BackgroundThe 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 ResultsUnchallenged 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.ConclusionGut 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.

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.

The concept of heterogeneity among obese individuals in their risk for developing metabolic dysfunction and associated complications has been recognized for decades. At the origin of the heterogeneity idea is the acknowledgement that individuals with central obesity are more prone to developing type 2 diabetes and cardiovascular disease than those with peripheral obesity. There have been attempts to categorize subjects according to their metabolic health and degree of obesity giving rise to different obese and non-obese phenotypes that include metabolically unhealthy normal-weight (MUHNW), metabolically healthy obese (MHO), and metabolically unhealthy obese (MUO). Individuals belonging to the MHO phenotype are obese according to their body mass index although exhibiting fewer or none metabolic anomalies such as type 2 diabetes, dyslipidemia, hypertension, and/or unfavorable inflammatory and fribinolytic profiles. However, some authors claim that MHO is only transient in nature. Additionally, the phenotype categorization is controversial as it lacks standardized definitions possibly blurring the distinction between obesity phenotypes and confounding the associations with health outcomes. To add to the discussion, the factors underlying the origin or protection from metabolic deterioration and cardiometabolic risk for these subclasses are being intensely investigated and several hypotheses have been put forward. In the present review, we compare the different definitions of obesity phenotypes and present several possible factors underlying them (adipose tissue distribution and cellularity, contaminant accumulation on the adipose tissue, dysbiosis and metabolic endotoxemia imposing on to the endocannabinoid tone and inflammasome, and nutrient intake and dietary patterns) having inflammatory activation at the center.

Autologous fat grafting is widely used for soft-tissue augmentation and replacement in reconstructive and aesthetic surgery providing a biocompatible, natural and inexpensive method. Multiple approaches have been developed in the past years, varying in the location of adipose tissue donor-sites, use of wetting solutions, harvesting, processing and placing techniques. Despite many advances in this subject, the lack of standardization in the protocols and the unpredictability of the resorption of the grafted tissue pose a significant limitation for graft retention and subsequent filling. In this review, we discuss several approaches and methods described over the last years concerning the harvesting of autologous fat grafts. We focus on contents such as the best donor-site, differences between existing harvesting techniques (namely tissue resection, hand aspiration or liposuction techniques), recommended harvesting cannula diameters, pressure application and volume of wetting solution injected prior aspiration. Results and comparisons between methods tend to vary according to the outcome measured, thus posing a limitation to pinpoint the most efficient methods to apply in fat grafting. Additionally, the lack of a standard assay to determine viability or volume augmentation of fat grafting remains another limitation to obtain universally accepted grafting procedures and protocols.

The interaction between the mammalian host and its resident gut microbiota is known to license adaptive immune responses. Nutritional constituents strongly influence composition and functional properties of the intestinal microbial communities. Here, we report that omission of a single essential amino acid - tryptophan – from the diet abrogates CNS autoimmunity in a mouse model of multiple sclerosis. Dietary tryptophan restriction results in impaired encephalitogenic T cell responses and is accompanied by a mild intestinal inflammatory response and a profound phenotypic shift of gut microbiota. Protective effects of dietary tryptophan restriction are abrogated in germ-free mice, but are independent of canonical host sensors of intracellular tryptophan metabolites. We conclude that dietary tryptophan restriction alters metabolic properties of gut microbiota, which in turn have an impact on encephalitogenic T cell responses. This link between gut microbiota, dietary tryptophan and adaptive immunity may help to develop therapeutic strategies for protection from autoimmune neuroinflammation.

Objective: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain–containing adapter-inducing interferon-β) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. Conclusions: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

In most organisms, heme biosynthesis is strictly controlled so as to avoid heme and heme precursor accumulation, which is toxic. Escherichia coli regulates heme biosynthesis by a feedback loop involving heme-induced proteolytic cleavage of HemA, glutamyl-tRNA reductase, which is the first enzyme in the heme biosynthetic pathway. We show here that heme homeostasis can be disrupted by overproduction of YfeX, a cytoplasmic protein that captures iron from heme that we named deferrochelatase. We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor). In both cases, we established that there is an increased PPIX concentration and we demonstrate that this compound is expelled by the MacAB-TolC pump, an efflux pump involved in E. coli and Salmonella for macrolide efflux. The E. coli macAB and tolC mutants accumulate PPIX and are sensitive to photo-inactivation. The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages. We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD) are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents.The aim of the present work was to evaluate the expression of feeding-related neuropeptides in hypothalamic progenitor cells and their capacity to differentiate to functional neurons which have been described to be affected by hypothalamic dysfunction.Our study shows that hypothalamic progenitor cells from rat embryos grow as floating neurospheres and express the feeding-related neuropeptides Neuropeptide Y (NPY), Agouti-related Protein (AGRP), Pro-OpioMelanocortin (POMC), Cocaine-and-Amphetamine Responsive Transcript (CART) and Orexin-A/Hypocretin-1. Moreover the relative mRNA expression of NPY and POMC increases during the expansion of hypothalamic neurospheres in proliferative conditions.Mature neurons were obtained from the differentiation of hypothalamic progenitor cells including NPY, AGRP, POMC, CART and Orexin-A positive neurons. Furthermore the relative mRNA expression of NPY, CART and Orexin-A increases after the differentiation of hypothalamic neurospheres. Similarly to the adult hypothalamic neurons the neurospheres-derived neurons express the glutamate transporter EAAT3. The orexigenic and anorexigenic phenotype of these neurons was identified by functional response to ghrelin and leptin hormones, respectively.This work demonstrates the presence of appetite-related neuropeptides in hypothalamic progenitor cells and neurons obtained from the differentiation of hypothalamic neurospheres, including the neuronal phenotypes that have been described by others as being affected by hypothalamic neurodegeneration. These in vitro models can be used to study hypothalamic progenitor cells aiming a therapeutic intervention to mitigate feeding dysfunction that are associated with hypothalamic neurodegeneration.