Putative pathogen-selected polymorphisms in the PKLR gene are associated with mycobacterial susceptibility in Brazilian and African populations.

Pyruvate kinase (PK), encoded by the PKLR gene, is a key player in glycolysis controlling the integrity of erythrocytes. Due to Plasmodium selection, mutations for PK deficiency, which leads to hemolytic anemia, are associated with resistance to malaria in sub-Saharan Africa and with susceptibility to intracellular pathogens in experimental models. In this case-control study, we enrolled 4,555 individuals and investigated whether PKLR single nucleotide polymorphisms (SNPs) putatively selected for malaria resistance are associated with susceptibility to leprosy across Brazil (Manaus-North; Salvador-Northeast; Rondonópolis-Midwest and Rio de Janeiro-Southeast) and with tuberculosis in Mozambique. Haplotype T/G/G (rs1052176/rs4971072/rs11264359) was associated with leprosy susceptibility in Rio de Janeiro (OR = 2.46, p = 0.00001) and Salvador (OR = 1.57, p = 0.04), and with tuberculosis in Mozambique (OR = 1.52, p = 0.07). This haplotype downregulates PKLR expression in nerve and skin, accordingly to GTEx, and might subtly modulate ferritin and haptoglobin levels in serum. Furthermore, we observed genetic signatures of positive selection in the HCN3 gene (xpEHH>2 -recent selection) in Europe but not in Africa, involving 6 SNPs which are PKLR/HCN3 eQTLs. However, this evidence was not corroborated by the other tests (FST, Tajima's D and iHS). Altogether, we provide evidence that a common PKLR locus in Africans contribute to mycobacterial susceptibility in African descent populations and also highlight, for first, PKLR as a susceptibility gene for leprosy and TB.

Evaluation of monocyte subsets and markers of activation in leprosy reactions.

Understanding host immune pathways associated with tissue damage during reactions are of upmost importance to the development of immune intervention strategies. The participation of monocytes in leprosy reactions was evaluated by determining the frequency of monocyte subsets and the degree of cellular activation through the expression of MHCII and the co-stimulatory molecules CD40, CD80, CD86. Leprosy subjects with or without reactions were included in this cross-sectional study. Peripheral blood mononuclear cell were isolated and stained ex vivo to determine monocyte subsets and the degree of cellular activation by flow cytometry. Intermediate monocytes were increased in leprosy patients with reactions when compared to patients without reactions. Although no difference was detected in the frequency of monocyte subsets between type 1 and 2 reactions, the expression of CD80 was increased in monocytes from patients with type 1 reactions and CD40 was higher in paucibacillary subjects presenting type 1 reactions. Moreover, CD86 and MHC II expression were higher in intermediate monocytes when compared to the other subsets in leprosy reaction types 1 and 2. Intermediate monocyte activation with CD86 and MHCII expression is involved with both type 1 and 2 reactions, whereas CD80 and CD40 expression is related to type 1 reactions.

Whole blood profiling of leprosy type 1(reversal) reactions highlights prominence of innate immune response genes.

BACKGROUND: The major factors contributing for nerve damage and permanent disabilities in leprosy are type 1 or reversal reactions (RR) and type 2 or erythema nodosum leprosum (ENL). Gene profiling of leprosy reactions have shown that different pathways are activated during the course of reactions, which is consistent with the exacerbated immune response exhibited by these patients. METHODS: We used qPCR to screen a panel of 90 genes related to the immune response in leprosy in RNA-derived peripheral leukocytes of patients with (N = 94) and without leprosy reactions (N = 57) in order to define expression signatures correlated to RR or ENL. RESULTS: Our results show that there is a marked signature for RR in the blood, comprising genes mostly related to the innate immune responses, including type I IFN components, autophagy, parkins and Toll like receptors. On the other hand, only Parkin was differentially expressed in the ENL group. CONCLUSIONS: The data put together corroborates previous work that brings evidence that an acute uncontrolled exacerbated immune response designed to contain the spread of M. leprae antigens might be cause of RR pathogenesis. Identifying a blood profile useful to predict leprosy reactions prior to its development might help to reduce the morbidity associated to this disabling disease.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy

BACKGROUND Leprosy or hansen’s disease is a spectral disease whose clinical forms mostly depends on host’s immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. OBJECTIVES To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. METHODS Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. FINDINGS Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. MAIN CONCLUSIONS All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host’s production of key cytokines and chemokines involved in the pathogenesis of this disease.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy.

BACKGROUND: Leprosy or hansen's disease is a spectral disease whose clinical forms mostly depends on host's immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. OBJECTIVES: To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. METHODS: Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. FINDINGS: Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1ß was related to TLR4 genotypes. MAIN CONCLUSIONS: All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host's production of key cytokines and chemokines involved in the pathogenesis of this disease.

The -308 bp TNF gene polymorphism influences tumor necrosis factor expression in leprosy patients in Bahia State, Brazil.

Leprosy or Hansen's disease is a debilitating chronic granulomatous disease caused by Mycobacterium leprae, with high incidence and prevalence in Brazil. The -308 bp G/A single nucleotide polymorphism (SNP rs1800629) in the tumor necrosis factor (TNF) gene promoter is a proposed risk factor for leprosy. In Brazil, Northern India, Egypt and Nepal, the common G allele was associated with leprosy. In Eastern India, Thailand and Malawi the minor A allele was the risk factor. Allele A was previously associated with high TNF. We genotyped rs1800629 in 326 leprosy cases from Bahia State, Brazil, including 72 paucibacillary (PB) and 47 multibacillary (MB) without reactions, and 69 reversal reaction (RR) and 78 erythema nodosum leprosum (ENL) with reactions. Logistic regression was used to compare patient groups with 331 healthy controls. Relative TNF mRNA was determined in peripheral blood leukocytes by QRTPCR, and serum TNF levels measured by ELISA. We found that TNF mRNA expression was higher (P=0.03) in leprosy patients compared to endemic controls, but did not differ significantly between clinical subgroups. Carriage of the minor A allele was associated (P=0.003) with low TNF mRNA across leprosy patients. Nevertheless, we found no evidence for either allele at this SNP as a risk factor for leprosy per se (OR=1.12, 95% CI 0.79-1.60, P=0.52), PB (OR=0.99, 95% CI 0.54-1.81, P=0.97), MB (OR=0.86, 95% CI 0.40-1.83, P=0.70), RR (OR=1.37, 95% CI 0.79-2.38, P=0.27) or ENL (OR=0.76, 95% CI 0.40-1.45, P=0.42) when compared to endemic controls. Further studies are required to determine whether the influence of the minor A allele on TNF mRNA levels determines response to treatment, particularly in the context of ENL reaction treatment with anti-TNF therapies and RR reactions where treatment with prednisolone is known to reduce TNF levels. Our findings contribute to understanding TNF as an important determinant of leprosy immunopathology in Brazil.

Viral Co-infection and Leprosy Outcomes: A Cohort Study.

BACKGROUND: The role of the host immunity in determining leprosy clinical forms and complications is well recognized, implying that changes in the immune status may interfere with several aspects of the disease. Therefore, we hypothesized that the presence of viral co-infections and associated immunological changes will have a clinical impact on leprosy outcomes. The aim of our study was to determine the clinical impact of human immunodeficiency virus (HIV), human T cell lymphotrophic virus type 1 (HTLV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection on the development of reactions, neuritis, neuropathy and relapses. METHODOLOGY/PRINCIPAL FINDINGS: Cohort study in 245 leprosy subjects from Bahia, Brazil. Patients were followed from the time of diagnosis until at least the end of multidrug therapy. Viral co-infection was detected in 36 out of the 245 patients (14.7%). Specific co-infection rates were 10.6% for HBV, 2.9% for HIV, 2.5% for HTLV-1 and 0.8% for HCV. All four groups of co-infected patients had higher rates of neuritis and nerve function impairment compared to non co-infected leprosy subjects. The relapse rate was also higher in the co-infected group (8.3%) versus patients without co-infection (1.9%); relative risk 4.37, 95% confidence interval 1.02-18.74. CONCLUSIONS/SIGNIFICANCE: Leprosy patients should be screened for HBV, HCV, HIV and HTLV-1 co-infections. Besides contributing to better health care, this measure will facilitate the early detection of severe complications through targeting of higher risk patients.

The role of ERBB2 gene polymorphisms in leprosy susceptibility.

Mycobacterium leprae infects skin and peripheral nerves causing deformities and disability. The M. leprae bacterium binds to ErbB2 on the Schwann cell surface causing demyelination and favoring spread of the bacilli and causing nerve injury. Polymorphisms at the ERBB2 gene were previously investigated as genetic risk factors for leprosy in two Brazilian populations but with inconsistent results. Herein we extend the analysis of ERBB2 variants to a third geographically distinct population in Brazil. Our results show that there is no association between the genotyped SNPs and the disease (p>0.05) in this population. A gene set or pathway analysis under the genomic region of ERBB2 will be necessary to clarify its regulation under M. leprae stimulus.