Abstract
Humans respond differently toward exposure against pathogens and some individuals are completely resistant against transmission due to a genetically determined susceptibility. A rising number of such, so-called, host factors have been described during the last years, but their role for diagnostic or therapeutic application is still to be clarified. Here, we describe the biology of the chemokine receptor CCR5 and its polymorphism in the context of host adaptation and immune system function. Furthermore, the first clinical applications exploiting our knowledge of this chemokine receptor as a host factor are described.
Introduction
In 1949, J B S Haldane published the speculative review ‘Disease and Evolution’. Therein, he presented for the first time the thesis that host factors may play a crucial role in adaptation against infectious diseases. 1 Actually, individuals respond remarkably differently toward exposure against the same pathogens and even more, some of them are completely resistant against transmission. This can partly be explained by alterations in the genome, leading to a decreased susceptibility of transmission, or an increased ability in elimination of the pathogen. In this context, probably the best-known example of such a beneficial mutation is found in the association of malaria and the hemoglobinopathies. For instance, individuals heterozygote for the beta-globulin sickle cell anemia, display a strong protection against death and severe course of disease after infection with Plasmodium species. Although there are hundreds of structural variants of hemoglobin, only those coding for the malaria-resistant variants have polymorphic frequencies reaching 15–20% in some parts of sub-Saharan Africa and exceeding 60% in some south-east Asian populations. Furthermore, this mutation implies such a positive selective benefit that there is no disadvantage for maintaining a population, although homozygotes display serious consequences of this variation, including early death of the individual. 2
In contrast, today, several genetic polymorphisms are described while the best beneficial effect is achieved if the mutation or disposition is homozygous. One of the best-investigated gene polymorphism has been found for the CC chemokine receptor 5 (CCR5). Chemokine receptors are transmembrane cell surface molecules that were originally identified in the context of leukocyte trafficking. By binding several peptides, receptor-bearing cells migrate toward tissues that secrete chemokines. CCR5 in combination with CD4 gained substantial interest as a crucial host factor for cell entry exploited by the human immunodeficiency virus type 1 (HIV-1). 3 Indeed, the interaction of CCR5 and HIV-1 does not represent the natural function of this chemokine receptor. To date, the exact physiological role of CCR5 is speculative and we have achieved insights into CCR5 function rather in individuals or cell systems where the receptor is depleted. Several mutations in the promoter region as well as a 32-base pair deletion (CCR5-delta32) in the exon of CCR5 lead to a decreased expression of up to a complete deficiency of this receptor. The high allelic frequency of CCR5-delta32 of 10–15% in Caucasians and the misbalance in geographical distribution of this mutation is suggestive to assume a beneficial role for the CCR5 depletion and confers adaptation of the host toward (unknown) environmental threats. 4
The CCR5 chemokine receptor
Structure and function
The chemokine receptor CCR5 belongs to the superfamily of the seven-transmembrane G-protein-coupled receptors (GPCRs). 5 It interacts with chemokines that mediate the trafficking and functions of memory/effector T-lymphocytes, macrophages and immature dendritic cells toward sites of inflammation. When bound by their chemokine ligands, these receptors are internalized, impairing the subsequent ability to bind their ligands. Once internalized, these receptors tend to recycle to the cell surface in time. Most chemokines activate more than one receptor subtype and like other chemokine receptors, CCR5 can bind several chemokines. 6 After activation with small ligands, GPCRs are rapidly phosphorylated at serine and threonine residues within the C-tail and the third intracellular loop. 7
Gene regulation
The CCR5 gene is mapped to the short arm of chromosome 3 among a group of genes that encode multiple chemokine receptors. 8,9 CCR5 up-regulation has been proposed by nuclear factor kappa-light-chain-enhancer (NF-κB), but recently it has been suggested that gene regulated is modified by a cyclic adenosine monophosphate (cAMP)/cAMP response element-binding protein (CREB) pathway. 10,11 A selection of previously described factors that may regulate CCR5 are given in Table 1.
Regulation of CCR5 expression
IFN, interferon; IL, interleukin; cAMP, cyclic adenosine monophasphate; CREB, cAMP response element-binding protein
Physiological function
The exact physiological function of CCR5 has been entirely unknown for a long time. Individuals lacking CCR5 display no remarkable illness and no increased susceptibility toward infectious diseases could be observed until Lim et al. 12 figured out a possible role for CCR5 during infection with the West Nile virus (WNV). They found an increased risk for individuals with the CCR5-delta32-mutation developing fatal encephalitis and therefore suggest that the functional receptor acts by recruiting leukocytes into the infected central nervous system. Nevertheless, CCR5 deficiency is not a risk factor for WNV infection per se, but is a risk factor for both early and late clinical manifestations after WNV-infection. 13
CCR5 and HIV cell entry
HIV-1 uses the highly conserved host elements of both CD4 and CCR5 for cell entry. The HIV envelope protein is comprised of three heterodimeric glycoproteins; each consists of a transmembrane glycoprotein 41 (gp41) non-covalently associated with glycoprotein 120 (gp120). In the first step, gp120 binds to cellular CD4, causing a conformational change in the envelope glycoprotein. In the second step, the conformation change exposes previously inaccessible domains permitting binding to the CCR5 co-receptor. This new association provides a second confirmation change in the third step as it induces the uncovering of the free amino terminal-fusion domain of gp41 consequently embedded into the host cell membrane, to link the viral and host cell membrane. The fusion of virion and host cell is mediated by forming a 6-helix bundle of gp41 bringing the viral membrane and host cell membrane together. 14
CCR5 polymorphism
Soon after the detection of CCR5 as a co-factor for HIV-1 cell entry, several mutations in this gene have been found. To date, there are at least 74 mutations described. 15–17 Several studies have demonstrated that polymorphism at the CCR5 locus both in the coding and the regulatory regions may affect susceptibility toward HIV infection. The much-studied CCR5-delta32 allele is a 32-base pair deletion that introduces a premature stop-codon into the CCR5 chemokine-receptor locus and thus obliterates the receptor. 18 Epidemiological studies of the Caucasian population demonstrated that the CCR5-delta32 deletion shows the highest frequency of 10–20% among the heterozygous, and 1% among the homozygous karyotype. Interestingly, the deletion cannot be found in the Asian, Middle East, African and the American Indian population. 19 It is supposed that the imbalanced distribution of this allele is caused by environmental-based selective pressure, resulting in a privilege for the delta32 deletion. 4,20
Suggested candidates for this positive selection pressure include protection from plague caused by Yersinia pestis, smallpox or other related poxviruses. Nevertheless, there is no laboratory evidence for this thesis in addition to the unrevealed physiological role of this chemokine receptor. 21–23
Consequences of CCR5 polymorphism
CCR5-delta32 and HIV infection
Homozygosity for CCR5-delta32 deletion is associated with a high, but not complete, protection against HIV-1. 8,24 Although CCR5-delta32 results in a high degree of resistance toward HIV-1-transmission, several individuals tested to be homozygous for this mutation acquired HIV-infection. 25–28 Under certain circumstances, HIV-1 quasispecies are able to use CXCR4 as an alternative co-receptor instead of CCR5. The responsible mutation occurs at V3 of the HIV-1 genome encoding an envelope glycoprotein (gp120) binding either to CCR5 (R5 type) or CXCR4 (X4 type). 29 The switch from R5 to X4 occurs in the natural course of infection and the rate of X4 increases to approximately 50% during antiretroviral therapy. 30 A trial with 979 HIV+ and antiretroviral-naïve individuals demonstrated that 18.2% were harboring an X4-variant of HIV-1. 31
Interestingly, although several individuals were frequently exposed to X4, the R5 variant dominates during transmission and early infection, irrespective of the course of transmission. 32 Furthermore, despite the fact that CCR5-delta32 homozygous individuals can be infected with X4 or R5X4 quasispecies, this practically never happens. 33 A ‘gatekeeper’ function preferring CCR5 using R5 is postulated during first infection showing an almost perfect negative selection of X4. 34
Recently, we performed the successful stem cell transplantation (SCT) of an HIV-1-infected patient suffering from acute myeloid leukemia, applying homozygous CCR5-delta32 donor cells. These hematopoietic progenitor cells engrafted, proliferated and differentiated to produce mature myeloid and lymphoid cells that were highly resistant to HIV-1 infection. Currently, nearly four years after the allogeneic SCT and in the absence of any antiretroviral treatment, no plasma viral load or proviral DNA was detectable in peripheral blood cells or certain tissue samples, including rectal mucosa and brain tissue. 35–37
Chronic viral infection
Murine models with CCR5 deficiency mice have demonstrated a robust T-cell response to several infectious agents. 38 A vigorous T-cell response is required to recover from acute hepatitis B virus (HBV) infection. Interestingly, Thio et al. 39 found that CCR5-delta32 increases the likelihood of recovery form HBV infection and reduces the development of chronic HBV infection by nearly 50%. Furthermore, this protective effect was exclusively mediated by the CCR5-delta32 deletion and not by any of the other neighboring polymorphisms.
Allogeneic SCT and graft versus host disease
Chemokines play a crucial role in the pathogenesis of graft versus host disease (GvHD) disease after allogeneic SCT. In experimental models, due to the redundancy of receptor − ligand interaction, the deficiency or blockade of a single chemokine does not protect the allograft from acute rejection. 40 However, recent studies have demonstrated that the blockade or absence of a single chemokine receptor does prolong allograft survival in a fully major histocompatibility complex (MHC) mismatched model. 41
In a study with CCR5-knockout and wild-type mice, animals were lethally irradiated and underwent full MHC-mismatch bone marrow transplantation. Observing more cases of GvHD in the group of CCR5-knockout mice, Kuziel and colleagues 42 concluded that the absence of CCR5 results in donor T-cell expansion with a consecutive higher rate of GvHD. In another animal model, the authors demonstrated that CCR5 and CXCR3 combined chemokine blockade is effective in prolonging allograft survival and limiting acute rejection concurrently. 43
A study investigating the CCR5 polymorphism from donors of 186 allografted recipients demonstrated contradicting results to those of Kuziel. Here, the authors suggested that the presence of the CCR5-delta32 allele represents a protective factor regarding the risk of developing GvHD after allogeneic SCT. 44 Previously, Murai et al. 45 described the recruitment of CCR5-expression CD8+ T-cells during acute liver GvHD in patients after allogeneic SCT.
Most recently, a significant association of the common CCR5 haplotype (H1/H1) and advantage of disease-free survival and overall survival in recipients of allogeneic SCT has been found. The authors suggested CCR5 genotyping as a new diagnostic and therapeutic strategy for therapy optimization. 46
Organ transplantation and graft rejection
Recipients of organ allografts homozygous for CCR5-delta32 show longer survival of transplant function than those with other genotypes. This has been shown for renal and liver transplants suggesting that patients with CCR5-delta32 might be candidates for a reduced immunosuppressive therapy. 40,47 Furthermore, interaction and blockade of the CCR5 receptor may also reduce alloantigen-specific T-lymphocyte proliferation and may be effective in preventing acute and chronic rejection of allograft. 48
CCR5-targeted therapy
Based on the challenges of the pharmacological interaction with CCR5 during HIV-1 therapy, we have gained deep insights into the action of CCR5 blocking agents. 49 These experiences can also be useful for other applications besides HIV-1 infection, for example, transplantation medicine. Interference with CCR5 function or expression in terms of manipulation of the immune system and responses seems to be a promising approach. Both CCR5 blockade and down-regulation with rapamycin are effective in modulating transplant immunity and lead to prolonged allograft survival in the animal model. 50
Currently, two clinical trials are investigating the effect of interaction with the CCR5 receptor system in patients with rheumatoid arthritis and patients following allogeneic SCT (NHI ClinicalTrials.gov Identifier: NCT00948753 & NCT00979771), but supposable applications of this approach are numerous. 51
Conclusions
Taken into account the actual technology in comparison with earlier times, we are now able to identify host factors in a more sufficient way. Additionally, new biotechnical methodology has improved our ability to generate a patient-optimized therapy based on individual host factors. This enables us to predict, prevent, diagnose and treat subtypes of diseases in a more efficient way. 52 Our diagnostic and therapeutic tools in individualized medicine have rapidly advanced in the last few years, such as the automated Sanger method, which has been considered as a ‘first-generation’ technology, and even more recent methods are referred to as next-generation sequencing. These new technologies include a mixture of several techniques: bacterial cloning or polymerase chain reaction, template purification, labeling of DNA fragments using the chain termination method with energy transfer, dye-labeled dideoxynucleotides and a DNA polymerase, capillary electrophoresis and fluorescence detection providing four-color plots to reveal the DNA sequence. 53 Most recently, the 454 Sequencing, as a next-generation sequencing technology, features a unique mix of long reads, exceptional accuracy and ultra-high throughput. 54
Studying host factors and their genetic contribution to infectious and other diseases presumes fundamental understanding of pathogenesis (Table 2). The association of candidate gene polymorphisms in several circumstances, not only for infectious diseases, has provided new strategies of intervention. In the future, genomic testing will allow us to perform both prognostic and predictive sub-typing of patient populations. Patients may benefit from therapeutics targeted to their specific disease processes and will (probably) therefore have a reduced risk regarding the development of life-threatening adverse events caused, for example, by genetic differences in drug metabolism. Finally, this kind of revolution in regenerative medicine and therapeutics offers the possibility to transform the efficiency of managing disease from palliation up to cure.
Host factors and infectious diseases
*Cytokine-inducible Src homology 2 (SH2) domain protein
SARS, severe acute respiratory syndrome
Footnotes
ACKNOWLEDGEMENTS
We thank Kate Krauss and Stephen LeBlanc from the AIDS Policy Project for supporting our work.