New Insights into the Immunological Changes in IL-10-Deficient Mice during the Course of Spontaneous Inflammation in the Gut Mucosa

IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut and this is illustrated by the fact that IL-10−/− mice develop spontaneous colitis. In this study, IL-10−/− mice were analyzed for immunological changes during colitis development. We found a reduced frequency of regulatory T cells CD4+CD25+Foxp3+ and higher frequency of activated T cells in the colon that precedes the macroscopic signs of the disease. Production of IL-17 and IFN-γ was higher in the colon. Colitis progression culminates with the reduction of CD4+LAP+ regulatory T cells in the intestine. Frequency of B1 cells and the secretory IgA production were both elevated. Despite these alterations, 16-week-old IL-10−/− mice could be rendered tolerant by a continuous feeding protocol. Our study provides detailed analysis of changes that precede colitis and it also suggests that oral tolerance could be used to design novel alternative therapies for the disease

Comparação do padrão inflamatório intestinal crônico antígeno específico entre camundongos C57BL/6J e BALB/C

Food allergy is an adverse immunological reaction that occurs in sensitized individuals after ingesting the antigenic food. The reactions that take place in the intestine are considered to be a type of inflammatory bowel disease and may be a consequence of tolerance rupture to food antigens leading to uncontrolled immune responses. The majority of murine models of food allergy are due to T helper 2 like immune responses, with IgE production and immediate inflammatory reactions. But allergies can also be induced in the absence of IgE, with a T helper 1 response. This discrepancy seems to be due to many factors such as genetic differences, timing of food intake and others. Our objective in this work is to compare the immune responses of two different murine inbred strains C57BL/6J and BALB/c using an antigen-specific inflammatory bowel disease model induced by peanut sensitization. Female mice of both strains were divided into normal or immune groups. Immune animals received a first subcutaneous immunization with 100ug of peanut protein extract with 1mg alum and a booster immunization only with the protein extract. Normal animals were sham immunized twice. After the second immunization the animals were subdivided in 4 groups and challenged with a diet-containing peanut for two, three or four weeks and the last group (control animals) were maintained with conventional chow. We analyzed antipeanut total IgG and IgG1 titers, the histological pattern of duodenal mucosa, the intestinal epithelial cell/intraepithelial leukocytes ratio (IEC/IEL) of duodenal villi and the CD4+ and CD8+ T cell patterns in Peyer patches and mesenteric lymph nodes of these animals. Our results show that BALB/c immune mice presented higher titers of antipeanut total IgG, but not IgG1, when compared to immune mice from C57BL/6J strain. BALB/c, but not C57BL/6J, normal mice presented increasing antipeanut IgG and IgG1 titers after eating peanuts without important changes in the mucosa. Two weeks of challenge diet was sufficient to induce an increase of intraepithelial leukocytes numbers (drop in IEC/IEL ratio) of BALB/c immune animals while in C57BL/6J mice this feature only takes place after the 3rd 4th week of challenge. BALB/c immune animals presented other intestinal inflammatory parameters, such as, edema, loss of villi integrity, which were more intense than in C57BL/6J immune mice after the 2nd week of challenge. After two weeks of peanut exposition, C57BL/6J immune animals presented an arise of CD8+ T cell population in Peyer patches while in mesenteric lymph nodes CD8+ T cell number decreased. Interestingly, C57BL/6J normal mice presented an increased number of CD4+ T cells in mesenteric lymph nodes after two weeks of being exposed to peanut for the first time. We concluded that there are differences in the intestinal inflammatory patterns between C57BL/6J and BALB/c strains. BALB/c strain is more susceptible to the acute reactions of the intestinal inflammation and the triggering of inflammatory signals is quicker and more intense in these mice. The higher titers of IgGs observed in BALB/c normal mice that eat peanuts could be explained by an active process of oral tolerization and not oral sensitization to this seed and that this phenomenon is characteristic of BALB/c strain. We also conclude that, at least in C57BL/6J strain, the probable T cell type, which is responsible for the mucosa damage in immune animals in our model of inflammatory bowel disease, are the CD8+ T cells which are presented in higher number in theirs Peyer patches.A alergia alimentar é uma reação imunológica adversa que acomete indivíduos sensibilizados quando ingerem o alimento alergênico. As reações que ocorrem no intestino são consideradas como um tipo de inflamação intestinal crônica e podem ser conseqüência da ruptura da tolerância aos antígenos alimentares, levando a repostas imunes descontroladas. A maioria dos modelos de alergia alimentar em camundongos é causada por resposta imune mediada por células T helper do tipo 2 com produção de IgE e reações inflamatórias imediatas. Porém as alergias podem ser induzidas também na ausência de IgE, com um padrão do tipo Th1. Essa discrepância parece ser dependente de muitos fatores como diferenças genéticas, o tempo de introdução do alimento entre outros. Portanto, o objetivo desse trabalho foi comparar as respostas imunes de duas linhagens isogênicas diferentes - C57BL/6J e BALB/c - no modelo de inflamação intestinal crônica antígeno-específico induzida pela sensibilização com amendoim. Camundongos fêmeas de ambas as linhagens foram divididos em grupos imunes ou normais. Animais imunes receberam uma primeira imunização por via subcutânea com 100µg de extrato protéico de amendoim com 1mg de hidróxido de alumínio e uma imunização secundária somente com o extrato protéico. Animais normais foram imunizados duas vezes com salina. Após a secunda imunização os animais foram subdivididos em 4 grupos e tratados com uma dieta desafio (contendo amendoim) por duas, três ou quatro semanas ou mantidos com a ração convencional (animais controle). Nós analisamos os títulos de IgG total e IgG1 antiamendoim, o padrão histológico da mucosa duodenal, a razão entre células epiteliais intestinais e leucócitos intraepiteliais das vilosidades duodenais e o padrão de células T CD4+ e CD8+ nas placas de Peyer e linfonodos mesentéricos desses animais. Nossos resultados mostram que animais BALB/c imunes apresentaram títulos de IgG total e IgG1 antiamendoim mais altos do que os animais C57BL/6J imunes. Ao contrário de animais C57BL/6J normais, os camundongos BALB/c normais apresentaram títulos crescentes de ambas as IgGs antiamendoim após o início da ingestão dessa semente sem apresentar mudanças na mucosa intestinal. Duas semanas de dieta desafio foram suficientes para induzir um aumento no número de leucócitos intraepiteliais em animais BALB/c imunes enquanto que em camundongos C57BL/6J esse sinal só foi observado após a terceira ou quarta semana de desafio. Os animais BALB/c imunes apresentaram outros parâmetros de inflamação intestinal, como edema e perda da integridade das vilosidades que foram mais intensos do que nos animais C57BL/6J imunes após a segunda semana de desafio. Os animais imunes C57BL/6J apresentaram um aumento de células T CD8+ nas placas de Peyer e uma queda dessas células nos linfonodos mesentéricos com duas semanas de inflamação. Curiosamente, os animais normais C57BL/6J apresentaram aumento da população de células T CD4+ tanto nas placas de Peyer quanto nos linfonodos mesentéricos após ingerirem o amendoim por duas semanas. Nós concluímos que há diferença no perfil de inflamação intestinal entre os animais C57BL/6J e BALB/c sendo que este último é mais susceptível às reações agudas da inflamação intestinal e os sinais de inflamação são mais intensos e aparecem mais rapidamente nesses animais. Os altos títulos de IgG total e IgG1 antiamendoim observados nos animais BALB/c normais que comem amendoim podem ser explicados por um processo ativo de tolerização oral e não de imunização oral a essa semente e que esse fenômeno é característico da linhagem BALB/c. Nós também concluímos que, pelo menos nos animais C57BL/6J, as células que provavelmente são responsáveis pelos danos causados à mucosa em nosso modelo de inflamação intestinal crônica, são os linfócitos T CD8+ que estão presentes em maior número nas placas de Peyer desses animais

Aspectos sistêmicos da tolerância oral

Exportado OPUSMade available in DSpace on 2019-08-12T02:00:25Z (GMT). No. of bitstreams: 1 tese__archimedes_castro_junior_para_diploma_ufmg.pdf: 5011860 bytes, checksum: 6b2cadfbf6381c6534993f048b84b067 (MD5) Previous issue date: 18A Tolerância Oral é tradicionalmente definida pela inibição da reatividade imune específica contra proteínas previamente ingeridas, devido a um decréscimo da atividade linfocitária. Pouca atenção tem sido dada à atividade imunológica sistêmica global em animais tolerantes orais. Por atividade imunológica global, entendemos a ativação concomitante de clones de linfócitos não específicos ao antígeno em questão, além dos clones antígeno-específicos. Paradoxalmente, nós observamos que a inibição da reatividade específica coexiste com um aumento da atividade global de linfócitos. Camundongos C57BL/6 tolerantes a ovalbumina apresentaram a supressão característica das respostas de anticorpos séricos específicos para todas as classes de Imunoglobulinas avaliadas: IgG1, IgE, IgM e IgA. Entretanto, o nível de IgM total sérica estava mais aumentado em animais tolerantes do que em imunizados. Os níveis de IgA total sérica estavam aumentados em ambos os grupos. Por consequência, animais tolerantes possuíam números elevados de células secretoras de imunoglobulinas (CSI) no baço e na medula óssea. No baço de animais tolerantes prevaleceu CSI produzindo IgA e IgM enquanto que na medula óssea predominam CSI produzindo IgG e IgM. Animais imunizados mostraram maior número de CSI secretando IgG no baço. Apesar de possuírem o mesmo número e frequência de linfócitos T ativados e reguladores no baço após a imunização secundária, animais tolerantes exibiram uma cinética particular de aparecimento dessas células após a imunização primária. Camundongos tolerantes mostraram uma expansão precoce de células T efetoras/memória CD4+CD44high um dia após a imunização, enquanto que animais imunizados tiveram esta população aumentada após o quarto dia. Similarmente, células T reguladoras CD4+Foxp3+ aparecem antes no baço de animais tolerantes, no segundo dia da cinética. Estas células expandem tardiamente em animais imunizados, porém alcançam uma frequência e número maior do que a de animais tolerantes após 7 dias. Digno de nota é o aumento no número de células T CD4+CD69+ (recém ativadas) e CD4+CD25+LAP+ (reguladoras) no baço de animais tolerantes mesmo antes da imunização parenteral, sete dias após a administração oral do antígeno. Neste período, animais tolerantes também possuem uma expressão aumentada de citocinas reguladoras (TGF- e IL- 10) e uma expressão transitória de citocinas efetoras (IL-2 e IFN-). Assim, concluímos que os fenômenos imunológicos subjacentes à tolerância oral vão além da inibição da reatividade específica, envolvendo uma ativação linfocitária mais distribuída e uma rápida mobilização de linfócitos ativados e reguladores.Oral tolerance is traditionally understood as a reduction of specific immune responsiveness to ingested proteins due to lymphocyte activity decrease. Few attention has been given to systemic immunological activity in orally tolerant organisms. Paradoxically, we found that specific lymphocyte activity inhibition was parallel to an increased global lymphocyte activity. C57BL/6 mice rendered orally tolerant to ovalbumin showed the characteristic suppression of antigen specific antibody responses in all serum immunoglobulin classes analyzed: IgG1, IgE, IgM and IgA. Nevertheless, these mice presented total IgA levels similar to immunized mice and higher levels of total IgM in serum. Accordingly, orally tolerant mice had higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow. Moreover, tolerant mice Ig-secreting cells in spleen were mainly IgM and IgA producers, whereas immunized mice had mostly IgG-secreting cells. Despite having the same number of regulatory and activated T cells in spleen after booster immunization, tolerant animals exhibit a different cells appearance kinetics in the spleen after the primary intraperitoneal immunization with antigen and adjuvant. Tolerant mice showed an earlier expansion of CD4+CD44high effector/memory T cell as soon as one day after immunization while immunized mice had these cells expanded at the fourth day. Similarly, CD4+Foxp3+ regulatory T cells appeared sooner in tolerant mice, two days after immunization. Immunized mice showed a CD4+Foxp3+ Treg cells expansion delay, achieving higher cells frequencies and numbers 7 days after immunization. Importantly, orally tolerant mice presented higher numbers of CD4+CD69+ T cells and CD4+CD25+LAP+ regulatory T cells in spleen even before parenteral immunization. Also, at this time tolerant animals showed a higher expression of regulatory cytokines (TGF- and IL-10) and a transient expression of lymphocyte activation cytokines (IL-2 and IFN-). Thus, over inhibiting specific responsiveness, orally-tolerant mice display an early and widespread mobilization of activated and regulatory lymphocytes

New Insights into the Immunological Changes in IL-10-Deficient Mice during the Course of Spontaneous Inflammation in the Gut Mucosa

IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut and this is illustrated by the fact that IL-10−/− mice develop spontaneous colitis. In this study, IL-10−/− mice were analyzed for immunological changes during colitis development. We found a reduced frequency of regulatory T cells and higher frequency of activated T cells in the colon that precedes the macroscopic signs of the disease. Production of IL-17 and IFN-γ was higher in the colon. Colitis progression culminates with the reduction of regulatory T cells in the intestine. Frequency of B1 cells and the secretory IgA production were both elevated. Despite these alterations, 16-week-old IL-10−/− mice could be rendered tolerant by a continuous feeding protocol. Our study provides detailed analysis of changes that precede colitis and it also suggests that oral tolerance could be used to design novel alternative therapies for the disease.Peer Reviewe

Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbiota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4+Foxp3+ and CD4+LAP+ regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD

Consumption of conjugated linoleic acid (CLA)-supplemented diet during colitis development ameliorates gut inflammation without causing steatosis in mice.

Dietary supplementation with conjugated linoleic acid (CLA) has been proposed for weight management and to prevent gut inflammation. However, some animal studies suggest that supplementation with CLA leads to the development of nonalcoholic fatty liver disease. The aims of this study were to test the efficiency of CLA in preventing dextran sulfate sodium (DSS)-induced colitis, to analyze the effects of CLA in the liver function, and to access putative liver alterations upon CLA supplementation during colitis. So, C57BL/6 mice were supplemented for 3 weeks with either control diet (AIN-G) or 1% CLA-supplemented diet. CLA content in the diet and in the liver of mice fed CLA containing diet were accessed by gas chromatography. On the first day of the third week of dietary treatment, mice received ad libitum a 1.5%?2.5% DSS solution for 7 days. Disease activity index score was evaluated; colon and liver samples were stained by hematoxylin and eosin for histopathology analysis and lamina propria cells were extracted to access the profile of innate cell infiltrate. Metabolic alterations before and after colitis induction were accessed by an open calorimetric circuit. Serum glucose, cholesterol, triglycerides and alanine aminotransaminase were measured; the content of fat in liver and feces was also accessed. CLA prevented weight loss, histopathologic and macroscopic signs of colitis, and inflammatory infiltration. Mice fed CLA-supplemented without colitis induction diet developed steatosis, which was prevented in mice with colitis probably due to the higher lipid consumption as energy during gut inflammation. This result suggests that CLA is safe for use during gut inflammation but not at steady-state conditions