Leprosy is a complex, chronic, infectious dermato-neurological disease that affects the skin and peripheral nerves especially during acute immune-inflammatory episodes known as type 1/T1R and type 2/T2R reactions. There is no experimental model for leprosy and leprosy skin lesions have been extensively used to unravel its multifaceted immunopathological mechanisms.This study investigated in situ expression of two distinct cell populations with important immunoregulatory roles: T regulatory (Treg) cells and mast cells (MC) in diverse skin diseases with emphasis on leprosy T1R and T2R. For the Treg cell study, 154 skin biopsies from 114 participants belonging to 3 groups were investigated: 1. Leprosy (n=74), 56 T1R (28-paired biopsies reactionfree/reactional from the same patient, 28 single reactional biopsy), 18 T2R (12 pairedreaction-free/reactional lesions, 6 single reactional biopsy); 2. Dermatoses: (n=29) noninfectious and cutaneous infectious diseases; 3. Normal controls: skin fragment of mammoplasty from healthy females that had cosmetic surgery. Double immunohistochemical detection of Treg cells was performed with automated platform for CD25 and Foxp3 staining. Quantifications of double immunostained Treg cells was performed (values expressed by mm2 ) blinded to the participants’ clinical status. For the mast cell study 80 skin biopsies from 3 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 T1R, 11 T2R), 29 patients with other dermatoses (the same as for Treg study) and 11 normal skins. Toluidine blue stained intact and degranulated MC counts/mm2 ; streptavidin-biotin-peroxidase immunostaining was used to detect tryptase/try+ and chymase/chy+ MCs and their density (median optical density) was evaluated. Results: Treg study: Not one CD25+ Foxp3+ Treg cell was seen in any of the 11 normal skin sections while variable numbers were detected in skin diseases (p<0.0001); the number of double stained cells was higher in infectious compared to non-infectious diseases (p=0.008). Treg cell numbers were comparable between leprosy and other infectious dermatoses (p=0.157) Treg cell counts in reactional lesions were higher than in reaction-free leprosy lesions (p<0.002). Paired biopsies of T1R or T2R reactional/reaction-free lesions showed xxvii increased numbers of Treg during T1R compared to reaction-free lesions from the same patient (p< 0.001). Treg cell median in T1R developed during MDT was slightly higher compared to T1R developed at diagnosis in naïve patients (p=0.047). There was a trend in increasing Treg cell numbers from the tuberculoid to borderline-lepromatous form, which showed the highest median value of Tregs, however this difference was not significant (p>0.8). Mast cell study: Infectious and non-infectious skin lesions showed higher numbers of degranulated than intact MC both for leprosy and other dermatoses, compared to normal skin. The numbers of degranulated MC were higher than intact MC regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), regardless of the occurrence of leprosy reactions (reactional and reaction-free) and regardless of the type of reaction (T1R/T2R). Try+ MC numbers and density were higher than chy+ MC in leprosy, in reaction-free and reactional lesions, particularly in T2R, but not in other dermatoses. Conclusions: Higher Treg numbers seen in T1R suggest Treg role in suppressing the exacerbated cell-mediated phenomenon that causes T1R. Differential expression/ of try+ and chy+ MC subsets was seen in leprosy compared to other skin diseases and to normal skin. However, neither leprosy form nor leprosy reaction was associated with MC changes in lesions suggesting that the Mycobacterium leprae infectious process per se dictates MC expression in leprosy skin lesions.

O sistema imune pode ser dividido em dois ramos principais, imunidade inata e imunidade adaptativa. Enquanto a imunidade inata reconhece padrões conservados em microrganismos ao longo da evolução, a imunidade adaptativa atua de forma especializada, através de linfócitos B e T. Estas células são capazes de reconhecer moléculas de patogénios com maior especificidade e combate-los mais eficazmente, eliminando-os na maior parte dos casos. Além desta função, uma característica fundamental da imunidade adaptativa e a geração de memoria imunológica. Desta forma, se um organismo for reinfectado com um patogénio, a resposta imune contra este será muito mais rápida e poderá decorrer sem doença. Inicialmente, as células T de memoria eram divididas em dois grupos de acordo com os órgãos pelos quais circulavam: células T de memoria efetora, que circulam em órgãos não linfoides, e células T de memoria central, assim conhecidas por apresentarem uma maior longevidade e terem um maior tropismo para órgãos linfoides. Mais recentemente novos grupos de células de memoria foram descritos, nomeadamente as células T de memoria residentes em tecidos (Trm). Como o nome indica, estas células estão destinadas a permanecer dentro dos tecidos onde foram geradas, expressando marcadores como CD69, que previne a sua saída, e CD103 ou CD49a, que lhes permite a integração no tecido e o seu patrulhamento. Para que a memoria imunológica seja gerada, uma resposta imune efetora tem de ser estabelecida previamente. A resposta de células T pode ser dividida classicamente em três grupos, de acordo com o tipo de patogénio encontrado. No caso de infeção por vírus ou bactérias intracelulares, há um contexto inflamatório que promove a diferenciação do linfócito T CD4 em linfócito T auxiliar do tipo 1 (Th1). Este processo e desencadeado por um programa transcricional mediado pelo fator de transcrição T-box expresso em células T (Tbet), que leva a produção de interferão gama (IFN-g). Por sua vez, o IFN-g ativa macrófagos e recruta linfócitos T CD8 citotóxicos, de forma a eliminar células infetadas. No contexto duma infeção helmíntica, ou em caso de alergia, uma resposta mediada por IL-4 induz o linfócito T a iniciar um programa de diferenciação em célula T auxiliar do tipo 2 (Th2). Este programa e orquestrado pelo fator de transcrição GATA-3, que promove a produção de interleucina (IL)- 4, IL-5 e IL-13, e também o recrutamento de eosinófilos, basófilos e mastócitos, que desencadeiam mecanismos de reparação tecidual. Se o patogénio tiver uma natureza extracelular, seja bactéria ou fungo, ira desencadear uma resposta mediada por IL-6 e TGF-b, que levara um linfócito T a diferenciar-se numa célula T auxiliar produtora de IL-17 (Th17). Este fenómeno traduz-se através do recetor retinoide órfão gama t (RORgt), que despoleta a expressão de IL-17 e IL-22 na célula Th17, promovendo o recrutamento de neutrófilos para o local de infeção e induzindo a fagocitose do patogénio.

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro-inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ-T-cell-based cancer therapies. Thus, the regulation of γδ-T-cell activity is of great biological and clinical relevance. Here, we show that murine CD4+CD25+ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro-inflammatory cytokines, IFN-γ and IL-17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid-induced TNF receptor-related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro-inflammatory functions of γδ T cells are regulated.

Peripheral T-cell expansion is of major relevance for immune function after lymphopenia. In order to promote regeneration, the process should result in a peripheral T-cell pool with a similar subpopulation structure as before lymphopenia. We investigated the repopulation of the CD8+ central-memory T cells (TCM) and effector-memory T cells (TEM) pools after adoptive transfer of sorted CD8+ T cells from naïve, TCM and TEM subsets into T-cell-deficient hosts. We show that the initial kinetics of expansion are distinct for each subset and that the contribution to the repopulation of the CD81+ T-cell pool by the progeny of each subset is not a mere function of its initial expansion. We demonstrate that CD4+ CD25+ Treg play a major role in the repopulation of the CD8+ T-cell pool and that CD8+ T-cell subsets impact on each other. In the absence of CD4+ CD25+ Treg, a small fraction of naïve CD81 T cells strongly proliferates, correlating with further expansion and differentiation of co-expanding CD8+ T cells. CD4+ CD25+ Treg suppress these responses and lead to controlled repopulation, contributing decisively to the maintenance of recovered TCM and TEM fractions, and leading to repopulation of each pool with progeny of its own kind.

It is well established that therapeutic impairment of Foxp3+ Treg in mice and humans favors immune rejection of solid tumors. Less explored is the impact Foxp3 allelic variants may have on tumor incidence, progression and therapy. In this work, we tested and demonstrate that the Foxp3fgfp reporter allele, found previously to either enhance or reduce Treg function in specific autoimmunity settings, confers increased anti-tumor immunity. Our conclusions stem out of the analysis of three tumor models of different tissue origin, in two murine genetic backgrounds. When compared to wild type animals, mice carrying the Foxp3fgfp allele spontaneously delay, reduce or prevent primary tumor growth, decrease metastasis growth, and potentiate the response to anti-CTLA4 monotherapy. These findings suggest allelic variances at the Foxp3 locus may serve as predictive indicators for personalized therapy and prognostics, and point at possible new therapeutic targets.

Root bark infusions of the plant Cissampelos sympodialis Eichl (Menispermaceae) are used in folk medicine, in Northeast Brazil, for the treatment of diseases of the respiratory and digestive tracts. Previous studies showed that the hydroalcoholic extract of the leaves (AFL) of the plant and warifteine (W), alkaloid bisbenzylisoquinolinic, presented anti-inflammatory and anti-allergic effects. This study evaluated the effect of the oral treatment of mice with W and methyl warifteine (MW) in the paw edema formation induced by phlogistic agents, vascular leakage and cell migration in acute inflammatory models and the effect of oral treatment with AFL and its alkaloids (W and MW) in chronic inflammation represented by the experimental model of food allergy (BALB / c mice sensitized with ovalbumin - OVA). Oral treatment with W reduced the paw edema induced by carrageenan, histamine and prostaglandin E2, an effect not presented in MW treatment. The warifteine and methyl-warifteine also reduced the vascular leakage, however without inhibiting cell migration associated with inflammation. In the experimental model of food allergy the treatment with W induced weight gain in animals with decreased of diarrhea. Methylation of warifteine did not induce weight gain nor inhibited allergic diarrhea during the allergen challenge. However treatment with AFL did not induce weight gain nor inhibited allergic diarrhea. In contrast, treatment with the AFL or its alkaloids reduced the IgE specific for ovalbumin (OVA) titer, increased the proportion of CD4 + or CD8+ T lymphocytes in mesenteric lymph nodes. The proportion of regulatory T lymphocytes in the mesenteric lymph nodes was also increased by the treatments. In vitro experiments, with cells from mesenteric lymph nodes of sensitized animals, demonstrated that W and MW inhibited the secretion of interleukin (IL-) 12 and IL-10 with no change in the interferon-γ (IFN-γ) and IL- 13 levels. These results demonstrated that the oral treatment with warifteine presented anti-inflammatory activity by inhibiting the action of mediators of inflammation and the methylation of the molecule did not improve this effect. Also, the treatment with AFL, W and MW showed immunomodulatory effects in food allergy with increased of Treg cells and decreased of cytokines derived from cells of the innate immune mechanism independent of that of the adaptive immune mechanism.

INTRODUÇÃO: Helicobacter pylori (H. pylori) é uma bactéria altamente prevalente em nosso meio e está diretamente envolvida em várias doenças do trato digestivo superior como gastrite, úlcera péptica e câncer gástrico. Neste estudo foram pesquisados dados preditivos do ponto de vista clínico, endoscópico, anatomopatológico bem como os mecanismos da resposta imune frente à presença desta bactéria Várias moléculas ativadoras do sistema imune têm sido descritas e estudadas na tentativa de conter a infecção pelo H pylori. OBJETIVOS: Estudar a presença de infecção por H. pylori na mucosa gástrica com a história clínica, antecedentes clínicos, doenças concomitantes, dados no exame de endoscopia digestiva alta (EDA) do exame anatomopatológico (EAP). Na resposta imune, analisar nos fragmentos do corpo gástrico por qPCR, a expressão mRNA das citocinas IFN- , IL-17, IL-10, TGF- , IL-6, IL-22, IL-23, IL-33; dos fatores de transcrição T-bet, ROR t e FOXP3; das enzimas ARG1, ARG2 e NOS2; dos neuropeptídeos VIP e TAC, e seus respectivos receptores VIP-R1 e TAC-R1. Comparar a expressão desses genes com a presença ou ausência da bactéria H. pylori. MATERIAL E MÉTODOS: foram avaliados 126 pacientes que apresentavam sintomas do trato digestivo superior e com indicação clínica de exame de Endoscopia Digestiva Alta (EDA). Antes da realização do exame de endoscopia digestiva alta foi feito um questionário clínico detalhado e durante o exame endoscópico realizaram-se biópsias múltiplas do esôfago, corpo e antro gástrico e teste rápido da urease. Fragmentos da mucosa do corpo e do antro gástrico foram coletados para o exame anatomopatológico e para análise da expressão das enzimas, citocinas e fatores de transcrição por meio de PCR em tempo real, qPCR. RESULTADOS: Não foi encontrado nenhum dado clínico que pudesse estar relacionado à presença da infecção do H. pylori no estômago. Os achados endoscópicos que tiveram relevância com a presença da bactéria foi a gastrite tanto no antro como no corpo gástrico (p<0,05). No exame anatomopatológico (EAP) os pacientes que usaram Inibidores de Bomba de Prótons (IBP) apresentaram uma diminuição da positividade da bactéria no antro e no corpo gástrico (p<0,05). A presença da gastrite no corpo gástrico ou antro gástrico teve significância com a positividade da bactéria (p<0,05). A expressão do gene ARG1 foi significativamente maior no grupo de pacientes com gastrite crônica sem atividade quando comparada ao grupo controle (Kruskal Wallis; p=0.02). A expressão do gene do TGF- e seu fator de transcrição FOXP3 foram significativamente maiores no grupo de pacientes com gastrite crônica sem atividade, quando comparada ao grupo controle. Ao comparar a expressão dos genes IFN- , IL-17, IL-10 e TGF- e os fatores de transcrição T-bet e ROR, com a presença ou ausência do H.pylori, não houve diferença significativa. No entanto, a expressão do FOXP3 foi significativamente menor nos pacientes H. pylori positivo quando comparado com os pacientes H. pylori negativos (Mann-Whitney; p=0.01). CONCLUSÕES: Infecção por H. pylori não mostrou nenhum fator clínico preditivo da sua presença. Na EDA destacou-se a presença da gastrite no antro como dado de provável infecção da bactéria. O uso de IBPs deverá ser suspenso por pelo menos duas semanas antes da EDA para evitarmos falsos negativos da presença do H. pylori no estômago. O EAP mostrou-se necessário para classificar os diferentes tipos gastrites e/ou alterações histopatológicas provocadas pela bactéria. ARG-1 e perfil Treg parecem estar modulando o processo inflamatório, protegendo esses pacientes de lesões teciduais com gastrite crônica sem atividade. Ainda, sugerimos que a IL-33 pode ser um mediador crucial da resposta imune à infecção após dano na mucosa gástrica.

ABSTRACT Objective The objective of the present study was to investigate the effect of vitamin A supplementation on serum Th17 (IL-6, IL-17, IFNγ) and Treg (TGF-β, IL-10) related cytokines in obese and non-obese women. Subjects and methods In a randomized double blind placebo controlled design, 56 obese women were randomly assigned to receive either an oral dose of 25,000 IU retinyl palmitate or placebo per day for 4 months. Twenty eight ages matched non-obese women were also received vitamin A. At the study entry, anthropometric variables were measured and serum Th17 and Treg related cytokine profile were determined at baseline and 4 months after intervention. Results Significantly higher baseline concentrations of IL-6 were observed in obese compared with non-obese women (P < 0.05). However, the initial concentrations of other cytokines were not significantly different between groups. The mean concentrations of IL-17 and TGF-β were significantly decreased after vitamin A supplementation in non-obese and obese women respectively. Positive relationships between IL-17 and IL-10 (r = 0.42, P < 0.001), TGF-β and IL-17 (r = 0.35, P < 0.001) and between IL-10 and IFN-γ (r = 0.41, P = 0.002) in total participants were also observed. Conclusions The results of the present study showed for the first time that vitamin A supplementation reduces serum concentrations of IL-17 and TGF-β in reproductive age women. Further studies are needed to explore the possible underlying mechanisms.

Abstract Objective: Maintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB). This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods: 250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls) were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs) in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. Results: The percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31 ± 1.35% vs. 1.58 ± 0.71% or 1.15 ± 0.49%, p < 0.05; Treg: 11.44 ± 2.69% vs. 7.54 ± 1.56% or 4.10 ± 0.99%, p < 0.05). The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85 ± 9.68 vs. 7.23 ± 5.19 or 8.21 ± 5.51 pg/mL, p < 0.05; IL-10: 28.70 ± 11.27 vs. 20.25 ± 8.57 or 13.94 ± 9.00 pg/mL, p < 0.05). Conclusions: Our study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.