RESUMO
Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.
Assuntos
Colite/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mucosa Intestinal/metabolismo , Microbiota/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Colite/genética , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/genética , Homeostase , Humanos , Imunidade Inata , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/imunologia , Queratinócitos/imunologia , Transdução de Sinais/imunologia , Animais , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Epiderme/patologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/imunologia , Transdução de Sinais/genéticaRESUMO
Pulmonary diseases are common in horses and have a major economic impact on the equine industry. Some of them could be associated with an inadequate immune response in the lung, but methods to evaluate this response in horses are lacking. The aim of this study was to develop and validate an experimental model that could be applied in several physiological and pathological conditions to assess the innate immune response of equine pulmonary cells. Equine alveolar macrophages (AMs) obtained from bronchoalveolar lavages were isolated from other cells by adhesion. TLR2, 3, and 4 expression in AMs was studied and their responses to commercial ligands (respectively FSL-1, Poly(I:C), and LPS) were evaluated after determination of the appropriate dose and time of incubation. TLR responses were assessed by measuring cytokine production using (1) gene expression of TNFα, IFNß, Il-1ß, and IFNα by qPCR (indirect method); and (2) cytokine production for TNFα and IFNß by ELISA (direct method). TLR 2, 3, and 4 were expressed by AMs. TLR 2 stimulation with 10 ng/mL of FSL-1 during 3h significantly increased IL-1ß and TNFα gene expression. TLR 3 stimulation with 1000 ng/mL of Poly(I:C) during 1h increased IFNß, IFNα, Il-1ß and TNFα expression. TLR 4 stimulation with 100 ng/mL of LPS during 3h increased TNFα, IFNß, and Il-1ß expression. Results obtained by ELISA quantification of TNFα and IFNß produced by AMs following stimulation during 6h were similar: FSL-1 increased TNFα production but not IFNß, Poly(I:C) and LPS increased production of IFNß and TNFα. In conclusion, pulmonary innate immunity of horses can be assessed ex vivo by measuring cytokine production following stimulation of AMs with TLR agonists. This experimental model could be applied under several conditions especially to improve the understanding of equine respiratory disease pathogenesis, and to suggest novel therapeutic opportunities.