CXCR1 and CXCR2 lie on a 26 kb region adjacent to each other on human Chromosome 2q35. CXCR2 is encoded on the positive strand and lies proximal to CXCR1 encoded on the negative strand. DNA samples were genotyped for SNPs (Table ​(Table2)2) CXCR2 (rs4674259) and CXCR1 (rs3138060) that tag major linkage disequilibrium (LD) blocks (r² > 0.8; as determined for available data from HapMap populations CEU/JPT/CHB; Additional File 1; Figure S1) across the two genes, as well as the non-synonymous exon 1 SNP rs2234671 in CXCR1. For the primary family-based study, SNPs were genotyped in 836 individuals using ABI predesigned Taqman assays (ABI, Mulgrave, Victoria, Australia). For the replication study, SNPs were genotyped using Sequenom iPLEX platform (Sequenom, San Diego, CA, USA) for 2022 individuals comprising 990 cases, 1029 controls (1168 males, 850 females). All 3 SNPs met minimum quality control checks for call rate (> 99.5%) across all individuals, and all were in Hardy Weinberg equilibrium (HWE) in unrelated founders of families and in the control replication sample. Twelve individuals were removed from further analysis where data was missing for two or more SNPs.

Family-based allelic association tests were performed within FBAT which is based on the transmission disequilibrium test (TDT) but allows for different genetic model analyses with incomplete parental data [22,23]. Analyses were performed using an additive model and under the null hypothesis of "no linkage and no association". Family-based haplotype TDT was performed using TRANSMIT [24]. Robust tests were performed to take account of multiple trios within some pedigrees (Table ​(Table1).1). Family based TDT power approximations [25] show that the 313 VL trios had 49% power to detect an odds ratio ≥ 1.5 at P = 0.01 for markers with MAF ≥ 0.1. Single marker and haplotype association tests for the case-control sample were carried out using logistic regression analysis performed in PLINK [26] under an additive model. Inclusion of caste as a covariate, which we have shown to provide a good surrogate for genetic substructure in genome-wide analyses in this population (unpublished data), was used to take account of population substructure. Religion was also analysed as a covariate. The 1933 individuals (941 cases and 992 controls) which passed quality control had 93.5% power to detect associations with an odds ratio of 1.5 for markers with MAF ≥ 0.1 at P = 0.01. Nominal two-tailed P-values are presented throughout, i.e. without correction for multiple testing. Application of a strict Bonferroni correction for 3 independent (r² < 0.8) SNPs provides a significance cut-off of P ≤ 0.017 (i.e. P = 0.05/3). Combined P-values using Fisher's Trend Test were calculated using MetaP [27]. LD plots for D' and r² were generated in Haploview [28].

Evidence for an association between Indian VL and SNPs at CXCR1, which was consistent with data for CL/ML from Brazil [16], prompted us to pursue two further avenues of investigation. In so doing, we did not discount the possibility that CXCR2 might play a role, since the D' measure (Additional File 2; Figure S2) indicates strong LD across the CXCR1/2 SNPs for this sample (D' = 0.98 and 0.91). First, we looked at expression levels of CXCR1/CXCR2 in mRNA from splenic aspirates from 19 patients as paired samples taken pre- and post-treatment for VL (Figure ​(Figure1).1). This demonstrated that CXCR2, but not CXCR1, is significantly (P = 0.021) reduced in expression in pre-treatment samples compared to post-treatment recovery samples, indicating that a deficiency in expression of CXCR2 might contribute to VL disease. Down regulation of CXCR2, but not CXCR1, was similarly observed in PMN from patients with pulmonary tuberculosis [31]. Secondly, we carried out a comprehensive replication of the association study in a much larger population-based case-control sample (Table ​(Table1)1) from the same region of Bihar State in India. Logistic regression analysis (Table ​(Table4)4) under an additive model of inheritance showed association between VL and SNPs CXCR2_rs4674259 (OR = 1.15, 95%CI = 1.01-1.31, P = 0.027) and CXCR1_rs3138060 (OR = 1.25, 95%CI = 1.02-1.53, P = 0.028), but not with CXCR1_rs2234671. Effect sizes (OR) are small, as is common in complex communicable or non-communicable diseases [32]. Significance was retained when either religion (CXCR2_rs4674259: OR = 1.15, 95%CI = 1.02-1.31, P = 0.028; CXCR1_rs3138060: OR = 1.26, 95%CI = 1.03-1.53, P = 0.026) or caste (CXCR2_rs4674259: OR = 1.15, 95%CI = 1.01-1.32, P = 0.039; CXCR1_rs3138060: OR = 1.25, 95%CI = 1.01-1.54, P = 0.037) was used as a covariate to take account of population substructure. Interestingly, although CXCR1_rs3138060 was the only SNP directly replicated across primary and replication samples, the same 3-SNP haplotype T_G_C (called on the negative strand; frequency 0.42) was shown to be the risk haplotype in both family- (Table ​(Table5;5; TRANSMIT; P = 0.014) and population- (Table ​(Table5;5; PLINK; OR = 1.16, P = 0.028) samples (combined P = 0.002). The 2- and 3-SNP over-transmitted haplotypes were more apparent in the family-based sample, which likely reflects over-relatedness for this set of families from this region of India where we showed previously that consanguineal marriages were common [19]. These haplotype results indicate that associations seen at CXCR1 might be due to LD with regulatory polymorphisms that influence CXCR2 expression. Together, the genetic and functional analyses favour a role for CXCR2 in contributing to susceptibility to VL in this region of India, although a role for CXCR1 cannot be discounted.

The involvement of either CXCR1 or CXCR2 in VL is of interest given recent observations on the different roles they may play in chemotaxis and activation of PMN in sites of infection [33], and the increasing recognition of the importance of PMN in the VL disease process [12,17]. Both are high affinity receptors for the CXC chemokine IL-8, but CXCR2 is more promiscuous in also binding a range of other CXC chemokines (CXCL-1, 2, 3, 5, 6, and 7) which may play important roles in directing a broader array of immune cells to sites of infection in the visceral organs. Recent studies [34] also show that, while neutralizing antibodies against CXCR2 abolish neutrophil extracellular trap (NET) formation, antibodies against CXCR1 have no effect. NETs are formed by DNA fibers decorated with antimicrobial proteins released from PMN upon activation. The release of DNA NETs, decorated with elastases and histones, by human PMN upon interaction with Leishmania parasites has been shown to ensnare the parasite and is leishmanicidal [10], adding to the potential importance of PMN in innate immunity to leishmanial infection.