Pênfigo Foliáceo Endêmico : avaliação celular e humoral de epítofos imunodominantes da proteina desmogleína 1 e sua relação com os genótipos HLA-DRB1 associados a doença

Abstract

O pênfigo foliáceo (PF) é uma doença autoimune órgão-específica, caracterizada pela presença de autoanticorpos IgG, predominantemente da subclasse IgG4, contra ectodomínios da proteína desmogleína 1 (DSG1), levando ao processo de acantólise. A doença é desencadeada pela exposição a fatores ambientais, que nas áreas endêmicas possivelmente são insetos hematófagos, especialmente Simulium nigrinanum. Há uma maior susceptibilidade ao PFE nos indivíduos que apresentam os alelos HLA-DRB1 pertencente aos grupos *01 e *04 e menor em indivíduos cujos genótipos incluem alelos dos grupos *07, *08 e *11, bem como o alelo *0301. O mimetismo antigênico é o possível mecanismo desencadeador da doença, iniciada por estímulos ambientais, levando a geração de autoanticorpos não patogênicos contra o quinto ectodomínio extracelular da DSG1 (EC5). Em indivíduos geneticamente predispostos ocorre a geração de autoanticorpos patogênicos contra o primeiro (EC1) e segundo (EC2) ectodomínios da DSG1, através do espalhamento intramolecular de epítopos. O presente trabalho teve por objetivo realizar um mapeamento de epítopos imunodominantes da proteína desmogleína 1 e correlacionar o perfil das respostas celular e humoral da proteína DSG1 e peptídeos sintéticos em pacientes e indivíduos sadios com os genótipos HLA-DRB1 associados à susceptibilidade ou resistência ao PF. Estudos anteriores mostraram que linhagens celulares e clones de LT CD4+ derivados do sangue periférico de pacientes com pênfigo foliáceo possuem resposta proliferativa aos ectodomínios da proteína recombinante da desmogleína 1. Essa resposta é restrita à apresentação de peptídeos por moléculas HLA-DR e promove a secreção de citocinas do tipo Th2 que estimulam a produção de autoanticorpos por LB autorreativos. Porém, até o presente momento nenhum peptídeo da DSG1 reconhecido por essas células T havia sido identificado. Através desse estudo identificamos epítopos imunodominantes da proteína DSG1, tanto da fase pré-clínica quanto clínica da doença. O reconhecimento celular e humoral preferencial de peptídeos por pacientes ou indivíduos sadios está associado aos genótipos HLA-DRB1 de susceptibilidade ao PF. Os peptídeos EC5 (524-540) e EC5 (530-549), mais freqüentemente reconhecidos por indivíduos sadios e também presentes em pacientes não tratados e em tratamento, compartilham uma sequência de aminoácidos que pode ser parte de um epítopo imunodominante reconhecido por autoanticorpos do EC5 durante a reação cruzada originada por mimetismo molecular com o agente ambiental. O peptídeo EC2 (235-258), reconhecido preferencialmente por anticorpos e células T de pacientes não-tratados, é um epítopo imunodominante, possivelmente alvo de reconhecimento por LT CD4+ durante o espalhamento intramolecular de epítopos em PF. Observamos também que pacientes em tratamento, durante a fase de remissão da doença mantêm LT CD4+ de memória para esse epítopo, porém apresentam resposta humoral preferencial a um terceiro epítopo de EC5, resíduos 499 a 518. Dessa forma postula-se que os autoanticorpos não patogênicos gerados durante a fase de remissão clínica do PF são direcionados a um novo epítopo do EC5 não reconhecido preferencialmente por indivíduos sadios. Estudos mais aprofundados do papel dessas sequências imunodominantes poderão contribuir para o desenvolvimento de novas imunoterapias visando à indução de tolerância em indivíduos geneticamente susceptíveis ao desenvolvimento do PF.

Pemphigus foliaceus is an epidermal autoimmune disease, characterized by presence of IgG antibodies, predominantly of the IgG4 subclass, against desmoglein 1 (DSG1) protein ectodomains. Acantholysis is the hallmark of the disease, which clinically is characterized by superficial blisters. Also known as fogo selvagem (meaning wild fire), pemphigis foliaceus is endemic in certain regions of Brazil. Genetic and environmental factors generate and modulate the autoimmune response. The disease is triggered by exposure to one or several possible environmental factors, that in endemic areas probably are hematophagous insects, especially Simulium nigrinanum. Susceptibility to endemic pemphigus foliaceus is igher among individuals that carry HLA-DRB1 alleles belonging to lineages *01 and *04 (susceptible alleles) and lower for individuals whose genotypes include alleles of lineages *07, *08 and *11, as well as allele *0301 (protective alleles). Antigenic mimicry with a viral or salivary protein transmitted by the insect possibly leads to the production of non-pathogenic autoantibodies against the fifth ectodomain (EC5) of DSG1. In those genetically predisposed individuals, pathogenic autoantibodies against the first (EC1) and second (EC2) ectodomains of DSG1 are generated through intramolecular epitope spreading. The aim of present study was to map the imunodominant epitopes of the DSG1 protein and to verify if the profile of humoral and cellular immune responses to the autoantigen and synthetic peptides correlate with HLA-DRB1 genotypes associated to increased susceptibility or resistance to the disease. Previous studies showed that CD4+ lymphocyte T lineages and lones derived of peripheral blood of patients with pemphigus foliaceus proliferate in response to ectodomains of recombinant DSG1. This response was restricted to presentation of peptides by HLA-DR molecules and eaded to secretion of Th2 cytokines that stimulate the production of antibodies by autorreactive B lymphocytes. However, the epitopes recognized by these cells had thus far not been identified. In the present tudy, we identified imunodominant epitopes of pre-clinic and clinic phases. The peptides preferentially recognized during cellular and humoral responses by patients and healthy individuals seemingly differ according to the HLADRB1 genotypes related to higher or lower susceptibility to pemphigus foliaceus. The peptides EC5(524-530) and EC5(530-549), most frequently recognized by healthy individuals and also by patients in treatment and without therapy, share an aminoacid sequence that can be part of an immunodominant epitope recognized by EC5 autoantibodies during the initial response, possibly raised by molecular mimicry with the environmental antigen. The peptide EC2(235-258), preferentially recognized by autoantibodies and T cells of patients without therapy, is an immunodominant epitope, possibly target of CD4+ T lymphocytes during the intramolecular epitope spreading phase leading to overt disease. In addition, we observed that treated patients kept memory CD4+ T lymphocytes to this peptide during the remission phase of disease, but developed humoral response to a third peptide in the EC5 ectodomain, spanning from residues 499 to 518. This observation revealed that the non-pathogenic autoantibodies generated during the remission phase are driving to an epitope of EC5 not preferentially recognized by healthy individuals. Additional studies dissecting the role of these immunodominant peptides can contribute to development of new immunotherapies aiming to induce tolerance in genetically susceptible individuals, thus avoiding the development of pemphigus foliaceus.

Abstract: Pemphigus foliaceus is an epidermal autoimmune disease, characterized by presence of IgG antibodies, predominantly of the IgG4 subclass, against desmoglein 1 (DSG1) protein ectodomains. cantholysis is the hallmark of the disease, which clinically is characterized by superficial blisters. Also known as fogo selvagem (meaning wild fire), pemphigis foliaceus is endemic in certain regions of Brazil. Genetic and nvironmental factors generate and modulate the autoimmune response. The disease is triggered by exposure to one or several possible environmental factors, that in endemic areas probably are hematophagous insects, especially Simulium nigrinanum. Susceptibility to endemic pemphigus foliaceus is higher among individuals that carry HLA-DRB1 alleles belonging to lineages *01 and *04 (susceptible alleles) and lower for individuals whose genotypes include alleles of lineages *07, *08 and *11, as well as allele *0301 (protective alleles). Antigenic mimicry with a viral or salivary protein transmitted by the insect possibly leads to the production of non-pathogenic autoantibodies against the fifth ectodomain (EC5) of DS 1. In those genetically predisposed individuals, pathogenic autoantibodies against the first (EC1) and second (EC2) ctodomains of DSG1 are generated through intramolecular epitope spreading. The aim of present study was to map the imunodominant epitopes of the DSG1 protein and to verify if the profile of humoral and cellular immune responses to the autoantigen and synthetic peptides correlate with HLA-DRB1 genotypes associated to increased susceptibility or resistance to the disease. Previous studies showed that CD4+ lymphocyte T lineages and clones derived of peripheral blood of patients with pemphigus foliaceus proliferate in response to ectodomains of recombinant DSG1. This response was restricted to presentation of peptides by LA-DR molecules and leaded to secretion of Th2 cytokines that stimulate the production of antibodies by autorreactive B lymphocytes. However, the epitopes recognized by these cells had thus far not been dentified. In the present study, we identified imunodominant epitopes of pre-clinic and clinic phases. The peptides preferentially recognized during cellular and humoral responses by patients and healthy individuals seemingly differ according to the HLADRB1 genotypes related to higher or lower susceptibility to pemphigus foliaceus. The peptides EC5(524-530) and EC5(530-549), most frequently recognized by healthy individuals and also by patients in treatment and without therapy, share an aminoacid sequen e that can be part of an immunodominant epitope recognized by EC5 autoantibodies during the initial response, possibly aised by molecular mimicry with he environmental antigen. The peptide EC2(235-258), preferentially recognized by autoantibodies and T cells of patients without therapy, is an immunodominant epitope, possibly target of CD4+ T lymphocytes during the intramolecular epitope spreading phase leading to overt disease. In addition, we observed that treated patients kept memory CD4+ T lymphocytes to this peptide during the remission phase of disease, but developed humoral response to a third peptide in the EC5 ectodomain, spanning from residues 499 to 518. This observation revealed that the non-pathogenic autoantibodies enerated during the remission phase are driving to an epitope of EC5 not preferentially recognized by healthy individuals. Additional studies dissecting the role of these immunodominant peptides can contribute to development of new immunotherapies aiming to induce tolerance in genetically susceptible individuals, thus avoiding the development of pemphigus foliaceus.