Cynthia Chester Cardoso

Leprosy susceptibility: genetic variations regulate innate and adaptive immunity, and disease outcome.

The past few years have been very productive concerning the identification of genes associated with leprosy. Candidate gene strategies using both case-control and family-based designs, as well as large-scale approaches such as linkage and gene-expression genomic scans and, more recently, genome-wide association studies, have refined and enriched the list of genes highlighting the most important innate and adaptive immune pathways associated with leprosy susceptibility or resistance. During the early events of host-pathogen interaction identified genes are involved in pattern recognition receptors, and mycobacterial uptake (TLRs, NOD2 and MRC1), which modulate autophagy. Another gene, LTA4H, which regulates the levels of lipoxin A4 and possibly interacts with lipid droplet-related events, also plays a role in the early immune responses to Mycobacterium leprae. Together, the activation of these pathways regulates cellular metabolism upon infection, activating cytokine production through NF-κB and vitamin D-vitamin D receptor pathways, while PARK2 and LRRK2 participate in the regulation of host-cell apoptosis. Concomitantly, genes triggered to form and maintain granulomas (TNF, LTA and IFNG) and genes involved in activating and differentiating T-helper cells (HLA, IL10, as well as the TNF/LTA axis and the IFNG/IL12 axis) bridge immunological regulation towards adaptive immunity. Subtle variations in these genes, mostly single nucleotide polymorphisms, alter the risk of developing the disease or the severity of leprosy. Knowing these genes and their role will ultimately lead to better strategies for leprosy prevention, treatment and early diagnosis. Finally, the same genes associated with leprosy were also associated with autoimmune (Crohns disease, rheumathoid arthritis, psoriasis) or neurodegenerative diseases (Parkinsons and Alzheimers). Thus, information retrieved using leprosy as a model could be valuable to understanding the pathogenesis of other complex diseases.

Genetic, epidemiological and biological analysis of interleukin-10 promoter single-nucleotide polymorphisms suggests a definitive role for 819C/T in leprosy susceptibility

Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-α (LTA) and vitamin-D receptor (VDR). Here, we combined a case–control study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the −819T allele is associated with leprosy susceptibility either in the case–control or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the −819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that −819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy.

TNF -308G>A Single Nucleotide Polymorphism Is Associated With Leprosy Among Brazilians: A Genetic Epidemiology Assessment, Meta-Analysis, and Functional Study

Leprosy is an infectious disease caused by Mycobacterium leprae. Tumor necrosis factor (TNF) plays a key role in the host response. Some association studies have implicated the single nucleotide polymorphism TNF -308G>A in leprosy susceptibility, but these results are still controversial. We first conducted 4 association studies (2639 individuals) that showed a protective effect of the -308A allele (odds ratio [OR] = 0.77; P = .005). Next, results of a meta-analysis reinforced this association after inclusion of our new data (OR = 0.74; P = .04). Furthermore, a subgroup analysis including only Brazilian studies suggested that the association is specific to this population (OR = 0.63; P = .005). Finally, functional analyses using whole blood cultures showed that patients carrying the -308A allele produced higher TNF levels after lipopolysaccharide (LPS) (6 hours) and M. leprae (3 hours) stimulation. These results reinforce the association between TNF and leprosy and suggest the -308A allele as a marker of disease resistance, especially among Brazilians.

Toll-like Receptor 1 N248S Single-Nucleotide Polymorphism Is Associated With Leprosy Risk and Regulates Immune Activation During Mycobacterial Infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Ninjurin 1 asp110ala single nucleotide polymorphism is associated with protection in leprosy nerve damage

Leprosy is the major cause of non-traumatic neuropathy. Herein, we investigated the role of ninjurin 1, an adhesion molecule involved in nerve regeneration in leprosy. Our results demonstrated that M. leprae stimulates in vitro up-regulation of ninjurin mRNA in cultured Schwann and blood cells as well as in vivo mRNA and protein expression in leprosy nerve biopsies. A polymorphism (asp110ala) was investigated in a case-control study (1123 individuals) and no association was found with leprosy per se or with disseminated forms. Nevertheless, ala110 was associated with functional nerve impairment (OR=2.42; p=0.02 for ala/ala) and with lower mRNA levels. Our data suggests that asp110ala could be a valuable genetic marker of nerve damage in leprosy.

Immune Activation in Amniotic Fluid from Zika Virus-Associated Microcephaly.

Recent advances in the understanding of neuropathogenesis associated with Zika virus (ZIKV) infection has led to descriptions of neonatal microcephaly cases. However, none of these reports have evaluated the humoral response during ZIKV infection. We report here polyfunctional immune activation associated with increased interferon‐gamma‐inducible protein 10, interleukin (IL)‐6, IL‐8, vascular endothelial growth factor (VEGF), monocyte chemoattractive protein 1 (MCP‐1), and granulocyte colony‐stimulating factor (G‐CSF) levels in the amniotic fluid of ZIKV‐positive pregnant women with neonatal microcephaly. These cytokines have been associated not only with neuronal damage, but also with differentiation and proliferation of neural progenitor cells. Our results suggested that the immune activation caused by ZIKV infection in the uterine environment could also interfere with fetal development. ANN NEUROL 2017;81:152–156

Candidate gene case-control and functional study shows macrophage inhibitory factor (MIF) polymorphism is associated with cutaneous leishmaniasis.

American tegumentary leishmaniasis (ATL) is an infectious disease caused mostly by Leishmania(Viannia)braziliensis in Southeast Brazil. The clinical manifestations are vast, ranging from asymptomatic to severe mucosal leishmaniasis (ML). It has been suggested that variation of the pathogen does not fully explain the response spectrum and the variability of clinical manifestations. Previous data have shown that host genetics also play a role in disease outcome. Herein, we have tested the association of TNF, IL10, IL12 and MIF single nucleotide polymorphisms (SNPs) using a case-control study design including 110 cutaneous leishmaniasis (CL) patients and 682 healthy subjects. The genotype-phenotype correlation was also assessed using leishmania antigens to stimulate peripheral blood mononuclear cells obtained from cured CL patients. Results demonstrated that the MIF -173C allele is associated with leishmaniasis outcome and also with lower levels of MIF in culture supernatants. Also, the TNF -308AA genotype was statistically increased among leishmaniasis patients. The results showed here suggest that the lower levels of MIF produced by MIF -173C carriers could influence the host-Leishmania interaction, favoring infection and disease progression. On the other hand, high TNF levels can contribute to tissue damage, consequently leading to skin lesions.

Pre-miR-146a (rs2910164 G>C) Single Nucleotide Polymorphism Is Genetically and Functionally Associated with Leprosy

Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G>T), miRSNP-223 (rs34952329 *>T), miRSNP-196a-2 (rs11614913 C>T) and miRSNP-146a (rs2910164 G>C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100∶1) was able to induce miR-146a expression in THP-1 (p<0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LTA) single nucleotide polymorphisms: Importance in ARDS in septic pediatric critically ill patients

Accumulating evidence indicates that genetic background influences the outcome of sepsis, which despite medical advances continues to be a major cause of morbidity and mortality. This study aimed to evaluate the influence of SNPs LTA +252A>G, TNF-863C>A and TNF-308G>A on susceptibility to sepsis, acute respiratory distress syndrome (ARDS), septic shock and sepsis mortality. A prospective case-control study was carried out in a Brazilian pediatric intensive care unit and included 490 septic pediatric patients submitted to mechanical ventilation and 610 healthy children. No SNP association was found with respect to sepsis susceptibility. Nevertheless, a haplotype was identified that was protective against sepsis (+252A/-863A/-308G; OR=0.65; p=0.03). We further observed protection against ARDS in TNF-308 GA genotype carriers (OR=0.29; p=0.0006) and -308A allele carriers (OR=0.40; p=0.003). In addition, increased risk for ARDS was detectable with the TNF-863 CA genotype (OR=1.83; p=0.01) and the -863A carrier status (OR=1.82; p=0.01). After stratification according to age, this outcome remained significantly associated with the -308GA genotype in infants. Finally, protection against sepsis-associated mortality was found for the TNF-308 GA genotype (OR=0.22; p=0.04). Overall, our findings document a protective effect of the TNF-308 GA genotype for the ARDS and sepsis mortality outcomes, further providing evidence for an increased risk of ARDS associated with the TNF-863 CA genotype. Trial registration (www.clinicaltrials.gov): NCT00792883.

Association of IL10 Polymorphisms and Leprosy: A Meta-Analysis

Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal IL10 polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11–1.34) and P-value = 3x10–5 confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups (OR = 1.2, CI = 1.10–1.31, P-value = 2x10–5). Also, meta-analysis confirmed association of -592 A allele and leprosy outcome (OR = 1.24, CI = 1.03–1.50, P-value = 0.02). In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r2 = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other IL10 polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.