Role of Chemokines Genetic Polymorphisms in Diseases

1. Role of Chemokines Genetic Polymorphisms in Diseases Ali H. Ad’hiah (Ph.D. Human Immunogenetics) Tropical-Biological Research Unit College of Science University of Baghdad

2. Introductory Theme • The interest into chemokine polymorphisms came with the discovery of allelic variants of HIV co-receptors that confers protection against virus entry into target cells. • Since then, chemokines genetic background has been deeply studied in order to find associations between allelic variants and inflammation-related diseases as well as infectious diseases. • In addition, chemokines genetic variations have been involved in other infectious diseases as HCV and Malaria, and also in a variety of non-infectious diseases such as cancer, autoimmune and cardiovascular diseases. • This seminar aims to present genetic variations in chemokines encoding genes and discuss their role, sometimes controversial, in a variety of diseases.

3. What are Chemokines? • Chemokines are a large superfamily of small (~ 8–15 kDa) structurally related chemotactic cytokines that regulate cell traffickingof various types of leukocytes to areas of injury and play different roles in both inflammatory and homeostatic processes. • They selectively, and often specifically, control the adhesion, chemotaxis, and activation of many types of leukocyte populations and subpopulations. Consequently, they are major regulators of leukocyte traffic. • Besides leukocyte migration, chemokines can also influence leukocyte cell survival and effector functions such as degranulation. • Furthermore, chemokines act on other cell types and tumor cells, thereby contributing to angiogenesis, hematopoiesis, organogenesisand tumor growth and metastasis.

4. Chemokines recruit the cells to sites of infection

5. (a) The four sequential but overlapping steps in neutrophil extravasation. (b) Cell-adhesion molecules and chemokines involved in the first three steps of neutrophil extravasation. • Initial rolling is mediated by binding of E-selectin molecules on the vascular endothelium to Mucinlike CAMs. • A chemokine such as IL-8 then binds to a G-protein–linked receptor on the neutrophil, triggering an activating signal. • This signal induces a conformational change in the integrin molecules, enabling them to adhere firmly to Ig-superfamily molecules on the endothelium.

6. CHEMOKINES: A DOUBLE-EDGED SWORD Host defense Angiogenesis Inflammation Hematopoiesis chemokines Neoplasia Autoimmune Disease Atherogenesis Infection Chemokines and their receptors are a system which has evolved to protect the host and maintain homeostasis, but disordered function of the system or exploitation by pathogens can result in antagonistic negative effects. Hence the concept of a double-edged sword

7. Functional Classification of Chemokines • Chemokines can be either structurally or functionally classified into subgroups. • Based on their function, they are designated as inflammatory and homeostatic chemokines. • The inflammatory chemokinesare inducible, expressed in response to infection, tissue injury or stress factors. They are responsible for the recruitment of effector leukocytes to the site of inflammation. • In contrast, the homeostaticchemokines are constitutively produced by tissue cells and control basal leukocyte trafficking, such as lymphocyte homing to secondary lymphoid organs and lymphocyte recirculation through peripheral tissues. • Some chemokines exert both inflammatory and homeostatic functions and are therefore called dual-function chemokines.

8. CXCL8/IL-8 CXCL1/GROα CXCL4/PF4 CXCL10/IP-10 Microbesinflammatory cytokines CXC chemokines CC chemokines CCL2/MCP-1 leukocytesendothelial cells epithelial cells fibroblasts CCL3/MIP-1 CCL5/RANTES CCL11/Eotaxin lymphocytes neutrophils basophils lymphocytes eosinophils Endothelial cells Mononuclear phagocytes

9. Under normal conditions, homeostatic chemokines regulate cellular traffic by directing cells that express certain chemokine receptors to specific locations where their chemokine ligands are expressed

10. Structural Classification of Chemokines • Approximately 50 chemokines (and 20 receptors) are identified to date. • Structurally, they are divided into four families on the basis of the pattern of the first two of four cysteine residues of the ligand: • The large CCfamily (Most of them are clustered on chromosome 17q11–21 and 9p13). • The CXC family (Most of them are clustered on chromosome 4q12–13 and 4q21). • The CX3C family (with only one member, CX3CL1). • The XC family (consisting of two highly related chemokines, both binding to the XCR1 receptor).

11. Classification of chemokines

14. The chemokine wheel Chemokine receptors and their ligand-specificity

15. Atypical Chemokine Receptors • A subset of chemokine receptors, initially called “silent” on the basis of their apparent failure to activate conventional signalling events, has recently attracted growing interest due to their ability to internalize, degrade, or transport ligands and thus modify gradients and create functional chemokine patterns in tissues. • These receptors recognize distinct and complementary sets of ligands with high affinity, have evolved to fulfill fundamentally different functions to the classical signalling chemokine receptors. • Based on these considerations, these receptors [D6, Duffy antigen receptor for chemokines (DARC), CCX-CKR1 and CXCR7] are now collectively considered as an emerging class of ‘atypical’ chemokine receptors.

16. DUFFY BLOOD GROUP ANTIGEN • The Duffy antigen receptor for chemokines (DARC) has recently become the focus of studies investigating interactions of inflammatory chemokines with erythrocytes during systemic inflammatory responses, as well as, with venular endothelial cells during chemokine-induced leukocyte adhesion and emigration. These studies uncovered new functional scope of this rather “old” molecule. • DARC was first described in 1950 as the Duffy blood group antigen. Three “Duffy-positive” phenotypes were described: Fy(a+b−), Fy(a−b+), and Fy(a+b+), arising from combinations of the co-dominant FYA and FYB genes. However, some individuals, designated “Duffy-negative,” express neither Fya nor Fyb antigens, Fy(a−b−). • The Duffy-negative phenotype was first linked with resistance to malaria when Fy(a−b−) volunteers exposed to the bites of P. vivax-infected mosquitoes, in contrast to Duffy-positives, did not develop malaria. • Duffy blood group antigen was designated DARC after it was shown to mediate the binding of inflammatory CC and CXC chemokines to erythrocytes.

17. Viral chemokines • Several virus [human herpesviruses (Kaposi sarcoma-associated herpes virus, HHV-8) and pox viruses (Molluscumcontagiosum virus, MCV) have been shown to encode CK-like proteins. • In HHV-8 two viral proteins with homology to CCL3– CCL4 (MIP proteins), called vMIP-I and vMIP-II, are produced. Both expressed proteins are angiogenic, showing a pathogenic role in Kaposi sarcoma. In addition, vMIP-II has unique biological activities in that it blocks infection by several different HIV-1 strains. This occurs because vMIP-II binds to a wide range of CKRs, some of which are used by HIV to gain cell. The HHV-8 virus thus appears to have ‘hijacked’ a human MIP-like CK, modifying it so that it can bind to more than one CKR and thereby increasing the pathogenicity of the virus, helping it to spread and proliferate. • The poxvirus MCV CK proteins also closely resemble CCL3 and appear to share the inhibitory effect that this CK has on human haematopoietic progenitor cells. These proteins are potent antagonists and can inhibit the chemotactic response to the human CK. It is likely that their major function in the virus is to aid it in immune evasion during infection.

18. Map of genomic organization of human chemokinesCC chemokinesin red, CXC chemokines in green, CX3C chemokine in yellow and C chemokinesin blue.

19. Genetic Polymorphism of Chemokines • In evolution, diversification through the generation of multiple alleles is very common and the immune system contains several groups of genes with prominent allelic variations. • The CK superfamily constitutes a very revealing case of how, through evolution, a complex network of genes has acquired a very diverse set of related functions. • Most, if not all, CKs probably arose by gene duplication of a single ancestral gene and, consequently, many CKs (just as many CKRs) are clustered in defined chromosomal locations. • Two main clusters have been recognized, both of them codifying the essential inflammatory CKs: the CXC cluster, located in chromosome 4q12–21 and the CC cluster, located in chromosome 17q11.2. • Those CKs that map in the CXC and CC clusters seem to maintain some similar functions: CXC cluster CKs recruit mainly neutrophils, whereas CC cluster members typically attract mononuclear cells.

20. Genetic Polymorphism of Chemokines • Another important way by which CK variations increased during CK evolution at the genomic level is through the generation of polymorphisms, especially single nucleotide polymorphisms (SNPs). • Other types of polymorphisms such as deletion/ insertion polymorphisms (DIPs), copy number polymorphisms (CNPs) or those due to repeated elements (as minisatellites and microsatellites) also contributed importantly to the CK genomic variation, but their distribution is more restricted. • Additionally, beyond the contribution of polymorphisms to the overall variability in CK superfamily, some CK genes that are polymorphic have alleles that are found to be repeatedly associated with disease.

21. Infectious diseases: HIV Infection • The main relation between HIV and chemokines resides in the virus’s need to bind to a co-receptor in addition to CD4 at the surface of permissive cells to enter the cell. • The main co-receptors of HIV are CCR5, especially during primary infection, and CXCR4 during the asymptomatic/late stages of progression to AIDS. • Although no obvious polymorphism has been identified for CXCR4, several genetic variations in the CCR5 gene have been clearly associated with HIV restriction, viral control, and progression to AIDS.

22. CC-receptors and HIV infection

23. CCR5 Polymorphism and HIV Infection • The strongest effect identified, which restricts HIV, involves the CCR5Δ32 variant with 20 base pairs missing in the coding sequence. This absence produces an mRNA frame shift and the emergence of a stop codon. • In term of polymorphism, 13 single nucleotide polymorphisms (SNPs) have been identified in the CCR5 gene promoter: A29G, G208T, G303A, A612G, C626A, C627T, C630T, C647A, A676G, T684C, C714G, G811A, and C927T, that is, at least 10 promoter haplotypes (numbered CCR5-P1 to P10). The CCR5Δ32 mutants is associated in linkage disequilibrium with the CCR5-P1 haplotype.

24. Infectious diseases: HCV Infection • The association study scanned for polymorphisms in CCL5, CCR5, CCR2, CCL3, CCL2 and CCR3 and reported four main findings: • The CCR5Δ32 allele is associated with mild fibrosis and reduced portal inflammation. • Polymorphisms in a CCR5 promoter—the 2132-C allele—are associated with increased risk of HCV persistence and with better initial response to interferon. • The CCL5-403 promoter polymorphism is associated with portal inflammation. • The MCP-2 Q46K mutation is associated with more severe fibrosis.

25. Transplantation • The major factors associated with transplant rejection episodes are HLA mismatches, donor age, and delayed graft function. But, Some graft rejection episodes cannot be explained by these factors, and the hypothesis of Chemokine genetic polymorphisms responsible for these events has been an attractive hypothesis according to the following observations: • The outcome of kidney transplants showed that the CCR2-64I variant and the CCR5 59029-A allele both protect against acute rejection. • CCL2-2518-G/G homozygotes are more susceptible to renal graft rejection over the long term than either A/G heterozygotes or A/A homozygotes at this position. • The finding that neither the CCL2-2518 nor CCR264I polymorphisms are involved in acute graft rejection suggested that the CCL2-2518-G allele, which is correlated with higher CCL2 secretion levels in PBMCs isolated from kidneys, plays a role only in the long-term survival of the transplant organ. • Another chemokine ligand, CCL4L1, and more specifically, the number of its copies, might influence lung transplantation outcome. Patients with more than 2 copies of CCL4L1 had a greater risk of developing acute rejection.

26. Autoimmune diseases: SLE • SLE is one of the most common autoimmune diseases and is due mainly to genetic variations that can be enhanced by environmental factors. Among the genetic factors associated with lupus is a polymorphism in the CXCL8 gene, CXCL8-845C, which might predispose African-Americans with SLE nephritis to more severe renal damage, possibly by influencing CXCL-8 expression. • The gene most often studied in association with SLE is CCL2. It has been reported that A/Gor G/G genotype at position 2518 might predispose individuals to the development of SLE and further that SLE patients with these genotypes might be at higher risk of developing lupus nephritis (LN), a major cause of morbidity and mortality in patients with SLE. • A further finding revealed that the CCL2-2518-G allele is a significant risk factor for SLE among Whites but not African-Americans suggests that genetic differences in CCL2 expression play a role in SLE.

27. Autoimmune diseases: Sjogren syndrome • Sjogren syndrome (SS), which is characterized mainly by CD4+ T-cell infiltration of exocrine glands, appears to be due to a predisposition associated with multiple gene variations and environmental triggers. Several chemokines, including CCL3, CCL4 and CCL5, may play a role in this disease. • An initial study reported a significantly lower frequency among SS patients, compared with healthy controls, of the CCR5Δ32 allele and consequently of subjects heterozygous for it. • Gene targeting in mice revealed that Ccr7-deficient animals are severely impaired in the induction of central and peripheral tolerance. Due to these defects, Ccr7-deficient mice spontaneously develop multi-organ autoimmunity showing symptoms similar to those observed in humans suffering from connective tissue autoimmune diseases.

28. Autoimmune diseases: Kawasaki disease • Kawasaki disease (KD) is characterized by acute systemic vascularitis in young children and is the leading cause of acquired heart disease in North America and Europe. Its incidence, however, is 7–15 times higher in Japanese than American or European children, which suggests that genetic variations promote its occurrence. • In a Dutch cohort of KD patients, the disease was associated with common genetic variants of the chemokine receptor gene cluster CCR3-CCR2-CCR5. • Of note, the lower frequency of the CCR5Δ32 allele in KD subjects than in the control group provides some confirmation of this allele’s protective role in the context of autoimmune diseases, as seen in SS. • Two additional studies found, in confirmation of the Dutch findings, that genetic variations in CCR5/CCL5 genes are associated either with KD or its coronary complications.

29. Autoimmune diseases: Arthritis • Because of the important role chemokines play in pro-inflammatory diseases, their contribution to rheumatoid arthritis (RA) has been extensively evaluated. Early results showed that the CCR5Δ32 allele might influence RA variables including IgM rheumatoid factor. These data suggest that the CCR5 polymorphism has a specific effect on the severity of RA . • A recent genome-wide analysis study of about 7000 RA patients and 20,000 controls identified a SNP in CCR6 that is significantly associated with RA. This SNP, rs3093024, is in strong LD with a tri-allelic dinucleotide polymorphism (CCR6DNP) and influences CCR6 transcription: the CCR6DNP-T allele induces stronger gene expression than that seen with either the CCR6DNP-A or -C alleles. Furthermore, the observed correlation of the CCR6DNP genotype with IL-17 in the sera of RA patients suggests that CCR6 plays an important role in IL-17-driven autoimmunity.

30. Allergy/Asthma • Atopic dermatitis (AD): The first polymorphism identified was in the CCL5 promoter (−401-A/G), and children homozygous for the −401-A allele were present in the AD group only, not in the control group, but no association was identified with asthma. • Also, this allele showed racial variation (the frequency of −401-A/A genotype was 15% in Africans compared with about 2.1% in whites). • Further study reported that the CCL2-2518-G allele (which is associated with increased CCL2 transcription) might be associated with asthma, and children carrying the −2518-G/G genotype were significantly more susceptible to severe asthma, which is correlated with increased levels of eosinophils. • A similar subsequent work confirmed the role of the CCL2-2518-G allele and extended it to allergic phenotypes in general, particularly by showing an association with higher sensitization levels to allergens.

31. BreastBloodTarget Organ Chemokines & Breast Cancer Metastasis • Metastasis is an orderly, multistep process involving the movement of cancer cells from the primary tumor to specific organs under the guidance of specific chemokines. • First, cancerous mammary epithelial cells undergo clonal proliferation, invade local tissue, induce angiogenesis, and CXCR4 on their surface. Then, cancer cells detach from the primary tumor, migrate across lymphatic and vascular walls in the tumor, and enter the systemic circulation. • Cancer cells are arrested in vascular beds in organs that produce high levels of the CXCR4 ligand (CXCL12), which is expressed on the surface of vascular endothelial cells. Binding of CXCL12 to CXCR4 induces the migration of cancer cells into normal tissue, where the cells proliferate, induce angiogenesis, and form metastatic tumors. • Breast-cancer cells do not usually metastasize to organs that produce low levels of CXCL12, such as the kidney.

32. Repertoire of chemokines and chemokine receptors expressed in cancer tissues: Close interactions occur between cancer cells and cells of the tumormicroenvironment

33. Cancer: Leukemia • AML is characterized by uncontrolled proliferation of myeloid progenitors in the bone marrow, and CXCR4 exerts a central role in the trafficking of these malignant cells. The CXCL12/CXCR4 axis has thus been screened for polymorphisms related to blast dissemination. • It has been shown that the 801-G/A gene polymorphism (which leads to higher CXCL12 secretion) in CXCL12 is associated with the number of peripheral blood blasts (PBB) and the number of extra-medullary tumor sites. More specifically, individuals with the 801-A/A genotype had more PBB than 801-G/G carriers did, as well as almost triple the likelihood of developing extra-medullary tumors. • Chemokines are also used to assess drug efficacy in acute lymphoblastic leukemia (ALL). The presence of minimal residual disease (MRD) is a marker of this anti-leukemic efficacy currently used to assess risk status in children with ALL. A study has demonstrated the CCR5 243-A/A genotype is associated with a less favorable MRD status

34. Lung cancer • The first associations between chemokine polymorphisms and lung cancer were reported in 2004, when Campaet al. showed that an CXCL8 promoter polymorphism was associated with a protective effect against lung cancer in women: the risk of developing non-small-cell lung cancer (NSCLC) was drastically reduced among women carrying the CXCL8-251-A/A genotype. • Analysis of the genotype frequencies for the CXCL12-3 polymorphism has indicated that individuals with genotypes not carrying the A allele are nearly 3.5 times more likely to develop a long-distance metastasis of epidermoid NSCLC and thus suggests that the involvement of chemokine polymorphisms is not limited to primary tumor sites only. • Assessment of postoperative metastatic recurrence found significantly higher values of CXCR4 and CXCR7 expression in patients with these recurrences than in those without them. The 5-year disease-free survival rate for patients with high CXCR7 levels was significantly lower than that for patients with low CXCR7 levels (63.2 vs. 84.8%)

35. Functions of glioma produced chemokines

36. Glioblastoma • Patients homozygous for the allele CX3CR1-I249 survived for a substantially longer period (mean: 23.5 vs. 14.1 months; P < 0.0001) after surgical operation. • The common CX3CR1 allele was also associated with a reduction in infiltration by microglial cells. • Accordingly, the authors proposed that this polymorphism might be useful in predicting survival.

37. Cardiovascular and cerebrovascular diseasesHypertension • A study showed significantly elevated levels of soluble CCL2 in hypertensive patients with diffuse atheroma, due to overexpression of this gene by endothelial cells. • More recently, a further study confirmed that CCL2 polymorphisms might play a role in blood pressure. The authors showed that blood pressure values were associated with the CCL2-2518-A/G polymorphism, and subjects with the mutant G allele had higher levels of both systolic and diastolic blood pressure than individuals with allele A; the same was true among asymptomatic subjects.

38. Other cardiovascular and cerebrovascular diseases: Atherosclerosis • Atherosclerosis is a major public health problem, as a cause of both myocardial infarction (MI) and brain infarction (BI). The first robust study to associate chemokine polymorphisms with these diseases found the CX3CR1-I249 and -M280 alleles to be associated with an increased risk of BI independently of other established risk factors. In addition, BI patients homozygous for the rare alleles were much more frequent in the group with no previous cardiovascular events. • Finally, ex vivo monocyte adhesion was tested and found to be highest in blood from individuals carrying the rare CX3CR1 alleles, and such finding is consistent with the mechanisms leading to stroke. • It has also been suggested that eotaxin/CCL11, which is known to promote cell migration, plays a role in MI. The authors showed that the CCL11-23T allele was associated with an increased risk of MI.

39. Central and peripheral nervous system diseases: Alzheimer disease • Alzheimer disease (AD) is the principal cause of dementia in older people. The formation of β-amyloid plaques and neurofibrillary tangles are the main events that lead to neuron degeneration in the brain, but pro-inflammatory processes promote disease progression. • Until now, the only chemokines thought to contribute to AD were CCL2 and CCL3. The A-2518G polymorphism of CCL2 was initially reported not to be a risk factor for AD development, even though this genotype is correlated with higher serum levels of CCL2, which can contribute to the AD inflammatory process. Further study showed that the same CCL2 gene genotype was associated with AD in a homogenous Italian population. • A Chinese study suggested that the CCL3-906 (TA)(6)/(TA)(6) genotype contributes to elevated serum CCL3 levels in AD patients, which in turn play a role in the inflammatory process in AD.

40. Conclusions • Chemokinesand their receptors take part in the outcome of various diseases, from viral infections to autoimmune syndromes. • They are major players in inflammatory events, which most often involve the recruitment of leukocytes at the right sites to eliminate pathogens. In some cases, however, impaired regulation of gene expression or a structural mutation in the coding sequence can lead to chronic inflammation-related diseases. • Two genetic polymorphisms of chemokine receptors have an almost fully penetrant genetic effect for two pathogens: CCR5Δ32 for HIV, and Duffy antigen for the P. vivaxmalaria parasite. These polymorphisms confer almost complete protection from these pathogens on the people bearing the homozygous genotype. • Strong evidence indicates that chemokines play a role in autoimmune diseases, although controversy remains in some areas. The genetic variations that can lead to chronic inflammation-related dysregulation can be attributed, at least in part, to the genes encoding chemokines/receptors. • Racial differences can also contribute to inconsistent observations.