RESUMO
Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-ß1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-ß1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-ß1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.
RESUMO
Germ-free animals (GF) are those without a microbiome since birth. This particular biological model has become one of special interest with the growing evidence of importance of the microbiome in the life, development, adaptation, and immunity of humans and animals in the environments in which they live. Anatomical differences observed in GF compared with conventionally-reared animals (CV) has given rise to the question of the influence of commensal microflora on the development of structure and function (even immunological) of the bowel. Only recently, thanks to achievements in microscopy and associated methods, structural differences can be better evaluated and put in perspective with the immunological characteristics of GF vs. CV animals. This study, using a GF rat model, describes for the first time the possible influence that the presence of commensal microflora, continuously stimulating mucosal immunity, has on the collagen scaffold organization of the colon mucosa. Significant differences were found between CV and GF mucosa structure with higher complexity in the CV rats associated to a more activated immune environment. The immunological data suggest that, in response to the presence of a microbiome, an effective homeostatic regulation in developed by the CV rats in healthy conditions to avoid inflammation and maintain cytokine levels near the spontaneous production found in the GF animals. The results indicated that collagen scaffold adapted to the immune microenvironment; therefore, it is apparent that the microbiome was able to condition the structure of the colon mucosa.
Assuntos
Vida Livre de Germes , Microbiota , Animais , Colo , Imunidade nas Mucosas , Mucosa Intestinal , RatosRESUMO
Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets' intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.
Assuntos
Infecções por Escherichia coli/imunologia , Escherichia coli/fisiologia , Imunoglobulina A Secretora/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Polissacarídeos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Aglutinação , Animais , Animais Recém-Nascidos , Resistência à Doença , Feminino , Vida Livre de Germes , Glicosilação , Gravidez , SuínosRESUMO
Psoriatic patients have altered microbiota, both in the intestine and on the skin. It is not clear, however, whether this is a cause or consequence of the disease. In this study, using an experimental mouse model of psoriasis induced by imiquimod (IMQ), we show that oral treatment with a broad spectrum of antibiotics (MIX) or metronidazole (MET) alone mitigates the severity of skin inflammation through downregulation of Th17 immune response in conventional mice. Since some antibiotics, including MET, can influence immune system reactivity, we also evaluated the effect of MIX in the same model under germ-free (GF) conditions. GF mice treated with MET did not show milder signs of imiquimod-induced skin inflammation (IISI) which supports the conclusion that the therapeutic effect is mediated by changes in microbiota composition. Moreover, compared to controls, mice treated with MIX had a significantly higher abundance of the genus Lactobacillus in the intestine and on the skin. Mice treated with MET had a significantly higher abundance of the genera Bifidobacterium and Enterococcus both on the skin and in the intestine and of Parabacteroides distasonis in the intestine. Additionally, GF mice and mice monocolonized with either Lactobacillus plantarum or segmented filamentous bacteria (SFB) were more resistant to IISI than conventional mice. Interestingly, compared to GF mice, IMQ induced a higher degree of systemic Th17 activation in mice monocolonized with SFB but not with L. plantarum. The present findings provide evidence that intestinal and skin microbiota directly regulates IISI and emphasizes the importance of microbiota in the pathogenesis of psoriasis.
RESUMO
Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1-/- mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1-/- only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.
Assuntos
Açúcares da Dieta/metabolismo , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal , Intolerância à Glucose , Animais , Vias Biossintéticas , Dieta , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Açúcares da Dieta/administração & dosagem , Suplementos Nutricionais , Teste de Tolerância a Glucose , Humanos , Camundongos , RatosRESUMO
Psoriasis is a chronic inflammatory skin disease in which Th17 cells play a crucial role. Since indigenous gut microbiota influences the development and reactivity of immune cells, we analyzed the link among microbiota, T cells and the formation of psoriatic lesions in the imiquimod-induced murine model of psoriasis. To explore the role of microbiota, we induced skin inflammation in germ-free (GF), broad-spectrum antibiotic (ATB)-treated or conventional (CV) BALB/c and C57BL/6 mice. We found that both mice reared in GF conditions for several generations and CV mice treated with ATB were more resistant to imiquimod-induced skin inflammation than CV mice. The ATB treatment dramatically changed the diversity of gut bacteria, which remained stable after subsequent imiquimod application; ATB treatment resulted in a substantial increase in the order Lactobacillales and a significant decrease in Coriobacteriales and Clostridiales. Moreover, as compared to CV mice, imiquimod induced a lower degree of local and systemic Th17 activation in both GF and ATB-treated mice. These findings suggest that gut microbiota control imiquimod-induced skin inflammation by altering the T cell response.
Assuntos
Microbioma Gastrointestinal/fisiologia , Psoríase/imunologia , Psoríase/microbiologia , Pele/imunologia , Células Th17/imunologia , Actinobacteria/efeitos dos fármacos , Actinobacteria/fisiologia , Aminoquinolinas/farmacologia , Animais , Antibacterianos/farmacologia , Clostridiales/efeitos dos fármacos , Clostridiales/fisiologia , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Expressão Gênica , Vida Livre de Germes , Humanos , Imiquimode , Interleucina-17/genética , Interleucina-17/imunologia , Lactobacillales/efeitos dos fármacos , Lactobacillales/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Psoríase/induzido quimicamente , Psoríase/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Pele/efeitos dos fármacos , Pele/microbiologia , Pele/patologia , Especificidade da Espécie , Células Th17/efeitos dos fármacos , Células Th17/microbiologiaRESUMO
The microbiota is a crucial modulator of the immune system. Here, we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). We induced EAU in germ-free (GF) or conventionally housed (CV) mice and in CV mice treated with a combination of broad-spectrum antibiotics either from the day of EAU induction or from one week prior to induction of disease. The severity of the inflammation was assessed by fundus biomicroscopy or by histology, including immunohistology. The immunophenotyping of T cells in local and distant lymph nodes was performed by flow cytometry. We found that GF mice and mice where the microbiota was reduced one week before EAU induction were protected from severe autoimmune inflammation. GF mice had lower numbers of infiltrating macrophages and significantly less T cell infiltration in the retina than CV mice with EAU. GF mice also had reduced numbers of IFN-γ and IL-17-producing T cells and increased numbers of regulatory T cells in the eye-draining lymph nodes. These data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response.
Assuntos
Doenças Autoimunes/imunologia , Olho/imunologia , Microbiota , Retinite/imunologia , Uveíte/microbiologia , Imunidade Adaptativa , Animais , Antibacterianos/farmacologia , Autoantígenos/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/microbiologia , Carga Bacteriana/efeitos dos fármacos , Modelos Animais de Doenças , Olho/patologia , Proteínas do Olho/imunologia , Feminino , Citometria de Fluxo , Vida Livre de Germes , Interferon gama/biossíntese , Interleucina-17/biossíntese , Ativação Linfocitária , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/imunologia , Retina/imunologia , Retinite/induzido quimicamente , Retinite/etiologia , Retinite/microbiologia , Proteínas de Ligação ao Retinol/imunologia , Linfócitos T Reguladores/imunologia , Uveíte/induzido quimicamente , Uveíte/imunologiaRESUMO
The mammalian microbiota plays a crucial role in the pathogenesis of many diseases. Thanks to recent advances in metagenomics, proteomics, and metabolomics, microbiome composition and metabolic activity can now be studied in detail. Results obtained by such fascinating and provocative studies would be meaningless without considering the perspective of the whole organism. Our work using gnotobiology as the major tool to unravel the mechanisms of host-microbe interaction has demonstrated the crucial role of microbiota in the initiation and progression of inflammation-associated colorectal neoplasia. Carcinogenesis in the gut is driven by the presence of potentially harmful microbes or by lack of protective ones, by the production of carcinogens generated by microbes, and by the induction of inflammation and modulation of the immune system. Here, we review these mechanisms with special emphasis on those where gnotobiology has yielded important insights.
Assuntos
Neoplasias Colorretais/microbiologia , Modelos Animais de Doenças , Vida Livre de Germes , Microbiota , Animais , Humanos , Inflamação/microbiologia , Microambiente TumoralRESUMO
BACKGROUND: Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model. METHODS: Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and ß-glucuronidase activity was measured in intestinal content by fluorescence assay. RESULTS: ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased ß-glucuronidase activity. However, the ß-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in ß-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue. CONCLUSIONS: We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.
Assuntos
Colite/complicações , Neoplasias do Colo/etiologia , Trato Gastrointestinal/microbiologia , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Metagenoma , Animais , Azoximetano/toxicidade , Western Blotting , Carcinógenos/toxicidade , Colite/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.
Assuntos
Doenças Autoimunes/etiologia , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Inflamação/etiologia , Metagenoma/imunologia , Mucosa/imunologia , Neoplasias/etiologia , Animais , Doenças Autoimunes/microbiologia , Modelos Animais de Doenças , Humanos , Imunidade , Inflamação/microbiologia , Neoplasias/microbiologiaRESUMO
BACKGROUND AND AIMS: Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production. METHODOLOGY/PRINCIPAL FINDINGS: Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro. Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection. CONCLUSIONS: Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.
Assuntos
Bactérias/patogenicidade , Gliadina/farmacocinética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Animais , Toxinas Bacterianas/farmacologia , Bifidobacterium/patogenicidade , Doença Celíaca/etiologia , Citocinas/biossíntese , Enterobacteriaceae/patogenicidade , Vida Livre de Germes , Gliadina/farmacologia , Células Caliciformes/patologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interferon gama/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Intestinos/patologia , Permeabilidade , RatosRESUMO
AIM: The aim of our work was to determine the influence of intestinal bacteria on the development of atherosclerotic lesions using apolipoprotein E (ApoE)-deficient knockout mice. METHODS: The experiments were performed on ApoE-/--deficient mouse strain C57BL/6, bred under germ-free (GF) conditions for two generations or under conventional conditions with defined microflora (CV). The mice were fed a standard low cholesterol diet or cholesterol-rich diet for 3-4 months. We studied the development of advanced lesions in the thoracic and abdominal aorta by histological, morphometric and immunohistological methods. RESULTS: Conventionally reared ApoE-/- mice (containing no pathogenic intestinal microbiota) and fed a standard low cholesterol diet in contrast to a high cholesterol diet did not develop atherosclerotic aortic plaques. In contrast, ApoE-/- mice reared under germfree conditions for 2 generations and fed a low cholesterol diet exhibited atherosclerotic plaques in the aorta. Characteristic lipid deposition with foam cells and macrophages was found in their arterial walls. CONCLUSION: In contrast to the absence of atherosclerotic plaques in conventionally reared ApoE-deficient mice, germ-free ApoE-/- mice consuming the same low cholesterol standard diet developed atherosclerotic plaques in the aorta. Differences in atherosclerotic plaques between GF and CV ApoE-/- mice are not so apparent when mice are fed a high cholesterol diet. Our findings thus document the protective effect of microbiota (commensal bacteria) on atherosclerosis development.
Assuntos
Aorta Abdominal/patologia , Aorta Torácica/patologia , Apolipoproteínas E/fisiologia , Aterosclerose/prevenção & controle , Colesterol na Dieta/administração & dosagem , Metagenoma , Animais , Aorta Abdominal/metabolismo , Aorta Torácica/metabolismo , Aterosclerose/etiologia , Aterosclerose/metabolismo , Western Blotting , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Vida Livre de Germes , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.
Assuntos
Adenocarcinoma/imunologia , Colo/imunologia , Neoplasias Colorretais/imunologia , Vida Livre de Germes/imunologia , Imunidade Inata , Adenocarcinoma/microbiologia , Animais , Colo/microbiologia , Neoplasias Colorretais/microbiologia , Modelos Animais de Doenças , Vida Livre de Germes/efeitos dos fármacos , Humanos , Tolerância Imunológica , Imunidade nas Mucosas/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Ratos , Receptor Cross-Talk/imunologiaRESUMO
BACKGROUND: Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice. RESULTS: We have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-gamma. CONCLUSION: Our data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.
Assuntos
Vida Livre de Germes/imunologia , Sistema Imunitário/microbiologia , Intestinos/imunologia , Lipopolissacarídeos/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Dieta , Sistema Imunitário/crescimento & desenvolvimento , Tolerância Imunológica , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/citologia , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Intestinal microbiota are considered to play an important role both in colorectal tumor development and in the modulation of mucosal immunity. Studies on animals reared in germ-free (GF, without intestinal microbiota) versus conventional (CV, with regular microbiota colonization of the bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and the anticancer immune response. The capability of the intestinal environment to modulate anticancer immunity not only at the mucosal but also at the systemic level is still an open question. In our study we found that, following the same protocol of colorectal cancer induction, GF rats developed less and smaller tumors than CV rats. The GF rats that did not develop cancer also presented a better anticancer immune response with an increase in NK, NKT, CTL, B cells and cytotoxicity in peripheral blood. We hypothesize that the lower antigenic challenge and the absence of the 'physiological inflammation', caused by the commensal microbiota in the gut of CV rats, may enhance the capability of the GF rats to develop more efficacious anticancer immune responses. The different levels of tolerance/regulatory mechanisms in GF versus the CV animals may modulate the anticancer response not only at the mucosal but also at the systemic immunity level.
Assuntos
Carcinoma/patologia , Neoplasias Colorretais/patologia , Vida Livre de Germes/fisiologia , Imunidade Inata/fisiologia , Intestinos/imunologia , Animais , Animais Recém-Nascidos , Carcinoma/imunologia , Carcinoma/microbiologia , Linhagem Celular , Proliferação de Células , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/microbiologia , Testes Imunológicos de Citotoxicidade , Progressão da Doença , Feminino , Vida Livre de Germes/imunologia , Células Matadoras Naturais/patologia , Linfócitos/patologia , Masculino , Ratos , Ratos Endogâmicos , Ratos WistarRESUMO
The indigenous microflora plays an integrative role in the maintenance of immunological homeostasis. Several studies reported that immunological tolerance is dependent on microbial colonization of the gut. In the present study, we investigated whether the absence of the microflora influences the sensitization process to an allergen as well as the ability to develop mucosal tolerance in a mouse model of birch pollen allergy. Germ-free or conventional BALB/c mice were intranasally or intragastrically pre-treated with the major birch pollen allergen Bet v 1 prior to sensitization with this allergen. Both germ-free and conventional mice displayed comparable Th2 biased immune responses after allergic sensitization. Oral as well as intranasal tolerization led to suppression of allergen-specific serum antibodies (IgG1, IgE, IgA) as well as cytokine production by splenocytes (IL-5, IFN-gamma) in both germ-free and conventional animals. Peyer's patches of germ-free animals were approximately 20 times smaller than in conventional animals, but the relative distribution of lymphocyte subpopulations was equal. We conclude that the absence of the microflora does not influence the ability to mount Th2 responses nor to establish tolerance towards the aeroallergen Bet v 1. Our findings may challenge the view that the commensal microflora is a key factor for breakdown of physiological tolerance and allergy development.
Assuntos
Alérgenos/imunologia , Trato Gastrointestinal/microbiologia , Hipersensibilidade Imediata/imunologia , Tolerância Imunológica , Imunidade nas Mucosas , Proteínas de Plantas/imunologia , Administração Intranasal , Alérgenos/administração & dosagem , Animais , Anticorpos/sangue , Antígenos de Plantas , Citocinas/biossíntese , Vida Livre de Germes , Subpopulações de Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/microbiologia , Nódulos Linfáticos Agregados/anatomia & histologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Proteínas de Plantas/administração & dosagem , EstômagoRESUMO
BACKGROUND: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RB(high) subset of CD4+T cells isolated from the spleen of normal BALB/c mice. METHODS: A CD4+CD45RB(high) subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ-free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen-free (SPF) bacteria. Mice were evaluated 8-12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). RESULTS: After the transfer of the CD4+CD45RB(high) T-cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO-1 (zona occludens). CONCLUSIONS: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB.
Assuntos
Transferência Adotiva , Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Antígenos Comuns de Leucócito/administração & dosagem , Animais , Colite/microbiologia , Colite/prevenção & controle , Modelos Animais de Doenças , Citometria de Fluxo , Hiperplasia/patologia , Hipertrofia/patologia , Hibridização in Situ Fluorescente , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microscopia Eletrônica de Varredura , Baço/imunologiaRESUMO
In humans, intestinal antigen exposure during neonatal life influences the T-cell receptor (TCR) repertoire. To define the relative effects of bacteria and food antigens in early life, we examined TCR diversity in the intestine of SPF and GF mice. TCR repertoire was assessed at a single time point pre-, peri- and post-weaning in the small and large intestine of SPF and GF mice using spectratyping and/or TCR-beta-chain sequencing. There was good concordance of data obtained by the two techniques. In SPF mice, the repertoire was polyclonal shortly after birth in the small and large intestine. After weaning, there was a significant change towards an oligoclonal repertoire in the small intestine. There was some evidence that specific clones were shared between the small and large intestine. In contrast, in GF mice, the repertoire was oligoclonal after birth, and remained restricted. These data show: firstly, that under SPF conditions, the intestine is seeded with a diverse T-cell population that becomes oligoclonal around the time of weaning; secondly, that GF mice were oligoclonal at each time point.
Assuntos
Rearranjo Gênico do Linfócito T/imunologia , Vida Livre de Germes/imunologia , Imunidade nas Mucosas/fisiologia , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Células Clonais , Primers do DNA , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DesmameRESUMO
Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut-homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM-1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM-1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3(+)beta(7) (+) cells were present in similar numbers in CV and GF intestine at birth. They were CD62L(-) in CV mice and were accompanied by further CD3(+)beta(7) (+)CD62L(-) T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus-derived T cells seed the intestinal lamina propria before birth via beta-MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool.
Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Células Mieloides/imunologia , Subpopulações de Linfócitos T/imunologia , Envelhecimento/imunologia , Animais , Animais Recém-Nascidos , Subpopulações de Linfócitos B/imunologia , Moléculas de Adesão Celular/metabolismo , Vida Livre de Germes , Imunidade nas Mucosas , Imunofenotipagem , Mucosa Intestinal/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , MucoproteínasRESUMO
The CD40-CD154 pathway is important in the pathogenesis of inflammatory bowel disease. Here we show that injection of an agonistic CD40 mAb to T and B cell-deficient mice was sufficient to induce a pathogenic systemic and intestinal innate inflammatory response that was functionally dependent on tumor necrosis factor-alpha and interferon-gamma as well as interleukin-12 p40 and interleukin-23 p40 secretion. CD40-induced colitis, but not wasting disease or serum proinflammatory cytokine production, depended on interleukin-23 p19 secretion, whereas interleukin-12 p35 secretion controlled wasting disease and serum cytokine production but not mucosal immunopathology. Intestinal inflammation was associated with IL-23 (p19) mRNA-producing intestinal dendritic cells and IL-17A mRNA within the intestine. Our experiments identified IL-23 as an effector cytokine within the innate intestinal immune system. The differential role of IL-23 in local but not systemic inflammation suggests that it may make a more specific target for the treatment of IBD.