RESUMEN
The symbiotic gut microbiota play pivotal roles in host physiology and the development of cardiovascular diseases, but the microbiota-triggered pattern recognition signaling mechanisms that impact thrombosis are poorly defined. In this article, we show that germ-free (GF) and Toll-like receptor-2 (Tlr2)-deficient mice have reduced thrombus growth after carotid artery injury relative to conventionally raised controls. GF Tlr2-/- and wild-type (WT) mice were indistinguishable, but colonization with microbiota restored a significant difference in thrombus growth between the genotypes. We identify reduced plasma levels of von Willebrand factor (VWF) and reduced VWF synthesis, specifically in hepatic endothelial cells, as a critical factor that is regulated by gut microbiota and determines thrombus growth in Tlr2-/- mice. Static platelet aggregate formation on extracellular matrix was similarly reduced in GF WT, Tlr2-/- , and heterozygous Vwf+/- mice that are all characterized by a modest reduction in plasma VWF levels. Defective platelet matrix interaction can be restored by exposure to WT plasma or to purified VWF depending on the VWF integrin binding site. Moreover, administration of VWF rescues defective thrombus growth in Tlr2-/- mice in vivo. These experiments delineate an unexpected pathway in which microbiota-triggered TLR2 signaling alters the synthesis of proadhesive VWF by the liver endothelium and favors platelet integrin-dependent thrombus growth.
Asunto(s)
Microbioma Gastrointestinal , Hígado/metabolismo , Transducción de Señal , Trombosis/metabolismo , Receptor Toll-Like 2/metabolismo , Factor de von Willebrand/biosíntesis , Animales , Plaquetas/metabolismo , Plaquetas/patología , Vida Libre de Gérmenes , Hígado/patología , Ratones , Ratones Noqueados , Agregación Plaquetaria/genética , Trombosis/genética , Trombosis/patología , Receptor Toll-Like 2/genética , Factor de von Willebrand/genéticaRESUMEN
BACKGROUND: The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown. METHODS AND RESULTS: Unchallenged germ-free mice (GF) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised (CONV-R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T-box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV-R mice, angiotensin II (AngII; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic-acid receptor-related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)-17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G(+) neutrophils and Ly6C(+) monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII. CONCLUSION: Gut microbiota facilitate AngII-induced vascular dysfunction and hypertension, at least in part, by supporting an MCP-1/IL-17 driven vascular immune cell infiltration and inflammation.
Asunto(s)
Angiotensina II/farmacología , Presión Arterial/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Vida Libre de Gérmenes , Leucocitos/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Animales , Aorta/citología , Aorta/efectos de los fármacos , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Quimiocina CCL2/efectos de los fármacos , Quimiocina CCL2/genética , Endotelio Vascular/efectos de los fármacos , Hipertensión/microbiología , Ratones , Monocitos , NADPH Oxidasa 2/efectos de los fármacos , NADPH Oxidasa 2/genética , Infiltración Neutrófila/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/efectos de los fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In our recent article Hörmann and coworkers have reported a role for epithelial cell-intrinsic TLR2 signaling for proliferation and renewal of the small intestinal epithelium. In this study, MyD88 and TRIF expression in the small intestine were affected by gut microbiota. Here, we report that in contrast to TLR2 and its co-receptor TLR1, TLR5 transcripts are not changed by presence of gut microbiota nor regulated through TLR2 or TLR4. Similar to TLR2 also TLR5 depends on MyD88 and TRIF adaptors. Our results indicate that TLR adaptor molecules could be determinants of TLR expression in the small intestine.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Microbioma Gastrointestinal/inmunología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Microbiota/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 5/biosíntesis , Expresión Génica , Humanos , Modelos BiológicosRESUMEN
The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs). Here, we report that colonization of germ-free (GF) Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R) showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.
Asunto(s)
Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Apoptosis , Caspasa 3/metabolismo , Línea Celular , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Escherichia coli/fisiología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Intestino Delgado/microbiología , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Lipopéptidos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/genética , Receptor Toll-Like 6/genética , Receptor Toll-Like 6/metabolismo , Transcriptoma/efectos de los fármacos , Regulación hacia ArribaRESUMEN
BACKGROUND: Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study. METHODS: This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers. RESULTS: In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies. CONCLUSION: We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function.