Background

Visceral leishmaniasis (VL), also known as Kala-azar, is a life-threatening disease caused by protozoans belonging to Leishmania donovani complex. Population based epidemiological surveys suggest that 80-90% of individuals infected with L. donovani show no clinical symptoms [1–3]. Familial clustering, and the range of clinical outcomes from asymptomatic to fatal disease within and between ethnic groups sharing similar risk factors, support a contribution of host genotype to susceptibility [4–8]. Resistance to VL, as determined by a positive antigen-specific DTH response without clinical symptoms, is ~80% heritable in family-based studies [9]. Genetic variability in ability to mount an innate immune response, such as in the influx of polymorphonuclear neutrophils (PMN) during initial hours of infection, could be important in innate killing of the parasite [10], and in providing the cytokine environment in which parasite-specific T cells are primed [11]. Pro-inflammatory responses elicited by PMN as part of the response to the bite of the sand fly vector are important in initiation of infection [12]. The arrival and maintenance of infiltrating cells at bite sites is thought to be mediated by sand fly-derived factors that mimic a tissue damage signal and/or activate chemokine/chemokine receptor pathways [13–15]. Recently [16] we examined the role of polymorphisms at CXCR1 and CXCR2, which act as receptors for CXC chemokines that attract PMN to inflammatory sites, in determining susceptibility to cutaneous forms of leishmaniasis caused by infection with L. braziliensis. We found that cutaneous (CL) and mucocutaneous (ML) forms of disease were associated with opposing alleles for SNP rs2854386 at CXCR1. Studies in mice [17] show that selective depletion of PMN has a dramatic effect on the course of infection with L. donovani. Here we use genetic and functional approaches to evaluate the role of PMN in VL caused by L. donovani in humans through analysis of the receptors CXCR1, which is a specific receptor for IL-8 (= CXCL8), and CXCR2, which is promiscuous in binding a variety of CXC chemokines (CXCL-1, 2, 3, 5, 6, 7) in addition to CXCL8.

Methods

The study was conducted in the district of Muzaffarpur in Bihar State, India, where VL is highly endemic. Diagnosis of VL was made on the basis of clinical, parasitological and serological criteria as described [18, 19]. Further epidemiological and demographic details relating to the study samples and study site are described elsewhere [20, 21]. Informed written consent in Hindi was obtained from all participating individuals and from parents of children under 18 years old. Approval for the study was provided by the Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Collection of families was undertaken between 2004 and 2006 while for the case-control study collection was undertaken during 2009-2010. Table 1 provides details of primary and replication samples, indicating the number of extended families that are decomposed into nuclear families for genetic analysis. For the family-based primary study, DNA was prepared from buccal swabs by whole genome amplification as described [19]. For the replication case-control study, genomic DNA was extracted from saliva using the Oragene technology (DNA Genotek, Ontario, Canada).

Table 1 Baseline characteristics of (A) families for the primary sample of Indian multicase VL families, and (B) the Indian case-control cohorts
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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 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.

Table 2 Details of polymorphisms genotyped and the minor allele frequency (MAF) of variants in the Indian study population
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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 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].

Splenic biopsies were taken as part of routine diagnostic procedure at the Kala Azar Medical Research Centre, Muzaffarpur, Bihar State, India. Paired pre- (Day-0) and post- (Day-30) treatment splenic samples were collected from 19 VL patients in 5xRNA Later (AMBION Inc., Austin, Texas, USA) during 2009-2010, transported to Varanasi at 4°C and stored at -80°C until RNA was isolated. Details regarding age and sex, splenic parasites and drug administered were recorded for each patient. Total RNA was isolated using RNeasy tissue kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer's instructions. Sample quality and integrity was assessed by ND-2000 spectrophotometer (Thermo Fischer Scientific, Wilmington, DE, USA) and agarose (Sigma Aldrich Chemicals, St Louis, MO, USA) gel electrophoresis. 1 μg of RNA was reverse transcribed using the High Capacity cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Taqman predesigned gene expression assays (CXCR1_Hs00174146_m1 and CXCR2_Hs01011557_m1) were used to perform expression studies (7500 HT Real Time PCR system, Applied Biosystems, Foster City CA, USA) with 18S rRNA (P/N 4319413E) being used as an endogenous control to normalize the expression data. No RT and no template controls were included in each plate. All samples were run in duplicate. Results were analysed by 7500 software v.2.0.1 and GraphPad Prism (version 5.00 for Windows, Graph Pad Software, San Diego California USA, http://www.graphpad.com). The significance of differences between pre- and post-treatment groups was determined using paired Student's T test. Power calculations showed that N = 19 paired samples had 92.2% power to detect a difference in mean values pre- and post-treatment of 0.9 with standard deviation of 0.9 at alpha level 0.01; 98.2% power at alpha level 0.05.

Results and Discussion

To test the hypothesis that polymorphisms at CXCR1/CXCR2 might influence susceptibility to VL in India we initially genotyped 3 tagging SNPs (Table 2) in 176 nuclear families (Table 1) used in our previous studies [19, 21] that contain 313 offspring with VL collected in the area of Muzaffarpur, Bihar State, India, where L. donovani is endemic. Using the family-based association test (FBAT) [29, 30] in this primary family dataset (Table 3) we found evidence (nominal P-values ≤ 0.03) for associations between VL and SNPs CXCR1_rs2234671 (Z-score = 2.935, P = 0.003) and CXCR1_rs3138060 (Z-score = 2.22, P = 0.026), but not with CXCR2_rs4674259. Since these two positive markers are in quite strong LD with each other (Additional File 2; Figure S2: D' = 0.84; r2 = 0.46), these associations are likely to be measuring a single effect. The association at CXCR1_rs2234671 is robust to application of a strict Bonferroni correction for 3 independent (i.e. r2 < 0.8) SNPs genotyped, which requires a significance cut-off of P ≤ 0.017 (i.e. P = 0.05/3).

Table 3 Family-based association analysis between CXCR1/CXCR2 and VL
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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 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 1) from the same region of Bihar State in India. Logistic regression analysis (Table 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 5; TRANSMIT; P = 0.014) and population- (Table 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.

Figure 1
figure 1

Relative expression of CXCR1 (A) and CXCR2 (B) mRNA in paired splenic aspirates from VL patients before (Day 0) and after (Day 30) antileishmanial treatment. Individual as well as mean (± SEM) relative expression for each group is indicated. Paired Student's t tests show significant differences in expression of CXCR2 but not CXCR1 when Day 0 values were compared to Day 30 values.

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Table 4 Population-based association analysis between CXCR1/CXCR2 and VL
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Table 5 Haplotype association analyses between CXCR1/CXCR2 and VL
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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.

Here we examined CXCR1 and CXCR2 as candidate genes for susceptibility to VL in India. Whilst SNPs at both loci were associated with VL, functional analysis of expression in splenic aspirates together with a common risk haplotype favour CXCR2 as the etiological gene regulating susceptibility to disease. Our data contribute to increasing evidence for an important role for PMN in directing the outcome of leishmanial infections in humans.

Conclusions

From the results of this well-powered primary and replication genetic study, together with functional analysis of gene expression, we conclude that CXCR2 plays a role in determining outcome of VL in India.