RESUMO
Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.
Assuntos
Candida albicans/imunologia , Parede Celular/imunologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , beta-Glucanas/imunologia , Células CACO-2 , Candida albicans/metabolismo , Candida albicans/fisiologia , Linhagem Celular Tumoral , Parede Celular/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mucosa Intestinal/microbiologia , Fosforilação/imunologia , Quinase Syk/imunologia , Quinase Syk/metabolismo , Zimosan/imunologia , Zimosan/metabolismo , beta-Glucanas/metabolismoRESUMO
BACKGROUND & AIMS: In healthy individuals, interactions between intestinal epithelial cells and lamina propria lymphocytes give rise to a population of CD8(+) T cells with suppressor functions (Ts cells). Disruption of Ts cell activities can lead to mucosal inflammation. We investigated what factors were required for expansion of the Ts cell population or loss of their activity in patients with Crohn's disease (CD). METHODS: We developed a method to generate Ts cell lines from freshly isolated lamina propria lymphocytes from patients with ulcerative colitis (UC), patients with CD, or healthy individuals (controls). Cells were stimulated with a monoclonal antibody against CD3, interleukin (IL)-7, and IL-15. After 14 days in culture, CD8(+)T cells were purified and cultured with IL-7 and IL-15. The resulting Ts cells were analyzed for suppressor activity, expression of surface markers, and cytokine secretion profiles. RNA was isolated from the 3 groups of Ts cells and used in microarray analyses. RESULTS: Ts cells from patients with UC and controls suppressed proliferation of CD4(+) T cells; the suppression required cell contact. In contrast, Ts cells from patients with CD had a reduced capacity to suppress CD4(+) T-cell proliferation. The difference in suppressive ability was not associated with surface or intracytoplasmic markers or secretion of cytokines. Microarray analysis identified changes in expression of genes regulated by transforming growth factor (TGF)-ß that were associated with the suppressive abilities of Ts cells. We found that TGF-ß or supernatants from Ts cells of patients with CD reduced the suppressor activity of control Ts cells. CONCLUSIONS: Ts cells isolated from patients with CD have a reduced ability to suppress proliferation of CD4(+)T cells compared with control Ts cells. TGF-ß signaling reduces the suppressor activity of Ts cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica/imunologia , Mucosa Intestinal/imunologia , Fator de Crescimento Transformador beta/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linhagem Celular , Separação Celular , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica/genética , Interferon gama/imunologia , Intestinos/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND & AIMS: Janus kinase (JAK) inhibitors are used for treating inflammatory bowel diseases (IBD). We aimed to identify molecular effects of JAK inhibition in human intestinal mucosa, considering IBD location and phenotype. METHODS: Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), and non-IBD controls (NC) were assessed for phosphorylated signal transducers and activators of transcription (p-STAT) levels and Inflammatory genes expression panel in response to ex-vivo JAK inhibitor (tofacitinib). Cytokine production by lamina propria lymphocytes in response to tofacitinib was assessed. Human intestinal organoids were used to investigate JAK inhibitors' effects on iNOS expression. RESULTS: Explants were collected from 68 patients (UC=20; CD=20; NC=28). p-STAT1\3\5 inhibition rates varied, being higher in colonic compared to ileal explants. p-STAT1\3 inhibition rates negatively correlated with CRP levels. While significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including NOS2. JAK inhibition significantly decreased Th1\Th2\Th17-related cytokine production from lamina propria lymphocytes. Various JAK inhibitors reduced IFN-γ-induced increase in iNOS expression in organoids. CONCLUSIONS: Site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying personalized drug effects in patients with IBD.
RESUMO
BACKGROUND & AIMS: Interactions between lymphocytes and intestinal epithelial cells occur in the subepithelial space of the gastrointestinal tract. Normal human lamina propria lymphocytes (LPLs) induce differentiation of intestinal epithelial cells. The absence of LPLs in mice, such as in RAG1(-/-) mice, results in defects in epithelial cell differentiation. We investigated the role of lymphoepithelial interactions in epithelial differentiation and barrier function. METHODS: We used adoptive transfer to determine if CD4(+) T cells (CD4(+)CD62L(+)CD45Rb(Hi) and/or CD4(+)CD62L(+)CD45Rb(Lo)) could overcome permeability defect (quantified in Ussing chambers). Immunofluorescence staining was performed to determine expression of cleaved Notch-1, villin, and claudin 5 in colon samples from mice and humans. Caco-2 cells were infected with a lentivirus containing a specific Notch-1 or scrambled short hairpin RNA sequence. Tight junction assembly was analyzed by immunoblot and immunofluorescence analyses, and transepithelial resistance was monitored. RESULTS: Expression of cleaved Notch-1, villin, or claudin 5 was not detected in RAG1(-/-) colonocytes; their loss correlated with increased intestinal permeability. Transfer of CD45Rb(Hi) and/or CD45Rb(Lo) cells into RAG1(-/-) mice induced expression of cleaved Notch, villin, and claudin 5 in colonocytes and significantly reduced the permeability of the distal colon. Loss of Notch-1 expression in Caco-2 cells correlated with decreased transepithelial resistance and dysregulated expression and localization of tight junction proteins. Levels of cleaved Notch-1 were increased in colonic epithelium of patients with Crohn's disease. CONCLUSIONS: LPLs promote mucosal barrier function, which is associated with activation of the Notch-1 signaling pathway. LPLs maintain intestinal homeostasis by inducing intestinal epithelial cell differentiation, polarization, and barrier function.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Mucosa Intestinal/fisiologia , Receptor Notch1/fisiologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Células CACO-2 , Claudina-5 , Doença de Crohn/patologia , Doença de Crohn/fisiopatologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Selectina L/análise , Selectina L/imunologia , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/imunologia , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Receptor Notch1/análise , Transdução de Sinais/fisiologia , Junções Íntimas/imunologia , Junções Íntimas/fisiologiaRESUMO
To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn's disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Assuntos
Infecções por Clostridium/microbiologia , Doença de Crohn/microbiologia , Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Adiposidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Clostridioides difficile , Feminino , Homeostase , Humanos , Íleo/microbiologia , Sistema Imunitário , Doenças Inflamatórias Intestinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Pessoa de Meia-Idade , Mucosa/microbiologia , Fenótipo , RNA Ribossômico 16S/metabolismo , Especificidade da Espécie , Adulto JovemRESUMO
Background: A large-scale increase in microRNA (miRNA) expression was observed in patients with ulcerative colitis who underwent pouch surgery and developed inflammation of the pouch (pouchitis). In this study, we assessed miRNA expression in these patients and investigated how regulation of its expression changes in the setting of pouchitis. Methods: Autologous samples that included mucosal biopsies, peripheral blood cells, and plasma were collected from the patients. Candidate primary and mature miRNA expressions were analyzed by quantitative polymerase chain reaction. A human intestinal epithelial cell line was used to test DICER activity, and the expression of key miRNA processing factors was analyzed by Western blot. miRNA-424 and its potential target serotonin reuptake transporter (SERT) expressions were examined by quantitative reverse transcription polymerase chain reaction and Western blot in human pouch tissues and in a human intestinal epithelial cell line stimulated with inflammatory cytokines TNF-α, IL-1ß, and INF-γ. Results: Candidate miRNA expression and protein expression of DICER-1, EXPORTIN-5, and AGO-2 were increased in association with pouch inflammation. Similarly, inflammatory cytokines increased protein expression of DICER-1, EXPORTIN-5, and AGO-2 and DICER activity in the epithelial cell line. The miRNA-424 expression increased whereas SERT expression decreased in the patients' mucosa. Similarly, incubation of the epithelial cell line with inflammatory cytokines resulted in increased miRNA-424 and decreased SERT mRNA and protein expression. Conclusions: The miRNA expression and processing are augmented in the inflamed intestinal mucosa of patients with pouchitis. These alterations are accompanied by increased expression of proteins involved in miRNA processing, suggesting that pouch inflammation contributes to miRNA processing and expression.