1471-2350-11-10 1471-2350 Research article <p><it>CXCR1 </it>and <it>SLC11A1 </it>polymorphisms affect susceptibility to cutaneous leishmaniasis in Brazil: a case-control and family-based study</p> Castellucci Léa leacastel@hotmail.com Jamieson E Sarra sjamieson@ichr.uwa.edu.au Miller E Nancy enm21@cam.ac.uk Menezes Eliane ep_menezes@ig.com.br Oliveira Joyce joycemouraoliveira@yahoo.com.br Magalhães Andrea amag@ufba.br Guimarães Henrique Luiz luizhenriquesg@yahoo.com.br Lessa Marcus lessamm@terra.com.br de Jesus Ribeiro Amélia jesus-amelia@uol.com.br Carvalho M Edgar edgar@ufba.br Blackwell M Jenefer jmb37@cam.ac.uk

Federal University of Bahia, Salvador, Brazil

Cambridge Institute for Medical Research and Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, UK

Telethon Institute for Child Health Research, Centre for Child Health Research, The University of Western Australia, Subiaco, Western Australia, Australia

Instituto de Investigação em Imunologia, São Paulo, Brazil

Universidade Federal de Sergipe - Aracaju, Brazil

BMC Medical Genetics 1471-2350 2010 11 1 10 http://www.biomedcentral.com/1471-2350/11/10 20089160 10.1186/1471-2350-11-10
18 9 2009 20 1 2010 20 1 2010 2010 Castellucci et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

L. braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. Wound healing neutrophil (PMN) and macrophage responses made following the bite of the vector sand fly contribute to disease progression in mice. To look at the interplay between PMN and macrophages in disease progression in humans we asked whether polymorphisms at genes that regulate their infiltration or function are associated with different clinical phenotypes. Specifically, CXCR1 (IL8RA) and CXCR2 (IL8RB) are receptors for chemokines that attract PMN to inflammatory sites. They lie 30-260 kb upstream of SLC11A1, a gene known primarily for its role in regulating macrophage activation, resistance to leishmaniasis, and wound healing responses in mice, but also known to be expressed in PMN, macrophages and dendritic cells.

Methods

Polymorphic variants at CXCR1, CXCR2 and SLC11A1 were analysed using Taqman or ABI fragment separation technologies in cases (60 CL; 60 ML), unrelated controls (n = 120), and multicase families (104 nuclear families; 88 ML, 250 CL cases) from Brazil. Logistic regression analysis, family-based association testing (FBAT) and haplotype analysis (TRANSMIT) were performed.

Results

Case-control analysis showed association between the common C allele (OR 2.38; 95% CI 1.23-4.57; P = 0.009) of CXCR1_rs2854386 and CL, supported by family-based (FBAT; Z score 2.002; P = 0.045) analysis (104 nuclear families; 88 ML, 250 CL cases). ML associated with the rarer G allele (Z score 1.999; P = 0.046). CL associated with a 3' insertion/deletion polymorphism at SLC11A1 (Z score 2.549; P = 0.011).

Conclusions

The study supports roles for CXCR1 and SLC11A1 in the outcome of L. braziliensis infection in humans. Slc11a1 does not influence cutaneous lesion development following needle injection of Leishmania in mice, suggesting that its role here might relate to the action of PMN, macrophage and/or dendritic cells in the wound healing response to the sand fly bite. Together with the CXCR1 association, the data are consistent with hypotheses relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele suggests that PMN also have an important positive role to play in preventing this form of the disease.

Background

Leishmania infection is associated with a broad spectrum of clinical phenotypes and many studies have demonstrated that host genetic factors play a part in determining the outcome of infection (reviewed 123). L. braziliensis infection causes cutaneous leishmaniasis (CL) with prolonged time to lesion healing. Pro-inflammatory cytokines, including tumor necrosis factor (TNF) and interferon-γ (IFN-γ), and macrophage activation are important in eventual self-healing, but an exaggerated response is associated with mucosal leishmaniasis (ML) 45. Pro-inflammatory responses elicited by polymorphonuclear neutrophils (PMN) as part of the wound healing response to the bite of the sand fly vector are important in initiation of lesion development in mice 6. It has been hypothesized 7 that differences in the ability of macrophages and dendritic cells from different inbred mouse strains to respond to apoptotic versus necrotic PMN arising during the wound healing response to an infected sand fly bite determines disease progression. The arrival and maintenance of infiltrating cells at bite sites is thought to be mediated by sand fly-derived factors that either mimic a tissue damage signal or activate chemokine/chemokine receptor pathways 8910. Expression patterns for chemokines have been associated with the evolution of large and small lesions in mice following L. braziliensis infection, influenced by both the strain of parasite 10 and the mouse genetic background 8.

One way to look at the interplay between PMN and macrophages in disease progression in humans is to determine whether polymorphisms at genes that regulate their infiltration or function are associated with different clinical phenotypes following infection with Leishmania spp. CXCR1 (IL8RA) and CXCR2 (IL8RB) are receptors for chemokines that attract PMN to inflammatory sites. They lie on human Chromosome 2q25 230-260 kb upstream of SLC11A1, a gene that regulates macrophage activation and resistance to visceral leishmaniasis (reviewed reference 11) as well as wound healing responses in mice 12. Here we report on a small case-control study, underpinned by family-based analysis, which provides evidence for separate roles for CXCR1 and SLC11A1 in determining susceptibility to leishmaniasis caused by L. braziliensis in Brazil.

Methods

The study was conducted in the area of Corte de Pedra, Bahia, Brazil, where L. braziliensis is endemic. Corte de Pedra is in a region of rural rain forest, where agriculture underpins the local economy. Around 3300 subjects were interviewed during 4 years to select the study population. For this genetics study, both case-control and family-based cohorts were studied. Index cases of ML were ascertained from medical records of the Corte de Pedra Public Health Post. The case definition of ML is a characteristic mucosal lesion with either parasitological confirmation or two of the three following criteria: positive delayed-type hypersensitivity test (DTH), positive leishmania serology, and a histopathology suggestive of leishmaniasis. All cases in the current study also responded to antileishmanial therapy. The families and neighborhoods of these ML patients were revisited, to establish the study population. CL is defined as the presence of a single chronic ulcerative lesion at a skin site without evidence of mucosal involvement, and without evidence of dissemination to 10 or more sites (disseminated leishmaniasis), also confirmed by detection of parasites or two of the three criteria listed above. Past cases that have been treated in the health post of Corte de Pedra have their diagnoses confirmed by medical records using the same criteria defined above, and all cases were examined for detection of a characteristic well delimited scar. Informed consent was obtained from all the participants, and the research was approved by the ethical committee of the Hospital Universitário Professor Edgard Santos, Salvador, Brazil. Demographical, epidemiological and phenotype characteristics of these subjects were previously described in full 13.

DNA was available from 60 ML index cases (47 males: 13 females; mean age ± SD = 40 ± 17 years), 60 age- and gender-matched unrelated CL cases (47 males: 13 females; mean age ± SD = 41 ± 17.8 years), 60 age- and sex-matched unrelated individuals (47 males: 13 females; mean age ± SD = 38 ± 18 years) positive for the leishmanin delayed hypersensitivity skin-test response (DTH+) and with no current or previous history of CL or ML disease, and 60 unrelated neighbourhood controls (47 males: 13 females; mean age ± SD = 40 ± 18 years). These neighborhood controls (NC) also had no clinical history of disease or leishmaniasis scars, but their leishmanin skin test status was unknown. For some of the case-control analyses the two patient groups, CL and ML, were analysed together to determine susceptibility to L. braziliensis per se, and the DTH+ and NC groups pooled as the unaffected controls. This 120 cases compared to 120 controls analysis had ≥65% power to detect an odds ratio ≥2 at P = 0.01 for markers with minor allele frequency (MAF) ≥0.2. The smaller comparison of 60 cases compared to 60 controls had ≥55% power to detect an odds ratio ≥2 at P = 0.05 for markers with minor allele frequency (MAF) ≥0.2. The 60 ML index cases were also used to ascertain a total of 67 multi-case leishmaniasis (mixed for CL and ML) pedigrees (104 nuclear families), providing a total of 88 ML cases (i.e. 28 additional cases; 15 of the 88 cases had no observable scar for prior CL disease) and 250 CL cases (exclusive of the 60 CL cases used in the case-control study. Thus the family study is used to validate associations for ML seen in the case-control analysis, while the CL sample in families is independent and can be used to replicate observations made in the case-control analysis. TDT power approximations 14 show that the 250 CL cases in 104 nuclear families had ≥85% power to detect an odds ratio ≥2 at P = 0.01 for markers with minor allele frequency ≥0.2. It is possible that some CL cases in our study could progress to ML disease at a later date. Epidemiological studies show that this will affect <4% of CL patients 15, thus representing a small reduction in the power of our study to detect CL-specific genetic effects. Full demographic and epidemiological information in relation to multi-case families used in this study have been presented elsewhere 16.

Genotyping was performed in Cambridge using Taqman or ABI fragment separation technologies for polymorphisms at CXCR1, CXCR2 and SLC11A1 as presented in Table 1. Genotype data are available on request for meta-analysis. All were in Hardy Weinberg Equilibrium in genetically unrelated founders of the families, and in the unrelated neighborhood controls (data not shown). Case-control data were analysed using logistic regression analysis. PEDCHECK 17 was used to determine Mendelian inconsistencies within families. Inconsistencies due to mis-paternities had already been removed as part of previous studies 13 (L. Castellucci, unpublished PhD thesis). Mendelian inconsistencies for individual markers in this study were due to errors (<2%) that occur in calling Taqman genotyping and were set to zero for analysis. Family-based allelic association tests based on the transmission disequilibrium test (TDT) but generalized to allow analysis under additive and dominant models of inheritance were performed within FBAT 1819 under the null hypothesis of "no linkage and no association". Unaffected members of the pedigrees were included in the study, contributing genotype information to increase statistical power of the FBAT analysis, especially for families with missing parents. Family-based haplotype TDT was performed using TRANSMIT 20. Nominal P-values are presented throughout, i.e. without correction for multiple testing.

<p>Table 1</p>

Information on the polymorphic markers genotyped for CXCR1, CXCR2 and SLC11A1.

Gene/Marker

Physical Position (bp)

Alleles1

MAF

Caucasian

Asian

African


CXCR2_rs4674259

(5' UTR)

218699250

T>C

0.388

0.517

0.341

0.883


CXCR1_rs2854386

3' region

218735747

C>G

0.218

0.058

0.100

0.425


CXCR1_rs2234671

Exon 1 S276T

218737353

C>G

0.194

0.058

0.100

0.317


CXCR1_rs3138060

Intron 1

218739745

G>C

0.142

0.058

0.102

0.129


SLC11A1_rs7573065

(-237 bp 5' UTR)

218954951

C>T

0.0903

-

-

04


SLC11A1_rs2276631

(2742; exon 3, Phe66Phe)

218957257

C>T

0.289

0.224

0.102

0.127


SLC11A1_rs3731865

(469+14G/C2; intron 4)

218958247

G>C

0.227

0.272

0.082

0.1204


SLC11A1_rs17221959

(8232; exon 8, Gly249Gly)

218960874

C>T

0.266

0.022

0.152

0.3104


SLC11A1_rs2279015

(1465-85G/A2; intron 13)

218967514

C>T

0.379

0.342

0.307

0.942


SLC11A1_rs17235409

(17032; exon 15, D543N)

218967976

G>A

0.0893

0.012

0.182

0.1204


SLC11A1_ rs17235416

(1729+55del42; 3' UTR TGTG IN/DEL)

218968058

IN/DEL

0.176

0.012

0.182

0.1884

Physical positions of markers are given according to Build 36.3 of the human genome. Where available, allele frequencies for the minor allele (MAF) in Brazil are shown for Caucasian, Asian and African populations (Hapmap CEU, JPT and YRI unless otherwise referenced). 1 Major>minor alleles for this Brazilian population; 221, bp positions of variants relative to an arbitrary site 76 bp upstream of the methionine start codon; 3Allele frequency too low to be taken forward in the association analyses; 431.

Results

The case-control groups had similar demographics, including age, duration of residence in the endemic area, housing and main occupation. The environmental exposures surveyed were also similar between the two family cohorts, except for house distance from the forest that was different between the CL (265.2 meters) and ML groups (144.1 meters) (P = 0.04, unpaired t test), but not between the ML group and the NC or DTH-positive groups, as fully described elsewhere 13.

Table 2 presents the results of the case-control logistic regression analysis. SNP rs2854386 at CXCR1 was associated with susceptibility to CL, but not to ML, when each of these patient groups was compared with either the NC, the DTH+, or the combined NC+DTH+ control groups. CL is associated (OR 2.38; 95% CI 1.19-3.40; global P = 0.006) with the common C allele. Analysis for susceptibility to L. braziliensis per se (i.e. CL+ML compared to NC+DTH+) did not improve the significance (Table 2) suggesting that, although CL disease usually precedes ML disease, there was something different about the ML patient group which meant that they did not contribute to this association. Similarly, significance observed at rs2854386 for CL disease under a dominant model in the FBAT analysis (Table 3) was not improved when the data were analysed for susceptibility to L. braziliensis per se. CL disease was associated with the common C allele. Interestingly, in this analysis, transmission disequilibrium of alleles from heterozygous parents to ML disease patients only (Table 3) showed disease associated with the opposite G allele at rs2854386, suggesting opposing influences on the role of this gene in CL versus ML disease. This was principally determined by disease associated with the dominant G allele in heterozygotes (FBAT Z score 2.221; P = 0.026) in the genotype analysis, with the homozygous recessive CC genotype being protective for ML (FBAT Z score = -1.999; P = 0.04). This was measurable in the family-based analysis but the genotype-wise test (not shown) was not valid in the case-control analysis due to the smaller sample size. Similar but weaker allele-wise associations were observed in the case-control analysis for SNP CXCR1_rs2234671 (Table 2), which is in strong (but not complete) linkage disequilibrium with CXCR1_rs2854386 in the family founders (Figure 1) and unrelated NC controls (data not shown) in our study.

<p>Figure 1</p>

Haploview analysis for D' and r2 pairwise measures of LD between CXCR2, CXCR1 and SLC11A1 SNPs and IN/DEL in family founders from the study population in Brazil

Haploview analysis for D' and r2 pairwise measures of LD between CXCR2, CXCR1 and SLC11A1 SNPs and IN/DEL in family founders from the study population in Brazil. D' values and confidence levels (LOD) are represented as black for D' = 1, LOD>2; shades of pink for high D', LOD<2; white for D'<1, LOD<2. r2 values are represented as black for r2 = 1, white for r2 = 0, with intermediate values for 0<r2 < 1 indicated by shades of grey. The numbers within the squares represent the D' or r2 scores for pairwise LD.

<p>Table 2</p>

Results of logistic regression analysis

Gene/Marker

Groups Compared

Allele

OR

95% CI

Global P value


CXCR2_rs4674259

CL vs DTH

C vs T

1.03

0.60-1.76

0.908


CL vs NC

C vs T

0.91

0.53-1.58

0.752


ML vs DTH

C vs T

1.92

1.05-3.51

0.028


ML vs NC

C vs T

1.71

0.93-3.14

0.076


CL vs DTH+NC

C vs T

0.97

0.60-1.56

0.909


ML vs DTH+NC

C vs T

1.82

1.07-3.07

0.023


CL+ML vs DTH+NC

C vs T

1.26

0.85-1.88

0.239


CXCR1_rs2854386

CL vs DTH

C vs G

2.31

1.07-4.96

0.027


CL vs NC

C vs G

2.48

1.21-5.10

0.009


ML vs DTH

C vs G

1.55

0.72-3.35

0.254


ML vs NC

C vs G

1.72

0.84-3.54

0.127


CL vs DTH+NC

C vs G

2.38

1.23-4.57

0.006


ML vs DTH+NC

C vs G

1.63

0.84-3.14

0.132


CL+ML vs DTH+NC

C vs G

2.01

1.19-3.40

0.007


CXCR1_rs2234671

CL vs DTH

C vs G

2.06

0.93-4.56

0.069


CL vs NC

C vs G

1.91

0.89-4.10

0.087


ML vs DTH

C vs G

1.73

0.74-4.01

0.190


ML vs NC

C vs G

1.62

0.72-3.63

0.226


CL vs DTH+NC

C vs G

1.96

0.98-3.89

0.044


ML vs DTH+NC

C vs G

1.66

0.79-3.46

0.160


CL+ML vs DTH+NC

C vs G

1.84

1.04-3.23

0.030


SLC11A1_rs 17235416

CL vs DTH

DEL vs IN

1.35

0.63-2.87

0.428


CL vs NC

DEL vs IN

0.87

0.42-1.79

0.714


ML vs DTH

DEL vs IN

1.10

0.53-2.30

0.781


ML vs NC

DEL vs IN

0.73

0.35-1.48

0.383


CL vs DTH+NC

DEL vs IN

1.07

0.57-1.99

0.820


ML vs DTH+NC

DEL vs IN

0.88

0.47-1.66

0.709


CL+ML vs DTH+NC

DEL vs IN

0.97

0.58-1.62

0.929

Under an additive model for case-control comparisons of CL, ML and L. braziliensis per se (CL+ML) disease groups with NC, DTH+ or NC+DTH+ control groups. Only the data for markers where significant associations (P = 0.05; bold) were observed in this or the family-based (Table 3) analysis are shown. OR = odds ratio; CI = 95% confidence interval.

<p>Table 3</p>

FBAT analysis of family data

Gene/Marker

Phenotype

Model

Allele/Genotype

Allele/Genotype Frequency

# Families

Obs T

Exp T

Z score

P value


CXCR1_rs2854386

CL

Dominant

C

0.83

12

35

29

2.002

0.045


ML

Dominant

G

0.17

20

17

12

1.999

0.046


ML

Genotype

CC

0.69

20

7

12

-1.999

0.046


ML

Genotype

CG1

0.28

22

18

12

2.221

0.026


CL+ML

Dominant

C

0.83

12

35

29

2.002

0.045


CXCR1_rs2234671

CL

Dominant

C

0.85

12

36

32

1.563

0.118


ML

Dominant

G

0.15

18

14

11

1.604

0.108


ML

Genotype

CG

0.30

19

14

9

1.964

0.049


CL+ML

Dominant

C

0.85

12

36

32

1.563

0.118


SLC11A1_rs 17235416

CL

Additive

IN

0.86

27

98

88

2.549

0.011


ML

Additive

IN

0.86

21

32

32

0.209

0.834


CL+ML

Additive

IN

0.86

28

105

95

2.333

0.020


CL

Dominant

DEL

0.14

24

30

12

-2.198

0.028


ML

Dominant

DEL

0.14

19

9

10

-0.426

0.670


CL+ML

Dominant

DEL

0.14

25

25

32

-2.179

0.029


CL

Genotype

IN/IN

0.75

24

35

27

2.198

0.028


CL

Genotype

IN/DEL2

0.23

27

28

33

-1.206

0.228


ML

Genotype

IN/IN

0.75

19

12

11

0.426

0.670


ML

Genotype

IN/DEL2

0.23

21

8

10

-1.021

0.307


CL+ML

Genotype

IN/IN

0.75

25

38

30

2.179

0.029


CL+ML

Genotype

IN/DEL2

0.23

28

29

34

-1.409

0.159

FBAT analysis for transmission of alleles from heterozygous parents to CL, ML and L. braziliensis per se (CL and ML) individuals in families. # families = number of families informative for the FBAT analysis. A positive Z score indicates association with disease; a negative Z score indicates the non-associated or protective allele or genotype. Obs T = observed transmissions; Exp T = expected transmissions. 1Insufficient informative families (# = 4) for the GG genotype (frequency 0.03) to contribute to the analysis; 2Insufficient informative families (# = 5,4,5) for the DEL/DEL genotype (frequency 0.02) to contribute to the analysis for CL, ML or CL+ML.

CXCR1_rs2234671 did not achieve significance for CL disease in the FBAT analysis (Table 3), but the presence of the G allele in the heterozygous genotype conferred disease association for ML (FBAT score 1.964; P = 0.049). Haplotype analysis (Table 4) across the 3 CXCR2/CXCR1 markers rs467259_rs2854386_rs2234671 confirmed that the common haplotype T_C_C (frequency 0.47) was significantly over-transmitted to individuals in the families with CL only (χ21df = 6.62; P = 0.01) and significantly under-transmitted to individuals in the families with ML (χ21df = 4.42; P = 0.04), consistent with the case-control analysis (Table 2) that showed ML associated with the rarer C allele at CXCR2_rs4674259 (OR = 1.82; 95% CI 1.07-3.07; global P = 0.023). Inclusion of the CXCR2 SNP rs4674259 was necessary to observe the haplotype associations, which were not explained by over- or under-transmission of the shorter C_C haplotype for rs2854386_rs2234671 (Table 4). The opposing effects of polymorphism at CXCR1/2 on CL and ML was clearly neutralized in analysis of haplotype transmission to the combined CL+ML group (Table 4).

<p>Table 4</p>

Haplotype analysis of family data

Disease Phenotype

Haplotypes

Haplotype Frequency

Over/Under Transmitted

rs4674259

rs2854386

rs2234671


CL

T_C

0.47

Over

O = 160; E = 145; χ21df = 5.88; P = 0.015


C_C

0.16

-

O = 62; E = 67; χ21df = 2.02; NS


T_C_C

0.47

Over

O = 152; E = 136; χ21df = 6.62; P = 0.010


ML

T_C

0.47

Under

O = 41; E = 49; χ21df = 5.19; P = 0.023


C_C

0.16

-

O = 17; E = 15; χ21df = 0.08; NS


T_C_C

0.47

Under

O = 39; E = 46; χ21df = 4.22; P = 0.040


CL or ML

T_C

0.47

-

O = 201; E = 194; χ21df = 0.96; NS


C_C

0.16

-

O = 80; E = 82; χ21df = 1.36; NS


T_C_C

0.47

-

O = 190; E = 181; χ21df = 1.59; NS

TRANSMIT haplotype association analysis for SNPs rs4674259_rs2854386_rs2234671 across CXCR2_CXCR1 showing over- and under-transmission for the common haplotypes in families analysed for the CL only phenotype, ML only phenotype, and CL or ML. O = observed number of transmissions; E = expected number of transmissions.

At SLC11A1, none of the polymorphisms showed association with either CL or ML disease in the case-control logistic regression analysis (Table 2). Association between the SLC11A1 1729+55del4 21 IN/DEL and CL (FBAT Z score +2.198 for the IN/IN homozygous genotype, P = 0.028) but not ML disease was observed in the family-based analysis (Table 3), again with the ML group not apparently contributing to the association. The disease allele is the common insertion allele, which is recessive, while the deletion is protective under the dominant model. Given the low level of linkage disequilibrium between the 1729+55del4 IN/DEL and rs2854386 (Figure 1), the associations observed between CL and SLC11A1 and between CL and CXCR1 are likely to be independent. There was insufficient power in the sample to determine whether these two loci had independent effects, or whether there was any interaction between them.

Discussion

The study presented here has provided interesting preliminary data which support roles for both CXCR1 and SLC11A1 in determining the outcome of L. braziliensis infection. Although the FBAT analysis supported the case-control analysis for CXCR1, the power of our study was limited by small sample size and MAF < 0.2 for the markers (rs2854386, rs2234671) of particular interest. In the context of the admixed population found in Brazil, the family-based analysis provides some confidence that the results are not due to mismatch between case and control groups. For that reason, we also have some confidence in the association between SLC11A1 observed in the more powerful family-based analysis for CL disease, even though this was not replicated in the case-control analysis. Further replication studies in larger cohorts will be essential to validate our results. Nevertheless, the data provide interesting insight into the possible roles of PMN and macrophages in leishmaniasis caused by L. braziliensis.

At present we do not know the functional basis to the association between CXCR1 rs2854386 and CL versus ML disease. This SNP lies in the 3' region of the CXCR1 gene, adjacent to rs4674259 in the 5' region of the CXCR2 gene. Opposing over- and under-transmitted associations with the common ancestral T_C_C haplotypes for CL and ML, but not the shorter C_C haplotypes, suggests location of the functional variant in the regulatory region between the genes that could affect expression of either of them. Although CXCR1 rs2854386 is in strong linkage disequilibrium with rs2234671, which encodes a mis-sense mutation that alters the amino acid sequence from Ser-to-Thr at position 276 of the protein, the haplotype analysis did not support over- and under-transmission of the C_C haplotype between these two markers as the reason for the opposing associations. Interestingly, the allele frequencies for the minor allele (MAF) for rs2854386 (0.218) and rs2234671 (0.194) observed in Brazil were high compared to those observed in Caucasian (CEU) (0.058 for both) and Asian (JPT) populations (0.100 for both), perhaps reflecting the contribution of African haplotypes (e.g. YRI MAFs 0.425 and 0.317, respectively) to the Brazilian population near Salvador, a city with a significant population of African origin. Failure to see complete concordance between the results at these two markers in our analysis could be due to differences in genotyping success between individuals, or to the presence of African haplotypes. Association of CL with the common ancestral T_C_C haplotype suggests that disease is associated with a fully functional variant, and hence that influx of PMN is associated with CL disease. This is consistent with data 6 and hypotheses 7 relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele, which we assume to be the functionally compromised allele/haplotype, suggests that PMN may have an important positive role to play in preventing this form of the disease. Recent studies have shown, for example, that PMN can play an important role in initiating and regulating innate immune defences that protect mucosal surfaces from fungal infection 22. Failure to protect the mucosa from early invasion and injury could result in local presentation of antigen and amplification of the acquired T cell-mediated proinflammatory response that is associated with ML disease.

The association of CL disease with the common 1729+55del4 IN variant at SLC11A1 is also of interest in relation to the putative role of this molecule, which is expressed in mature macrophages 23, dendritic cells 24 and PMN 25, in regulating expression of secretory leukocyte protease inhibitor and hence affecting the wound healing response 12. Differences in lesion development have not been observed following subcutaneous needle injection of either L. major 26 or L. mexicana 27 into Slc11a1 congenic mice, suggesting that the genetic influence of SLC11A1 on susceptibility to CL following natural infection in humans might be mediated by the effect on the wound healing response to the sand fly bite. This means that the mechanism by which SLC11A1 influences CL disease may be different to its influence on visceral leishmaniasis in mice following intravenous needle injection 28, or in natural infection of dogs 2930 and humans 3132, consistent with its many pleiotropic effects 11. Our study was not sufficiently powered to look for interaction between the CXCR1 and SLC11A1 in this study, and further work will be needed to determine whether the association at SLC11A1 relates to its role in PMN, macrophages or dendritic cells at the site of infection. For the present, our study begins to provide novel insight to the possible role of PMN in lesion development of leishmaniasis caused by L. braziliensis infection in Brazil.

Conclusions

The study supports roles for CXCR1 (IL8RA) and SLC11A1 in the outcome of L. braziliensis infection in humans. Previous data in mice showing that Slc11a1 does not influence cutaneous lesion development following needle injection of Leishmania suggests that its role here might relate to the action of PMN, macrophage and/or dendritic cells in the wound healing response to the sand fly bite. Together with the CXCR1 association, the data are consistent with hypotheses relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele at CXCR1 rs2854386 suggests that PMN also have an important positive role to play in preventing this form of the disease.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LC carried out the field collection and preparation of the samples, performed the genotyping, and participated in the statistical analysis and interpretation of the data. SEJ and ENM trained LC in the laboratory for genotyping techniques, in database entry and use of the genetic database GenIE in Cambridge, and in genetic statistical analysis methods. SEJ cross-checked statistical analyses and carried out additional statistical tests. EM, JO, AM and LHG participated in the field collection of data, processing of DNA samples and database entry in Brazil. ML is the doctor responsible for confirmation of the ML cases by performing ENT exams. ARJ trained the field group, initial selection of cases from the health post, assisted with field collection of data and participated in the design of the study. EMC helped conceive the study, initial selection of cases from the health post, and provided the logistical support to make the study possible. JMB participated in the design of the study, conceived the specific hypothesis to be tested, made the final interpretation of the data, and prepared the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We acknowledge the support of NIH Grant AI 30639 for the field work in Brazil, and The Wellcome Trust for supporting the laboratory work and statistical analyses carried out in the UK. LC was supported by NIH/FIC 1 D43 TW007127-01 for her period of stay in UK. JO and AM were also supported by NIH/FIC 1 D43 TW007127-01 in Brazil.

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