Avaliação de polimorfismos nos receptores do tipo toll na resposta ao tratamento com talidomida e prednisona em pacientes com eritema nodoso hansênico

O eritema nodoso hansênico (ENH) é uma complicação inflamatória dolorosa causada por uma resposta imune humoral ao Mycobacterium leprae. Acomete especialmente pacientes classificados como multibacilares, com a presença de nódulos eritematosos subcutâneos que podem ulcerar. O tratamento do ENH tem como objetivo principal o controle da inflamação aguda e da neurite, aliviar a dor e o desconforto e, prevenir o desenvolvimento e extensão de lesões. No Brasil, os medicamentos mais utilizados são talidomida e prednisona. Receptores do tipo toll (TLRs) são receptores de membrana que atuam no reconhecimento de microrganismos para ativar eventos de sinalização de resposta imune a jusante. Após o contato com seus ligantes, vias de sinalização intracelular são ativadas, levando a ativação e transcrição de moléculas próinflamatórias. Alguns desses receptores foram identificados como capazes de reconhecer o M. leprae e, desencadear ampla resposta antimicrobiana e inflamatória. Polimorfismos em genes que codificam as proteínas TLRs, podem ser determinantes no curso da hanseníase e aparecimento de ENH. Assim, o objetivo desse trabalho foi avaliar a influência de variantes genéticas em genes TLR na resposta ao tratamento do ENH com talidomida e prednisona. 130 pacientes com ENH tratados com talidomida e/ ou prednisona foram selecionados em Porto Alegre (RS), Fortaleza (CE), Imperatriz (MA), São Luis (MA) e Monte Negro (RO). Foram analisadas até seis consultas previamente registradas no prontuário dos pacientes com a coleta de dados demográficos, histórico de hanseníase e ENH. O DNA dos pacientes foi extraído de amostras de saliva e sangue periférico. Os polimorfismos rs4833095/TLR1, rs3804099/TLR2, rs1927914/TLR4 e rs5743810/TLR6 foram genotipados usando a técnica de PCR em tempo real. O teste de qui-quadrado foi utilizado para avaliar o equilíbrio de Hardy-Weinberg para todos os polimorfismos. O método das equações de estimativa generalizada (GEE) foi utilizado para avaliar a influência dos polimorfismos na variação da dose de talidomida e prednisona. Todas as análises estatísticas foram realizadas com o SPSS versão 20. Descobrimos que a dose de talidomida nos genótipos CT e TT de TLR1/rs4833095 variou em torno de 48mg entre as regiões Norte e Sul (p=0.001). Para TLR2/rs3804099, a variação também ocorreu de acordo com a região do paciente. Em todos os genótipos desse SNP (CC, CT e TT), os pacientes da região Sul receberam uma média de 45,3mg a menos de talidomida em comparação à região Norte (p=0.001). Na prednisona, o efeito dos genótipos de CT e CC na variação da dose dependiam do tempo (p=0.018). Identificamos que o efeito do genótipo na variação da dose de talidomida nos genótipos AA e AG de TLR6/rs5743810 também depende do tempo. Durante o tratamento, pudemos observar uma redução de dose apenas para indivíduos com AG e GG. A análise da associação entre as variantes genéticas e a manifestação de efeitos adversos mostrou associação entre o genótipo TT de TLR1/rs4833095 e edema (p=0.019). Além disso, os genótipos TT de TLR2/rs3804099 (p=0.016) foram associados a efeitos dermatológicos adversos, prurido, pele seca e perda de cabelo. Ainda existem muitas lacunas sobre o mecanismo de interação entre os diferentes TLRs e M. leprae e, sobre como eles podem interferir no curso e no tratamento da doença. No entanto, este estudo mostra que a avaliação dos TLRs pode auxiliar no entendimento da resposta ao tratamento do ENH com talidomida e prednisona.Erythema nodosum leprosum (ENL) is a painful inflammatory complication caused by a humoral immune response to Mycobacterium leprae. It especially affects patients classified as multibacillary, with the presence of subcutaneous erythematous nodules that may ulcerate. The treatment of ENL has the main objective of controlling acute inflammation and neuritis, relieving pain and discomfort and preventing the development and extension of injuries. In Brazil, the most used drugs are thalidomide and prednisone. Toll-type receptors (TLRs) are membrane receptors that act in the recognition of microorganisms to activate signaling events of the downstream immune response. After contact with their ligands, intracellular signaling pathways are activated, leading to the activation and transcription of pro-inflammatory molecules. Some of these receptors have been identified as capable of recognizing M. leprae and, triggering a wide antimicrobial and inflammatory response. Polymorphisms in genes that encode TLR proteins may be determinants in the course of leprosy and the appearance of ENL. Thus, the objective of this work was to evaluate the influence of genetic variants on TLR genes in the response to treatment of ENL with thalidomide and prednisone. 130 patients with ENL treated with thalidomide and/or prednisone were selected in Porto Alegre (RS), Fortaleza (CE), Imperatriz (MA), São Luis (MA) and Monte Negro (RO). Up to six visits previously recorded in the patients' medical records were analyzed with the collection of demographic data, leprosy history and ENL. The patients' DNA was extracted from saliva and peripheral blood samples. The rs4833095/TLR1, rs3804099/TLR2, rs1927914/TLR4 and rs5743810/TLR6 polymorphisms were genotyped using the real-time PCR technique. The chi-square test was used to assess the Hardy-Weinberg balance for all polymorphisms. The method of generalized estimation equations (GEE) was used to evaluate the influence of polymorphisms on the variation of the dose of thalidomide and prednisone. All statistical analyzes were performed using SPSS version 20. We found that the dose of thalidomide in the CT and TT genotypes of TLR1/rs4833095 varied around 48mg between the North and South regions (p=0.001). For TLR2/ rs3804099, the variation also occurred according to the patient's region. In all genotypes of this SNP (CC, CT and TT), patients in the South region received an average of 45.3mg less thalidomide compared to the North region (p=0.001). In prednisone, the effect of CT and CC genotypes on dose variation depended on time (p=0.018). We identified that the effect of the genotype on the variation of the thalidomide dose in the AA and AG genotypes of TLR6/rs5743810 also depends on time. During treatment, we were able to observe a dose reduction only for individuals with AG and GG. Analysis of the association between genetic variants and the manifestation of adverse effects showed an association between the TT genotype of TLR1 rs4833095 and edema (p=0.019). There are still many gaps about the interaction mechanism between the different TLRs and M. leprae and about how they can interfere with the course and treatment of the disease. However, this study shows that the evaluation of TLRs can help in understanding the response to the treatment of ENL with thalidomide and prednisone

Role of gut microbiota in infectious and inflammatory diseases

Thousands of microorganisms compose the human gut microbiota, fighting pathogens in infectious diseases and inhibiting or inducing inflammation in different immunological contexts. The gut microbiome is a dynamic and complex ecosystem that helps in the proliferation, growth, and differentiation of epithelial and immune cells to maintain intestinal homeostasis. Disorders that cause alteration of this microbiota lead to an imbalance in the host’s immune regulation. Growing evidence supports that the gut microbial community is associated with the development and progression of different infectious and inflammatory diseases. Therefore, understanding the interaction between intestinal microbiota and the modulation of the host’s immune system is fundamental to understanding the mechanisms involved in different pathologies, as well as for the search of new treatments. Here we review the main gut bacteria capable of impacting the immune response in different pathologies and we discuss the mechanisms by which this interaction between the immune system and the microbiota can alter disease outcomes

Role of gut microbiota in infectious and inflammatory diseases

Thousands of microorganisms compose the human gut microbiota, fighting pathogens in infectious diseases and inhibiting or inducing inflammation in different immunological contexts. The gut microbiome is a dynamic and complex ecosystem that helps in the proliferation, growth, and differentiation of epithelial and immune cells to maintain intestinal homeostasis. Disorders that cause alteration of this microbiota lead to an imbalance in the host’s immune regulation. Growing evidence supports that the gut microbial community is associated with the development and progression of different infectious and inflammatory diseases. Therefore, understanding the interaction between intestinal microbiota and the modulation of the host’s immune system is fundamental to understanding the mechanisms involved in different pathologies, as well as for the search of new treatments. Here we review the main gut bacteria capable of impacting the immune response in different pathologies and we discuss the mechanisms by which this interaction between the immune system and the microbiota can alter disease outcomes

Evaluation of polymorphisms in toll-like receptor genes as biomarkers of the response to treatment of Erythema nodosum leprosum

Erythema nodosum leprosum (ENL) is an inflammatory complication caused by a dysregulated immune response to Mycobacterium leprae. Some Toll-like receptors (TLRs) have been identified as capable of recognizing antigens from M. leprae, triggering a wide antimicrobial and inflammatory response. Genetic polymorphisms in these receptors could influence in the appearance of ENL as well as in its treatment. Thus, the objective of this work was to evaluate the association of genetic variants of TLRs genes with the response to treatment of ENL with thalidomide and prednisone. A total of 162 ENL patients were recruited from different regions of Brazil and clinical information was collected from their medical records. Genomic DNA was isolated from blood and saliva samples and genetic variants in TLR1 (rs4833095), TLR2 (rs3804099), TLR4 (rs1927914), and TLR6 (rs5743810) genes were genotyped by TaqMan real-time PCR system. In order to evaluate the variants’ association with the dose of the medications used during the treatment, we applied the Generalized Estimating Equations (GEE) analysis. In the present sample, 123 (75.9%) patients were men and 86 (53.1%) were in treatment for leprosy during the ENL episode. We found an association between polymorphisms in TLR1/rs4833095, TLR2/rs3804099, TLR4/rs1927914, and TLR6/rs5783810 with the dose variation of thalidomide in a time-dependent manner, i.e.,the association with the genetic variant and the dose of the drug was different depending on the moment of the treatment evaluated. In addition, we identified that the association of polymorphisms in TLR1/rs4833095, TLR2/rs3804099, and TLR6/rs5783810 with the dose variation of prednisone also were time-dependent. Despite these associations, in all the interactions found, the influence of genetic variants on dose variation was not clinically relevant for therapeutic changes. The results obtained in this study show that TLRs polymorphism might play a role in the response to ENL treatment, however, in this context, they could not be considered as useful biomarkers in the clinical setting due small differences in medication doses. A larger sample size with patients with a more genetic profile is fundamental in order to estimate the association of genetic variants with the treatment of ENL and their clinical significance