Abcam, ab288715) were then applied at appropriate dilutions and incubated overnight, followed by incubation with a secondary antibody (Invitrogen, USA) for 1 h at room temperature. After immunoblotting, the membranes underwent three sequential 10-min washes to remove unbound antibodies. Enhanced chemiluminescence (Bio-Rad, Hercules, CA) facilitated the visualization of antigen-antibody complexes, and specific protein bands were imaged using a Luminescent image analyzer (Thermo Fisher Scientific, USA).

Quantification of citrullinated histone H3

Citrullinated histone H3 levels in BALF samples were determined using the Citrullinated Histone H3 (Clone 11D3) ELISA Kit (Cayman, 501620). The procedure followed the manufacturer’s instructions, involving the binding of specific antibodies to citrullinated histone H3, which is then detected via an enzyme-linked secondary antibody to produce a measurable signal.

Quantification of S100A8/A9

S100A8/A9 levels in BALF samples were determined using the S100A8/A9 ELISA Kit (Invitrogen, EM67RB). The procedure was followed the manufacturer’s instructions.

Quantification of double-stranded DNA

Double-stranded DNA (ds-DNA) levels in BALF were measured using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P11496). The assay was performed according to the manufacturer’s protocol, which involves binding of the PicoGreen reagent to dsDNA, resulting in a fluorescence signal proportional to the DNA concentration.

Quantification of MPO-DNA complexes

Myeloperoxidase-DNA (MPO-DNA) levels in BALF were quantified using Mouse Myeloperoxidase ELISA Kit (Abcam, ab275109) according to the manufacturer’s directions.

Quantification of inflammation cytokines

Serum and BALF samples were collected and sent to the Immune Monitoring Shared Resource at the University of Michigan (UMICH) for the assessment of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and the anti-inflammatory cytokine (IL-10).

Immunofluorescence

The mouse lung tissue frozen slides and Cells underwent a blocking step before being exposed overnight at 4°C to primary antibodies targeting CitH3 (Abcam, ab281584) and S100A8/A9 (Abcam, ab288715). Afterward, the sections were rinsed twice to eliminate any unbound antibodies and subsequently treated with the appropriate secondary antibodies (Invitrogen, USA) for 1 h at room temperature.

Hematoxylin and eosin staining

The degree of acute lung injury was also assessed based on the pathology analysis. The fixed lung tissues were embedded in paraffin wax, and then 4 μm-thick lung sections were cut and stained with hematoxylin and eosin (H&E). The morphology results were evaluated by a pathologist who was blinded to the experimental groups. The lung injury score was determined according to Suzuki’s method.¹⁶

Statistical analysis

GraphPad Prism 10 software facilitated the statistical analysis. Survival curve comparison and assessment of survival rate differences among experimental groups were performed using Mantel–Cox tests. For analyses involving three or more groups, one-way analysis of variance (ANOVA) alongside Bonferroni’s multiple comparison test was employed to identify significant differences among individual groups. Comparisons between two groups were conducted using the Student’s t-test, a non-parametric test suitable for small sample sizes or non-normally distributed data. Results are presented as mean ± standard error of the mean (SEM), depicting both the central tendency and measurement precision. Statistical significance was set at p < 0.05, indicating notable differences between the compared groups.

Results

Identification of CitH3-associated proteomic changes in an LPS-induced endotoxic model

CitH3 is a well-established blood biomarker for the diagnosis and treatment of sepsis.⁴ To enhance the accuracy of diagnostic and therapeutic applications, we developed a novel CitH3 monoclonal antibody (hCitH3-mAb) that differs from available commercial options. To investigate proteins associated with our novel CitH3 antibody in an LPS-induced endotoxemia model, mice were divided into vehicle and LPS-treated groups. After 18 h of LPS treatment, mouse serum samples were collected, and the co-immunoprecipitation protocol was meticulously executed following IgG depletion. The hCitH3-mAb was added to the pre-cleared serum and incubated overnight at 4°C with rotation. The eluted proteins were then analyzed using LC-MS/MS analysis. The results, as illustrated in Figure 1, revealed 52 upregulated proteins in the LPS-treated group. Notably, S100A8 and S100A9 emerged prominently among these proteins, signifying their substantial changes in abundance. These findings provide valuable insights into the alterations in the proteomic landscape associated with CitH3 in response to LPS-induced endotoxemia, further emphasizing the potential significance of S100A8 and S100A9 in this context.

Figure 1.

Analysis of CitH3-related protein abundance changes in plasma of mice with LPS-induced endotoxicity. C57BL/6 mice were divided into two groups (n = 3/group): vehicle-treated and LPS-treated. Serum samples were collected from each group 18 h after LPS changeling and subjected to co-immunoprecipitation using a CitH3 antibody. The eluted proteins were then analyzed using LC-MS/MS analysis. The volcano map illustrates the relationship between protein ratio (x-axis) and the p value of repeated test results (y-axis), with each point on the graph representing an individual protein. Upregulated proteins are depicted in the red region, while downregulated proteins are in the green region. Directing attention to the LPS treatment group, the volcano map emphasizes 52 upregulated proteins. Notably, S100A8 and S100A9 hold a prominent position among these, indicating their significant changes in abundance.

The presence of binding complexes between CitH3 and S100A8/A9 observed in peripheral blood

Pseudomonas aeruginosa (PA) remains a major cause of bloodstream infections associated with high mortality.¹⁷ To investigate the interaction dynamics of CitH3 and S100A8/A9 within peripheral blood in PA induced sepsis, co-immunoprecipitation was performed. Following a 24-h post-PA challenge, serum was collected, and the co-immunoprecipitation protocol was meticulously executed after IgG depletion. IgG or specific CitH3 antibody were added to the pre-cleared serum and incubate overnight at 4°C with rotation. The eluted proteins were analyzed with S100A8/A9 antibody by using western blotting. Our findings demonstrate a clear interaction between CitH3 and S100A8/A9 in the intricate environment of peripheral blood, emphasizing their association in response to PA infection (Figure 2).

Figure 2.

Interaction of CitH3 and S100A8/A9 in serum post-PA infection. C57BL/6J mice were intranasally inoculated with Pseudomonas aeruginosa (PA) at a concentration of 2.5 × 10⁶ colony-forming units. Serum was collected 24 h post-PA infection and subjected to IgG depletion followed by co-immunoprecipitation using IgG or CitH3 antibody. Subsequently, S100A8/A9 was quantified through western blot analysis. Data are representative of three independent experiments expressed as means ± SEM.

Elevated levels of CitH3 and S100A8/A9 detected in both lung tissue and BALF during PA infection-induced sepsis

During PA-induced sepsis, CitH3 and S100A8/A9 have emerged as promising biomarkers for identifying sepsis and related organ injury.^4,14 To elucidate their significance in PA induced acute lung injury, mice were nasal inoculation with PA bacteria. CitH3 and S100A8/A9 levels were measured in BALF by using ELISA, while immunofluorescence was employed to assess their presence in lung tissue. Within 24 h of PA infection, significant elevations in CitH3 and S100A8/A9 levels were detected in BALF and lung tissue (Figure 3(a) and (b)). Notably, immunofluorescence revealed a compelling co-localization (in white) of CitH3 and S100A8/A9 within lung tissue (Figure 3(b)).

Figure 3.

Expression of CitH3 and S100A8/A9 in BALF and lung samples. C57BL/6J mice were infected with 2.5 × 10⁶ CFU Pseudomonas aeruginosa (PA) 19660/mouse for 24 h. (a) Bronchoalveolar lavage fluid (BALF) was collected from all the treatment groups 24 h post-PA infection, and the levels of CitH3 and S100A8 were determined using ELISA. (b) Lung tissue samples were collected 24 h post-PA infection and subjected to immunofluorescence by using S100A8/A9 (in green) and CitH3 (in pink) antibody. Data are representative of three independent experiments expressed as means ± SEM.

The association of CitH3 and S100A8/A9 in neutrophils

CitH3 and S100A8/A9 are associated with neutrophil extracellular traps (NETs) formation and implicated in various inflammatory and autoimmune conditions due to its release by neutrophils during cell death.^6,18–20 Neutrophils play a crucial role in the body’s response to sepsis, as they are among the first responders to infections.²¹ To affirm the association of CitH3 and S100A8/A9 within neutrophils, mouse neutrophils were isolated and subjected to PMA treatment for 3 h. After this treatment, NETosis occurred, accompanied by elevated levels of co-localization between CitH3 and S100A8/A9 within the neutrophilic environment. This emphasizes their potential significance in shaping neutrophil function and modulating inflammatory responses. The observed co-localization (in white) further underscores their intricate involvement in the intricate processes associated with sepsis and inflammatory conditions (Figure 4(a)). To gain deeper insights into the interaction between CitH3 and S100A8/A9 within neutrophils during PA infection, we conducted an experiment involving nasal inoculation of mice with PA bacteria. Half an hour post-infection, the mice received a single tail vein injection of either hCitH3-mAb (10 mg/kg) or an equivalent dose of DMSO. After 24 h, we harvested bone marrow-derived neutrophils from the infected mice. These neutrophils were then subjected to co-immunoprecipitation using a CitH3-specific antibody to capture the CitH3 protein complex, followed by the quantification of S100A8/A9 proteins through western blot analysis. Our results provided compelling evidence of a significant interaction between CitH3 and S100A8/A9 within neutrophils. Notably, treatment with hCitH3-mAb reduced this interaction, underscoring their close association during the immune response to PA infection (Figure 4(b)). This interaction highlights the intricate molecular mechanisms at play and suggests a coordinated role of CitH3 and S100A8/A9 in modulating neutrophil functions and inflammatory responses during infection. The elucidation of this interaction adds valuable knowledge to our understanding of neutrophil behavior in the context of PA infection and broader inflammatory processes.

Figure 4.

Association of CitH3 and S100A8/A9 in BMDNs. (a) Bone marrow-derived neutrophils (BMDNs) isolated from C57BL/6 mice were subjected to treatment with or without PMA at 500 nM for 3 h. Following treatment, cells were fixed and subjected to staining for CitH3 (in pink), S100A8/A9 (in green), and DAPI (in blue). (b) C57BL/6J mice were intranasally inoculated with 2.5 × 10⁶ CFU of Pseudomonas aeruginosa (PA). 0.5 h post-infection, mice received a single tail vein injection of either hCitH3-mAb (10 mg/kg) or an equivalent dose of DMSO. Twenty-four hours post-infection, BMDNs were isolated from the mice and subjected to Co-immunoprecipitation using CitH3 antibody, subsequently, S100A8/A9 was quantified through western blot analysis.

The impact of CitH3 and S100A8/A9 interaction on alveolar neutrophil population and NETosis during PA infection

Having confirmed the interaction between CitH3 and S100A8/A9 during PA infection, we aimed to investigate the effect of this interaction on neutrophil recruitment and NETosis. To this end, C57BL/6J mice were intranasally inoculated with PA. Thirty minutes post-infection, the mice received a single dose of either hCitH3-mAb, paquinimod, or a combination of hCitH3-mAb and paquinimod to assess the impact of these treatments on the interaction between CitH3 and S100A8/A9 during the infection process.

After a 24-h period post-infection, alveolar cells were isolated from the mice. These cells were subjected to flow cytometry analysis to quantify the distribution of neutrophil populations, specifically CD45⁺, CD11b⁺, and Ly6G⁺ cells. Our results showed that neutrophil populations increased following PA infection. Both hCitH3-mAb and paquinimod significantly decreased the neutrophil population during PA infection, with the combination of hCitH3-mAb and paquinimod showing the greatest efficacy in diminishing neutrophil presence (Figure 5(a)). Additionally, BALF was collected from all treatment groups 24 h post-infection, and the levels of NETosis were determined using ELISA. The markers assessed included CitH3, ds-DNA, and MPO-DNA complexes. Our results demonstrated that NETosis levels increased after PA infection. Both hCitH3-mAb and paquinimod effectively reduced the PA infection-induced NETosis, with the combination treatment showing the highest efficiency in decreasing NETosis levels (Figure 5(b)). This comprehensive approach allowed us to evaluate both the recruitment of neutrophils to the site of infection and the extent of NETosis. The results contribute to our understanding of how CitH3 and S100A8/A9 interactions influence neutrophil functions and the broader inflammatory response during PA infection.

Figure 5.

C57BL/6J mice were intranasally inoculated with Pseudomonas aeruginosa (PA) at a concentration of 2.5 × 10⁶ colony-forming units per mouse). Subsequently, mice received a single dose of hCitH3-mAb (10 mg/kg of body weight), paquinimod (5 mg/kg of body weight), a combination of hCitH3-mAb and paquinimod, or an equivalent dose of DMSO was administered via tail vein injection 0.5 h post-infection. (a) Alveolar cells were then isolated 24 h after PA infection, cells were subsequently subjected to flow cytometry analysis to quantify the distribution of CD45⁺, CD11b⁺, Ly6G⁺ neutrophil populations (n = 3/group). (b) Bronchoalveolar lavage fluid (BALF) was collected from all the treatment groups of mice 24 h after PA infection, and the levels of NETosis (CitH3, ds-DNA, and MPO-DNA) were determined using ELISA (n = 5/group). Data are representative of three independent experiments expressed as means ± SEM.

A therapeutic approach concurrently targeting CitH3 and S100A8/A9 demonstrates significant efficacy in ameliorating PA-induced sepsis

The observed interaction between CitH3 and S100A8/A9 in peripheral blood, coupled with their co-localization in neutrophils, prompted us to investigate their functional relevance and potential interplay in lung injury and survival rates during PA-induced sepsis. To examine this hypothesis, mice were administered either the humanized CitH3 antibody hCitH3-mAb, the S100A8/A9 inhibitor paquinimod, or a combination of hCitH3-mAb and paquinimod, given 30 min after PA challenge. As depicted in Figure 6(a), both hCitH3-mAb and paquinimod treatments improved survival rates, with the combination treatment demonstrating superior efficacy compared to singular hCitH3-mAb or paquinimod treatments. Histological examination via HE staining revealed that compared to individual treatments, the combination treatment with paquinimod and hCitH3-mAb efficiently decreased lung injury (Figure 6(b)). Quantification of inflammatory cytokine levels in serum and BALF using ELISA (Figure 6(c) and (d)) showed a significant decrease in pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) following PA infection in the presence of hCitH3-mAb and paquinimod. Furthermore, the combination treatment exhibited notably higher efficacy in modulating cytokine levels compared to individual hCitH3-mAb or paquinimod treatments. Conversely, the anti-inflammatory cytokine IL-10 showed a remarkable increase in the group treated with the combination of hCitH3-mAb and paquinimod. The combined treatment significantly improved lung injury and survival rates and effectively modulated inflammatory cytokine levels compared to individual treatments, suggesting a synergistic effect. These findings underscore the potential of combined targeting of CitH3 and S100A8/A9 in mitigating lung injury and improving survival rates in PA-induced sepsis.

Figure 6.

Therapeutic efficacy of hCitH3-mAb and paquinimod combination treatment in PA-induced acute lung injury in mice. C57BL/6J mice were intranasally inoculated with Pseudomonas aeruginosa (PA) at a concentration of 2.5 × 10⁶ colony-forming units per mouse). Subsequently, mice received a single dose of hCitH3-mAb (10 mg/kg of body weight), paquinimod (5 mg/kg of body weight), a combination of hCitH3-mAb and paquinimod, or an equivalent dose of DMSO was administered via tail vein injection 0.5 h post-infection. (a) Survival rates were monitored for 10 days (n = 8/group), and analysis was conducted using Mantel–Cox test. (b) Lung tissue samples were collected 24 h after PA infection and subjected to HE staining (n = 3/group). (c) Serum was collected from all the treatment groups of mice 24 h after PA infection, and the levels of inflammation cytokines (TNF-α, IL-1β, IL-6, and IL-10) were determined using ELISA (n = 5/group). (d) Bronchoalveolar lavage fluid (BALF) was collected from all the treatment groups, and the levels of inflammation cytokines (TNF-α, IL-1β, IL-6, and IL-10) were determined using ELISA (n = 5/group). Data are representative of three independent experiments expressed as means ± SEM.

Figure 7.

Schematic representation of the CitH3-S100A8/A9 axis as a key driver of neutrophil-mediated pathology in sepsis.

Discussion

Sepsis remains a formidable global health challenge, characterized by dysregulated immune responses to infection that culminate in life-threatening organ dysfunction.^22–24 Despite advances in critical care, its high mortality persists due to the intricate interplay between hyperinflammation, immune exhaustion, and pathogen virulence.^25–27 Our study identifies citrullinated histone H3 (CitH3) and S100A8/A9 as pivotal mediators of neutrophil-driven pathology in sepsis, offering mechanistic insights into their synergistic roles and therapeutic potential.

CitH3 and the calcium-binding protein S100A8/A9 have emerged as promising indicators of sepsis and associated organ injury.^14,28–30 To comprehensively investigate their roles, we employed a two-step approach: (1) unbiased LC-MS/MS proteomic analysis using LPS-induced sepsis to identify key molecular interactions and (2) targeted validation in a clinically relevant Pseudomonas aeruginosa (PA)-infected model. The LPS model provided a controlled setting to delineate fundamental inflammatory pathways, whereas the PA infection model more accurately reflects the complex host-pathogen interactions in bacterial sepsis.

Using LC-MS/MS analysis, we demonstrated that CitH3 directly interacts with S100A8/A9 in peripheral blood and lung tissue during sepsis. This interaction was particularly pronounced in neutrophils undergoing NETosis, a programmed cell death mechanism central to antimicrobial defense and tissue injury.³¹ CitH3, a hallmark of chromatin decondensation during NET formation, collaborates with S100A8/A9—a calcium-binding protein that regulates chromatin remodeling and amplifies inflammatory signaling within neutrophil extracellular traps (NETs).^7,14,31,32 The transition from the LPS model to the PA model allowed us to validate these findings under conditions that more closely resemble human sepsis, where pathogen-derived virulence factors further modulate immune responses. The rapid upregulation of both proteins in lung tissue and bronchoalveolar lavage fluid (BALF) within 24 h of PA infection underscores their role in acute lung injury, likely through dual mechanisms: (1) promoting neutrophil recruitment and (2) exacerbating epithelial barrier disruption via cytotoxic NET release.

The therapeutic implications of this CitH3-S100A8/A9 axis are significant. Combinatorial inhibition using hCitH3-mAb and paquinimod synergistically reduced neutrophil infiltration and NETosis in our models. This aligns with prior evidence showing that anti-CitH3 strategies mitigate organ damage in sepsis, while paquinimod curtails neutrophil hyperactivation in infections.^33,34 The synergy likely arises from disrupting two critical nodes: CitH3 blockade limits NET-driven tissue injury, while S100A8/A9 inhibition dampens pro-inflammatory signaling (e.g. NF-κB/MAPK pathways) and neutrophil survival. Notably, this dual approach attenuated lung pathology and improved survival in PA sepsis, suggesting it may rebalance immune responses without compromising pathogen clearance—a critical advantage in sepsis therapy.

Our previous work demonstrated the protective effects of anti-CitH3 antibodies in septic mice, and this study demonstrates the efficacy of paquinimod in reducing neutrophil accumulation and activation during PA infections. The co-localization of CitH3 and S100A8/A9 in nuclear and cytoplasmic compartments of neutrophils, particularly during NETosis, highlights their intertwined roles in shaping neutrophil function. However, sepsis pathophysiology involves multifaceted interactions between immune cells, endothelial damage, and microbial factors. While our study focuses on PA-induced sepsis, the CitH3-S100A8/A9 axis likely extends to other sepsis etiologies, given their roles in sterile inflammation and NETosis.

Several limitations warrant consideration. First, our analysis focused on early sepsis (24-h window), and longitudinal studies are needed to define the temporal dynamics of CitH3 and S100A8/A9 during sepsis progression and resolution. Second, our study does not directly address whether S100A8/A9 inhibition compromises neutrophil function because is beyond the scope of the present study. However, we recognize its importance and will investigate it in future research. Lastly, the CitH3-S100A8/A9 interaction may intersect with broader pathways, such as platelet activation or complement cascades, which remain unexplored.

In conclusion, our study elucidates the CitH3-S100A8/A9 axis as a central driver of neutrophil-mediated pathology in sepsis. By demonstrating their synergistic contributions to NETosis and organ injury, we advance a novel therapeutic strategy: dual targeting of CitH3 and S100A8/A9 to disrupt hyperinflammation while preserving immune function. Future work should explore the diagnostic potential of CitH3-S100A8/A9 complexes as sepsis biomarkers and refine this approach in diverse clinical contexts, ultimately bridging the gap between mechanistic insight and improved patient outcomes.

Footnotes

Acknowledgements

Figure 7 was created with . We sincerely acknowledge Dr. Yonghong Luo, Dr. Yanhong Guo, Dr. Yuchen Chen, and Dr. Melina Kibbe for their valuable contributions to the final revision and discussion of the manuscript.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The hCitH3-mAb used in this study was generated by HTIC, Inc. Dr. Jianjie Ma, the founder of the company, holds the patent US20240262899A1.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by National Institutes of Health grants R01-HL155116.

Ethics approval

Ethical approval for this study was obtained from Institutional Animal Care and Use Committee (IACUC) at University of Michigan (PRO00010569).

Animal welfare

All experimental procedures and animal care practices adhered to the guidelines established by the University of Michigan Institutional Animal Care and Use Committee, ensuring compliance with approved protocols.

ORCID iD

Tao Dong

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