Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium

Background 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. Results 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. Conclusions 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

Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

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 p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox 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

Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection

An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.Fundação de Amparo à Pesquisa do Estado de São Paulo/[07/03336-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[02/13863-2]/FAPESP/BrasilUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages

The snake venom myotoxin (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.Fundación de Apoyo a la Investigación del Estado de Sao Paulo/[07/03336-9]/FAPESP/BrasilConsejo Nacional de Desarrollo Científico y Tecnológico/[306099/2008-0]/CNPq/BrasilFundación de Apoyo a la Investigación del Estado de Sao Paulo/[07/03337-5]/FAPESP/BrasilConsejo Nacional de Desarrollo Científico y Tecnológico/[202077/2008-0]/CNPq/BrasilFundación de Apoyo a la Investigación del Estado de Sao Paulo/[12/10653-9]/FAPESP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages

The effects of a snakevenom Lys-49phospholipaseA2 (PLA2) homolog named MT-II,devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2(COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages wit hMT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca2+ independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2induced by MT-II. Inhibition of nuclear factor kB (NF-kB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either proteinkinase C (PKC), proteintyrosinekinase (PTK),or extracellular signal-regulated kinase(ERK) pathways abrogated MT-II-induced NF-kB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibit or reduced MT-II-induced COX-2 expression and PGD2 production.Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-kB activation,but affected neither prostaglandins production nor COX-2expression.MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively,our data demonstrate that a catalytically-inactivePLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2expression in macrophages in both in vitro and in vivo models,indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-kB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.Fundação de Amparo à Pesquisa do Estado de São Paulo/[02/13863-2]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[07/03336-9]/FAPESP/BrasilInstituto Nacional de Ciência e Tecnologia em Toxinas/[2008/57898-0]/INCT-TOX/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico/[306099/2008-0]/CNPq/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[07/03337-5]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[06/58341-4]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[10/07630-1]/FAPESP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

DataSheet1_Reduced intestinal butyrate availability is associated with the vascular remodeling in resistance arteries of hypertensive rats.docx

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.</p

The results, expressed as the means with standard deviations, for the expression levels of PPAR-γ and C/EBPα in MSCs from control (n = 6) and malnourished (n = 6) animals as determined by Western blot analysis.

<p>The Western blot image was representative of six independent experiments with similar results. After densitometric analysis, results for PPAR-γ and C/EBPα protein expression were normalized to β-actin values. The number in brackets denotes the number of animals used in the experiment. *(p ≤ 0.05) indicates where there was a significant difference between the control group and the malnourished group.</p

Flow cytometry analyses of the bone marrow of control and malnourished mice.

<p>The selection of gates, R1, for the cell populations that were not labeled with fluorochromes are indicated (SSC in the y-axis vs. FSC in the x-axis). Immunophenotypical bone marrow analysis was carried out in gate R1 for CD117 and CD45 expression. The graphical results are expressed as the means with standard deviations for CD117⁺ and CD45⁺ expression by control (n = 6) and malnourished (n = 6) mice. The number in brackets denotes the number of animals used in the experiment. *(p ≤ 0.05) and ***(p ≤ 0.001) indicate where there was a significant difference between the control group and the malnourished group.</p