The −2518 bp promoter polymorphism at CCL2/MCP1 influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil

https://doi.org/10.1016/j.meegid.2010.04.006Get rights and content

Abstract

Mucosal leishmaniasis (ML) follows localized cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. Proinflammatory responses mediate CL self-healing but are exaggerated in ML. Proinflammatory monocyte chemoattractant protein 1 (MCP-1; encoded by CCL2) is associated with CL. We explore its role in CL/ML through analysis of the regulatory CCL2 −2518 bp promoter polymorphism in CL/ML population samples and families from Brazil. Genotype frequencies were compared among ML/CL cases and control groups using logistic regression and the family-based association test (FBAT). MCP-1 was measured in plasma and macrophages. The GG recessive genotype at CCL2 −2518 bp was more common in patients with ML (N = 67) than in neighborhood control (NC; N = 60) subjects (OR 1.78; 95% CI 1.01–3.14; P = 0.045), than in NC combined with leishmanin skin-test positive (N = 60) controls (OR 4.40; 95% CI 1.42–13.65; P = 0.010), and than in controls combined with CL (N = 60) patients (OR 2.78; 95% CI 1.13–6.85; P = 0.045). No associations were observed for CL compared to any groups. FBAT (91 ML and 223 CL cases in families) confirmed recessive association of ML with allele G (Z = 2.679; P = 0.007). Higher levels of MCP-1 occurred in plasma (P = 0.03) and macrophages (P < 0.0001) from GG compared to AA individuals. These results suggest that high MCP-1 increases risk of ML.

Introduction

Leishmania parasites cause a spectrum of disease phenotypes which differ according to clinical manifestations and immune response. Although distinct species of Leishmania can cause different forms of the disease, a single species can also be associated with two or more distinct clinical forms of leishmaniasis (Almeida et al., 1996, Barral et al., 1991, Carvalho et al., 1985). The immune response and genetic background of the host may be important in disease pathogenesis.

Following infection with Leishmania braziliensis, cutaneous leishmaniasis (CL) is the most common form disease involving the skin, characterized by one or more (<10) granular ulcers with elevated borders. Concomitantly, or months to years later, ∼3% of individuals affected by CL develop ML (Carvalho et al., 1985). This severe form is characterized by destructive lesions which may be incapacitating and disfiguring; occasionally becoming life-threatening when lesions of the pharynx and larynx obstruct the respiratory passages and/or cause difficulty in swallowing (Marsden et al., 1998). ML is characterized by a strong cell-mediated immune response with intense infiltrate, pronounced production of inflammatory cytokines such as interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) (Bacellar et al., 2002). There is also reduced response to the immunomodulatory cytokine interleukin(IL)-10 and lower expression of IL-10 receptor (Faria et al., 2005).

Chemokines are produced as one of the earliest responses against Leishmania parasites, providing a signal for the initiation of the immune response such as chemotaxis, cytokine production, and activation of different cellular subsets (Antoniazi et al., 2004). The chemokine, CC Motif, Ligand 2 (CCL2), also known as Monocyte Chemotactic Protein 1 (MCP-1), is produced by lymphocyte and monocyte lineages and plays a role in both cellular immune reactions and responses to acute tissue injury (Leonard and Yoshimura, 1990). Herein we refer to the gene as CCL2 and the protein as MCP-1. Several studies have reported putative roles for MCP-1 in leishmaniasis from infection studies in vitro (Bhattacharyya et al., 2002, Ritter and Moll, 2000) as well as by analysis of human (Ritter et al., 1996) and murine (de Moura et al., 2005) lesions, but it is unclear whether the proinflammatory response associated with this chemokine is potentially disease exacerbatory or protective. As for TNF-α, the balance between enough proinflammatory activity to promote parasite killing, but not to cause tissue damage, is a central feature of CL vs. ML disease, whilst induction of anti-inflammatory cytokines like IL-10 plays an important modulating role.

One way to explore the role of chemokines and other pro- and anti-inflammatory molecules in humans is to determine whether polymorphisms that affect expression or function are associated with disease outcome. In Venezuela, for example, polymorphisms at both TNF and LTA, the genes encoding TNF-α and lymphotoxin-α respectively, have been associated with increased susceptibility to ML caused by L. braziliensis infection (Cabrera et al., 1995). In Brazil, the −174 bp G/C single nucleotide polymorphism (SNP) in the promoter region of the gene (IL6) encoding IL-6 was also shown to be associated with ML (Castellucci et al., 2006), while the IL10 −819 bp C/T promoter region SNP is associated with susceptibility to CL (Salhi et al., 2008). Recently, Flores-Villanueva et al. (2005) demonstrated an association between the rarer G allele at the CCL2 −2518 bp A/G (rs1024611) promoter SNP and susceptibility to developing active pulmonary tuberculosis in populations from Mexico and Korea. Tuberculosis patients carrying the G allele had the highest plasma levels of MCP-1 and the lowest plasma levels of IL12p40. This led the authors to conclude that individuals with the CCL2 −2518GG genotype produce high concentrations of MCP-1, which inhibits IL-12p40 production in response to Mycobacterium tuberculosis and increases the likelihood of tuberculosis infection progressing to active disease by preventing T helper 1 immunity. Since the IL-12p40 chain is shared with IL-23, this cytokine might also be affected. On the other hand, Ramasawmy et al. (2006) showed an association between the common A allele, which is known to be associated with low transcriptional activity (Rovin et al., 1999), and chronic Chagas cardiomyopathy in Brazilians, suggesting that MCP-1 was required to prevent disease. Here we report on a small case–control study, underpinned by family-based analysis, which provides evidence for association between the CCL2 −2518 bp G allele with high plasma and macrophage MCP-1 levels, and susceptibility to ML but not CL disease caused by L. braziliensis.

Section snippets

Case patients, control subjects and study design

As previously (Castellucci et al., 2006), the study was conducted in the area of Corte de Pedra, Bahia, Brazil where L. braziliensis is endemic (Cuba-Cuba et al., 1984, Rosa et al., 1988). All participants were enrolled between January 2001 and November 2004. The majority of cases were retrospective cases that had not had parasites isolated. For all prospective cases of CL and ML studied in this endemic area over the period 2001–2007, L. braziliensis has been identified as the etiological agent

Population-based analysis of MCP-1 −2518 A/G bp polymorphism

There was no evidence of deviation from Hardy–Weinberg equilibrium using unrelated individuals in these families. Table 1 presents the frequency distribution for genotypes and alleles in the different population-based patient and control groups. To determine initially whether the CCL2 −2518 bp SNP was associated with susceptibility to leishmaniasis per se, we compared (Table 2) the ML and CL groups against NC and DTH+ groups. This comparison was significant in the global genotype-wise but not

Discussion

The data presented here provide both population-based and family-based evidence for an association between the G allele of the CCL2 −2518 bp promoter SNP and susceptibility to ML disease following infection with L. braziliensis. This correlated with elevated plasma levels of MCP-1 in GG individuals, and with higher release of MCP-1 by both un-stimulated and stimulated macrophages. Previous work has demonstrated that MCP-1 enhances the cytotoxic response against L. donovani amastigotes via a

Acknowledgements

We thank Ednaldo Lima do Lago and other staff at the Health Post of Corte de Pedra for helping with patient recruitment. We thank Paulo Machado and Albert Schriefer for help in diagnosis of patients. We thank Elbe M. Silva and Lúcia Reis for secretarial assistance. This work was funded in part by NIH grants P50 AI-30639 and R03AI070909 (LC, EMC, MEW), NIH/FIC 1 D43 TW007127-01 (JO, AM), R01 AI076233 (JMB and MEW), R01AI067874 (JMB and MEW), CNPq (ARJ, RA, EMC), a VA Merit Review grant (MEW),

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