The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We ...characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.
The snake venom miotoxin (MT)-III is a group IIA secreted phospholipase A2 (sPLA2) with pro-inflammatory activities. Previous studies have demonstrated that MT-III has the ability to stimulate ...macrophages to release inflammatory lipid mediators derived from arachidonic acid metabolism. Among them, we highlight prostaglandin (PG)E2 produced by the cyclooxygenase (COX)-2 pathway, through activation of nuclear factor (NF)-κB. However, the mechanisms coordinating this process are not fully understood. This study investigates the regulatory mechanisms exerted by other groups of bioactive eicosanoids derived from 12-lipoxygenase (12-LO), in particular 12-hydroxyeicosatetraenoic (12-HETE), on group IIA sPLA2-induced (i) PGE2 release, (ii) COX-2 expression, and (iii) activation of signaling pathways p38 mitogen-activated protein kinases(p38MAPK), protein C kinase (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB. Stimulation of macrophages with group IIA sPLA2 resulted in release of 12-HETE without modification of 12-LO protein levels. Pre-treatment of these cells with baicalein, a 12-LO inhibitor, decreased the sPLA2-induced PGE2 production, significantly reduced COX-2 expression, and inhibited sPLA2-induced ERK; however, it did not affect p38MAPK or PKC phosphorylation. In turn, sPLA2-induced PGE2 release and COX-2 expression, but not NF-κB activation, was attenuated by pre-treating macrophages with PD98059 an inhibitor of ERK1/2. These results suggest that, in macrophages, group IIA sPLA2-induced PGE2 release and COX-2 protein expression are distinctly mediated through 12-HETE followed by ERK1/2 pathway activation, independently of NF-κB activation. These findings highlight an as yet undescribed mechanism by which 12-HETE regulates one of the distinct signaling pathways for snake venom group IIA sPLA2-induced PGE2 release and COX-2 expression in macrophages.
•A group IIA sPLA2 isolated from snake venom activates 12-LO pathway inducing 12-HETE biosynthesis in macrophages.•12-HETE regulates group IIA sPLA2-induced COX-2 protein expression and PGE2 release.•12-HETE produced by group IIA sPLA2 amplifies inflammatory response through activation of ERK1/2 pathway.
Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to ...hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states.
The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in ...extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.
The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male ...mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNFα and IL-1β by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNFα production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.
How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a ...poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue.
Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum.
Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.
During hypertension an unbalance of short-chain fatty acids (SCFAs) production by intestinal bacteria is described. However, no data evaluate the association of SCFAs and vascular remodeling in ...hypertension, which is an important hallmark of this disease. Thus, the present study aims to evaluate the correlations between SCFAs availability and the resistance arteries remodeling in hypertension, as well as to identify the possible pathway by which the SCFAs could exert a structural and mechanical influence. Hence, male spontaneously hypertensive rats (SHR) and normotensive Wistar rats had blood pressure measured by tail-cuff plethysmography; fecal SCFAs content assessed by gas chromatography; gene expression of SCFAs-transporters in gut epithelium and SCFAs-sensing receptors on mesenteric resistance arteries (MRA) quantified by PCR; and MRA structural and mechanical parameters analyzed by pressure myograph. Reduced butyrate fecal content was found in SHR, with no changes in propionate and acetate, as well as decreased mRNA levels of SCFAs-transporters (MCT1, MCT4, and SMCT1) in the intestinal epithelium. In addition, lower gene expression of SCFAs-sensing receptors (GPR41, GPR43, and GPR109a, but not Olfr78) was identified in MRAs of SHR, which also shows inward eutrophic remodeling with stiffness. Butyrate content presented a negative correlation with systolic blood pressure and with the structural alterations found on MRAs, while a positive correlation between butyrate content and mechanical parameters was detected. Altogether the present study suggests that lower butyrate content due to ineffective SCFA bioavailability, associated with lower SCFAs-sensing receptors expression, could favor MRA remodeling, increasing peripheral vascular resistance and worsening hypertension prognosis.
We have previously demonstrated that reduced graphene oxide (rGO) administered intravenously in rats was detected inside the hippocampus after downregulation of the tight and adherens junction ...proteins of the blood-brain barrier. While down-regulators of junctional proteins could be useful tools for drug delivery through the paracellular pathway, concerns over toxicity must be investigated before clinical application. Herein, our purpose was to trace whether the rGO inside the hippocampus triggered toxic alterations in this brain region and in target organs (blood, liver and kidney) of rats at various time points (15 min, 1, 3 h and 7 days).
The assessed rGO-treated rats (7 mg/kg) were clinically indistinguishable from controls at all the time points. Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO single injection seemed to produce minimal toxicological effects at the time points assessed. Relative to control, the only change was a decrease in the blood urea nitrogen level 3 h post-treatment and increases in superoxide dismutase activity 1 h and 7 days post-treatment. While no alteration in leukocyte parameters was detected between control and rGO-treated animals, time-dependent leukocytosis (rGO-1 h versus rGO-3 h) and leukopenia (rGO-3 h versus rGO-7 days) was observed intra-treated groups. Nevertheless, no inflammatory response was induced in serum and hippocampus at any time.
The toxic effects seemed to be peripheral and transitory in the short-term analysis after systemic administration of rGO. The effects were self-limited and non-significant even at 7 days post-rGO administration.
Antibiotic-induced dysbiosis is a key predisposing factor for Clostridium difficile infections (CDIs), which cause intestinal disease ranging from mild diarrhea to pseudomembranous colitis. Here, we ...examined the impact of a microbiota-derived metabolite, short-chain fatty acid acetate, on an acute mouse model of CDI. We found that administration of acetate is remarkably beneficial in ameliorating disease. Mechanistically, we show that acetate enhances innate immune responses by acting on both neutrophils and ILC3s through its cognate receptor free fatty acid receptor 2 (FFAR2). In neutrophils, acetate-FFAR2 signaling accelerates their recruitment to the inflammatory sites, facilitates inflammasome activation, and promotes the release of IL-1β; in ILC3s, acetate-FFAR2 augments expression of the IL-1 receptor, which boosts IL-22 secretion in response to IL-1β. We conclude that microbiota-derived acetate promotes host innate responses to C. difficile through coordinate action on neutrophils and ILC3s.
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