Estrobolome dysregulation is associated with altered immunometabolism in a mouse model of endometriosis.

Introduction: Endometriosis is a painful disease that affects around 5% of women of reproductive age. In endometriosis, ectopic endometrial cells or seeded endometrial debris grow in abnormal locations including the peritoneal cavity. Common manifestations of endometriosis include dyspareunia, dysmenorrhea, chronic pelvic pain and often infertility and symptomatic relief or surgical removal are mainstays of treatment. Endometriosis both promotes and responds to estrogen imbalance, leading to intestinal bacterial estrobolome dysregulation and a subsequent induction of inflammation. Methods: In the current study, we investigated the linkage between gut dysbiosis and immune metabolic response in endometriotic mice. Ovariectomized BALB/c mice received intraperitoneal transplantation of endometrial tissue from OVX donors (OVX+END). Control groups included naïve mice (Naïve), naïve mice that received endometrial transplants (Naive+END) and OVX mice that received the vehicle (OVX+VEH). Colonic content was collected 2 weeks post-transplantation for 16s rRNA pyrosequencing and peritoneal fluid was collected to determine the phenotype of inflammatory cells by flow cytometry. Results: We noted a significant increase in the number of peritoneal fluid cells, specifically, T cells, natural killer (NK) cells, and NKT cells in OVX+END mice. Phylogenetic taxonomy analysis showed significant dysbiosis in OVX+END mice, with an increase in abundance of Phylum Tenericutes, Class Mollicutes, Order Aneroplasmatales, and Genus Aneroplasma, and a decrease in Order Clostridiales, and Genus Dehalobacterium, when compared to OVX+VEH controls. The metabolomic profile showed an increase in some tricarboxylic acid cycle (TCA)-related metabolites accompanied by a reduction in short-chain fatty acids (SCFA) such as butyric acid in OVX+END mice. Additionally, the mitochondrial and ATP production of immune cells was enforced to a maximal rate in OVX+END mice when compared to OVX+VEH mice. Conclusion: The current study demonstrates that endometriosis alters the gut microbiota and associated immune metabolism.

The relationship between OXT gene polymorphisms and reproductive hormones in pregnant and lactating Awassi Ewes.

BACKGROUND: Numerous genetic loci interact intricately to control reproduction in mammals. The oxytocin gene (OXT) is a promising candidate for reproductive traits in mammals. Previously, sheep and goats have been studied for the presence of the OXT polymorphism. As of yet, no polymorphisms have been identified in the OXT gene of Awassi sheep. Thus, this study was conducted to determine the effects of OXT polymorphism and litter size on reproductive hormones in pregnant and lactating Awassi ewes. METHODS AND RESULTS: This study evaluated 232 ewes aged 3 and 4 years (123 single-progeny ewes and 109 twin-producing ewes). Serum was collected to measure reproductive hormones using ELISA kits manufactured by ELK Biotechnology. DNA was extracted from sheep blood for genotyping and sequencing to identify variations in OXT gene (exon 2, 266 bp). Genotyping analysis revealed three genotypes within 266 bp: CC, CA, and AA. Sequence analysis revealed a novel mutation in exon 2: 188 C > A. Statistical analysis showed significant associations between the 188 C > A SNP and phenotypic traits. Twin-pregnant ewes carrying CC genotypes had higher estrogen, progesterone, and follicle-stimulating hormone/luteinizing hormone levels (65.86 ± 3.87) (pg/mL), (6.51 ± 0.39) (ng/mL), and (20.22 ± 1.27) (ng/mL)/( 23.37 ± 2.14) (ng/mL) respectively, compared to CA and AA genotypes in the fourth month of twin-pregnant ewes compared to single-pregnant ewes. CONCLUSIONS: This study found that the 188 C > A SNP negatively affected reproductive hormone levels in Awassi sheep. These findings provide breeders with a new insight into the sheep OXT gene, useful for future breeding.

Corrigendum: Resveratrol attenuates allergic asthma and associated inflammation in the lungs through regulation of miRNA-34a that targets FoxP3 in mice.

[This corrects the article DOI: 10.3389/fimmu.2018.02992.].

Resveratrol attenuates staphylococcal enterotoxin B-activated immune cell metabolism via upregulation of miR-100 and suppression of mTOR signaling pathway.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of insults, such as bacterial and viral infections, including SARS-CoV-2, leading to high mortality. In the murine model of ARDS induced by Staphylococcal enterotoxin-B (SEB), our previous studies showed that while SEB triggered 100% mortality, treatment with Resveratrol (RES) completely prevented such mortality by attenuating inflammation in the lungs. In the current study, we investigated the metabolic profile of SEB-activated immune cells in the lungs following treatment with RES. RES-treated mice had higher expression of miR-100 in the lung mononuclear cells (MNCs), which targeted mTOR, leading to its decreased expression. Also, Single-cell RNA-seq (scRNA seq) unveiled the decreased expression of mTOR in a variety of immune cells in the lungs. There was also an increase in glycolytic and mitochondrial respiration in the cells from SEB + VEH group in comparison with SEB + RES group. Together these data suggested that RES alters the metabolic reprogramming of SEB-activated immune cells, through suppression of mTOR activation and its down- and upstream effects on energy metabolism. Also, miR-100 could serve as novel potential therapeutic molecule in the amelioration of ARDS.

A novel deleterious oxytocin variant is associated with the lower twinning ratio in Awassi ewes.

This study aimed to assess the possible association of oxytocin (OXT) gene with reproductive traits in two groups of Awassi ewes that differ in their reproductive potentials. Sheep were genotyped using PCR-single-stranded conformation polymorphism approach. Three genotypes were detected in exon 2, CC, CA, and AA, and a novel SNP was identified with a missense effect on oxytocin (c.188C > A → p.Arg55Leu). A significant (p < 0.01) association of p.Arg55Leu with the twinning rate was found as ewes with AA and CA genotypes exhibited, respectively a lower twinning ratio than those with the wild-type CC genotype. The deleterious impact of p.Arg55Leu was demonstrated by all in silico tools that were utilized to assess the effect of this variant on the structure, function, and stability of oxytocin. Molecular docking showed that p.Arg55Leu caused a dramatic alteration in the binding of oxytocin with its receptor and reduced the number of interacted amino acids between them. Our study suggests that ewes with AA and CA genotypes showed a lower reproductive performance due to the presence of p.Arg55Leu, which caused damaging impacts on oxytocin and is binding with the OXT receptor. The utilization of the p.Arg55Leu could be useful for improving Awassi reproductive potential.

The Ability of Resveratrol to Attenuate Ovalbumin-Mediated Allergic Asthma Is Associated With Changes in Microbiota Involving the Gut-Lung Axis, Enhanced Barrier Function and Decreased Inflammation in the Lungs.

Asthma is a chronic respiratory disease highly prevalent worldwide. Recent studies have suggested a role for microbiome-associated gut-lung axis in asthma development. In the current study, we investigated if Resveratrol (RES), a plant-based polyphenol, can attenuate ovalbumin (OVA)-induced murine allergic asthma, and if so, the role of microbiome in the gut-lung axis in this process. We found that RES attenuated allergic asthma with significant improvements in pulmonary functions in OVA-exposed mice when tested using plethysmography for frequency (F), mean volume (MV), specific airway resistance (sRaw), and delay time(dT). RES treatment also suppressed inflammatory cytokines in the lungs. RES modulated lung microbiota and caused an abundance of Akkermansia muciniphila accompanied by a reduction of LPS biosynthesis in OVA-treated mice. Furthermore, RES also altered gut microbiota and induced enrichment of Bacteroides acidifaciens significantly in the colon accompanied by an increase in butyric acid concentration in the colonic contents from OVA-treated mice. Additionally, RES caused significant increases in tight junction proteins and decreased mucin (Muc5ac) in the pulmonary epithelium of OVA-treated mice. Our results demonstrated that RES may attenuate asthma by inducing beneficial microbiota in the gut-lung axis and through the promotion of normal barrier functions of the lung.

The Ability of AhR Ligands to Attenuate Delayed Type Hypersensitivity Reaction Is Associated With Alterations in the Gut Microbiota.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates T cell function. The aim of this study was to investigate the effects of AhR ligands, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), and 6-Formylindolo[3,2-b]carbazole (FICZ), on gut-associated microbiota and T cell responses during delayed-type hypersensitivity (DTH) reaction induced by methylated bovine serum albumin (mBSA) in a mouse model. Mice with DTH showed significant changes in gut microbiota including an increased abundance of Bacteroidetes and decreased Firmicutes at the phylum level. Also, there was a decrease in Clostridium cluster XIV and IV, which promote anti-inflammatory responses, and an increase in Prevotella copri that facilitates pro-inflammatory responses. Interestingly, treatment of mice with TCDD attenuated the DTH response, induced Tregs, suppressed Th17 cells in the mesenteric lymph nodes (MLNs), and reversed the gut microbiota composition toward normalcy. In contrast, FICZ exacerbated the DTH response, induced heightened Th17 cells, and failed to cause a major shift in gut microbiota. Furthermore, TCDD but not FICZ caused an increase in the levels of short-chain fatty acids (SCFA), n-butyric acid, and acetic acid. Administration of sodium butyrate into mice with DTH suppressed the response, increased Tregs, and reduced Th17 cells IL17. Butyrate also caused an increase in the abundance of Clostridium and a decrease in Prevotella. Lastly, TCDD, as well as butyrate but not FICZ, were able to inhibit proinflammatory Histone deacetylases (HDACs) class I and II. Together, our data suggest that AhR ligands, such as TCDD that suppress DTH response, may mediate this effect by reversing the gut dysbiosis induced during this inflammatory response, while FICZ may fail to suppress the DTH response because of its inability to overturn the dysbiosis.

The Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome by Downregulating miRNA that Target Inflammatory Pathways.

Acute respiratory distress syndrome (ARDS) is defined as a type of respiratory failure that is caused by a variety of insults such as pneumonia, sepsis, trauma and certain viral infections. In this study, we investigated the effect of an endocannabinoid, anandamide (AEA), on ARDS induced in the mouse by Staphylococcus Enterotoxin B (SEB). Administration of a single intranasal dose of SEB in mice and treated with exogenous AEA at a dose of 40 mg/kg body weight led to the amelioration of ARDS in mice. Clinically, plethysmography results indicated that there was an improvement in lung function after AEA treatment accompanied by a decrease of inflammatory cell infiltrate. There was also a significant decrease in pro-inflammatory cytokines IL-2, TNF-α, and IFN-γ, and immune cells including CD4+ T cells, CD8+ T cells, Vß8+ T cells, and NK+ T cells in the lungs. Concurrently, an increase in anti-inflammatory phenotypes such as CD11b + Gr1+ Myeloid-derived Suppressor Cells (MDSCs), CD4 + FOXP3 + Tregs, and CD4+IL10 + cells was observed in the lungs. Microarray data showed that AEA treatment in ARDS mice significantly altered numerous miRNA including downregulation of miRNA-23a-3p, which caused an upregulation of arginase (ARG1), which encodes for arginase, a marker for MDSCs, as well as TGF-ß2, which induces Tregs. AEA also caused down-regulation of miRNA-34a-5p which led to induction of FoxP3, a master regulator of Tregs. Transfection of T cells using miRNA-23a-3p or miRNA-34a-5p mimics and inhibitors confirmed that these miRNAs targeted ARG1, TGFß2 and FoxP3. In conclusion, the data obtained from this study suggests that endocannabinoids such as AEA can attenuate ARDS induced by SEB by suppressing inflammation through down-regulation of key miRNA that regulate immunosuppressive pathways involving the induction of MDSCs and Tregs.

Resveratrol-mediated attenuation of superantigen-driven acute respiratory distress syndrome is mediated by microbiota in the lungs and gut.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of agents, including Staphylococcal Enterotoxin B (SEB). Interestingly, a significant proportion of patients with COVID-19, also develop ARDS. In the absence of effective treatments, ARDS results in almost 40% mortality. Previous studies from our laboratory demonstrated that resveratrol (RES), a stilbenoid, with potent anti-inflammatory properties can attenuate SEB-induced ARDS. In the current study, we investigated the role of RES-induced alterations in the gut and lung microbiota in the regulation of ARDS. Our studies revealed that SEB administration induced inflammatory cytokines, ARDS, and 100% mortality in C3H/HeJ mice. Additionally, SEB caused a significant increase in pathogenic Proteobacteria phylum and Propionibacterium acnes species in the lungs. In contrast, RES treatment attenuated SEB-mediated ARDS and mortality in mice, and significantly increased probiotic Actinobacteria phylum, Tenericutes phylum, and Lactobacillus reuteri species in both the colon and lungs. Colonic Microbiota Transplantation (CMT) from SEB-injected mice that were treated with RES as well as the transfer of L. reuteri into recipient mice inhibited the production of SEB-mediated induction of pro-inflammatory cytokines such as IFN-γ and IL-17 but increased that of anti-inflammatory IL-10. Additionally, such CMT and L. reuteri recipient mice exposed to SEB, showed a decrease in lung-infiltrating mononuclear cells, cytotoxic CD8+ T cells, NKT cells, Th1 cells, and Th17 cells, but an increase in the population of regulatory T cells (Tregs) and Th3 cells, and increase in the survival of mice from SEB-mediated ARDS. Together, the current study demonstrates that ARDS induced by SEB triggers dysbiosis in the lungs and gut and that attenuation of ARDS by RES may be mediated, at least in part, by alterations in microbiota in the lungs and the gut, especially through the induction of beneficial bacteria such as L. reuteri.

AhR Activation Leads to Alterations in the Gut Microbiome with Consequent Effect on Induction of Myeloid Derived Suppressor Cells in a CXCR2-Dependent Manner.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent ligand for AhR and a known carcinogen. While AhR activation by TCDD leads to significant immunosuppression, how this translates into carcinogenic signal is unclear. Recently, we demonstrated that activation of AhR by TCDD in naïve C57BL6 mice leads to massive induction of myeloid derived-suppressor cells (MDSCs). In the current study, we investigated the role of the gut microbiota in TCDD-mediated MDSC induction. TCDD caused significant alterations in the gut microbiome, such as increases in Prevotella and Lactobacillus, while decreasing Sutterella and Bacteroides. Fecal transplants from TCDD-treated donor mice into antibiotic-treated mice induced MDSCs and increased regulatory T-cells (Tregs). Injecting TCDD directly into antibiotic-treated mice also induced MDSCs, although to a lesser extent. These data suggested that TCDD-induced dysbiosis plays a critical role in MDSC induction. Interestingly, treatment with TCDD led to induction of MDSCs in the colon and undetectable levels of cysteine. MDSCs suppressed T cell proliferation while reconstitution with cysteine restored this response. Lastly, blocking CXC chemokine receptor 2 (CXCR2) impeded TCDD-mediated MDSC induction. Our data demonstrate that AhR activation by TCDD triggers dysbiosis which, in turn, regulates, at least in part, induction of MDSCs.

Δ9-Tetrahydrocannabinol Prevents Mortality from Acute Respiratory Distress Syndrome through the Induction of Apoptosis in Immune Cells, Leading to Cytokine Storm Suppression.

Acute Respiratory Distress Syndrome (ARDS) causes up to 40% mortality in humans and is difficult to treat. ARDS is also one of the major triggers of mortality associated with coronavirus-induced disease (COVID-19). We used a mouse model of ARDS induced by Staphylococcal enterotoxin B (SEB), which triggers 100% mortality, to investigate the mechanisms through which Δ9-tetrahydrocannabinol (THC) attenuates ARDS. SEB was used to trigger ARDS in C3H mice. These mice were treated with THC and analyzed for survival, ARDS, cytokine storm, and metabolome. Additionally, cells isolated from the lungs were used to perform single-cell RNA sequencing and transcriptome analysis. A database analysis of human COVID-19 patients was also performed to compare the signaling pathways with SEB-mediated ARDS. The treatment of SEB-mediated ARDS mice with THC led to a 100% survival, decreased lung inflammation, and the suppression of cytokine storm. This was associated with immune cell apoptosis involving the mitochondrial pathway, as suggested by single-cell RNA sequencing. A transcriptomic analysis of immune cells from the lungs revealed an increase in mitochondrial respiratory chain enzymes following THC treatment. In addition, metabolomic analysis revealed elevated serum concentrations of amino acids, lysine, n-acetyl methionine, carnitine, and propionyl L-carnitine in THC-treated mice. THC caused the downregulation of miR-185, which correlated with an increase in the pro-apoptotic gene targets. Interestingly, the gene expression datasets from the bronchoalveolar lavage fluid (BALF) of human COVID-19 patients showed some similarities between cytokine and apoptotic genes with SEB-induced ARDS. Collectively, this study suggests that the activation of cannabinoid receptors may serve as a therapeutic modality to treat ARDS associated with COVID-19.

Protective effects of Δ9 -tetrahydrocannabinol against enterotoxin-induced acute respiratory distress syndrome are mediated by modulation of microbiota.

BACKGROUND AND PURPOSE: Staphylococcal enterotoxin-B (SEB) is one of the most potent bacterial superantigens that exerts profound toxic effects by inducing a cytokine storm. Inhaled SEB can cause acute respiratory distress syndrome (ARDS), which is often fatal and with no effective treatments. EXPERIMENTAL APPROACH: Efficacy of Δ9 -tetrahydrocannabinol (THC) was tested in a mouse model of SEB-mediated ARDS, in which lung inflammation, alterations in gut/lung microbiota and production of short-chain fatty acids (SCFAs) was measured. Gene dysregulation of lung epithelial cells was studied by transcriptome arrays. Faecal microbiota transplantation (FMT) was performed to confirm the role of microbiota in suppressing ARDS. KEY RESULTS: While SEB triggered ARDS and 100% mortality in mice, THC protected the mice from fatality. Pyrosequencing analysis revealed that THC caused significant and similar alterations in microbiota in the lungs and gut of mice exposed to SEB. THC significantly increased the abundance of beneficial bacterial species, Ruminococcus gnavus, but decreased pathogenic microbiota, Akkermansia muciniphila. FMT confirmed that THC-mediated reversal of microbial dysbiosis played crucial role in attenuation of SEB-mediated ARDS. THC treatment caused an increase in SCFA, of which propionic acid was found to inhibit the inflammatory response. Transcriptome array showed that THC up-regulated several genes like lysozyme1 and lysozyme2, ß-defensin-2, claudin, zonula-1, occludin-1, Mucin2 and Muc5b while down-regulating ß-defensin-1. CONCLUSION AND IMPLICATIONS: The study demonstrates for the first time that THC attenuates SEB-mediated ARDS and toxicity by altering the microbiota in the lungs and the gut as well as promoting antimicrobial and anti-inflammatory pathways.

Administration of Δ9-Tetrahydrocannabinol (THC) Post-Staphylococcal Enterotoxin B Exposure Protects Mice From Acute Respiratory Distress Syndrome and Toxicity.

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening complication that can ensue following Staphylococcus aureus infection. The enterotoxin produced by these bacteria (SEB) acts as a superantigen thereby activating a large proportion of T cells leading to cytokine storm and severe lung injury. Δ9Tetrahydrocannabinol (THC), a psychoactive ingredient found in Cannabis sativa, has been shown to act as a potent anti-inflammatory agent. In the current study, we investigated the effect of THC treatment on SEB-induced ARDS in mice. While exposure to SEB resulted in acute mortality, treatment with THC led to 100% survival of mice. THC treatment significantly suppressed the inflammatory cytokines, IFN-γ and TNF-α. Additionally, THC elevated the induction of regulatory T cells (Tregs) and their associated cytokines, IL-10 and TGF-ß. Moreover, THC caused induction of Myeloid-Derived Suppressor Cells (MDSCs). THC acted through CB2 receptor as pharmacological inhibitor of CB2 receptors blocked the anti-inflammatory effects. THC-treated mice showed significant alterations in the expression of miRNA (miRs) in the lung-infiltrated mononuclear cells (MNCs). Specifically, THC caused downregulation of let7a-5p which targeted SOCS1 and downregulation of miR-34-5p which caused increased expression of FoxP3, NOS1, and CSF1R. Together, these data suggested that THC-mediated alterations in miR expression in the lungs may play a critical role in the induction of immunosuppressive Tregs and MDSCs as well as suppression of cytokine storm leading to attenuation of SEB-mediated lung injury.

AhR Activation Leads to Massive Mobilization of Myeloid-Derived Suppressor Cells with Immunosuppressive Activity through Regulation of CXCR2 and MicroRNA miR-150-5p and miR-543-3p That Target Anti-Inflammatory Genes.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, is a potent ligand for aryl hydrocarbon receptor (AhR). In the current study, we made an exciting observation that naive C57BL/6 mice that were exposed i.p. to TCDD showed massive mobilization of myeloid-derived suppressor cells (MDSCs) in the peritoneal cavity. These MDSCs were highly immunosuppressive and attenuated Con A-induced hepatitis upon adoptive transfer. TCDD administration in naive mice also led to induction of several chemokines and cytokines in the peritoneal cavity and serum (CCL2, CCL3, CCL4, CCL11, CXCL1, CXCL2, CXCL5, CXCL9, G-CSF, GM-CSF, VEGF, and M-CSF) and chemokine receptors on MDSCs (CCR1, CCR5, and CXCR2). Treatment with CXCR2 or AhR antagonist in mice led to marked reduction in TCDD-induced MDSCs. TCDD-induced MDSCs had high mitochondrial respiration and glycolytic rate and exhibited differential microRNA (miRNA) expression profile. Specifically, there was significant downregulation of miR-150-5p and miR-543-3p. These two miRNAs targeted and enhanced anti-inflammatory and MDSC-regulatory genes, including IL-10, PIM1, ARG2, STAT3, CCL11 and its receptors CCR3 and CCR5 as well as CXCR2. The role of miRs in MDSC activation was confirmed by transfection studies. Together, the current study demonstrates that activation of AhR in naive mice triggers robust mobilization of MDSCs through induction of chemokines and their receptors and MDSC activation through regulation of miRNA expression. AhR ligands include diverse compounds from environmental toxicants, such as TCDD, that are carcinogenic to dietary indoles that are anti-inflammatory. Our studies provide new insights on how such ligands may regulate health and disease through induction of MDSCs.

Combination of cannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), mitigates experimental autoimmune encephalomyelitis (EAE) by altering the gut microbiome.

Currently, a combination of marijuana cannabinoids including delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) is used as a drug to treat muscle spasticity in patients with Multiple Sclerosis (MS). Because these cannabinoids can also suppress inflammation, it is unclear whether such patients benefit from suppression of neuroinflammation and if so, what is the mechanism through which cannabinoids act. In the currently study, we used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to study the role of gut microbiota in the attenuation of clinical signs of paralysis and inflammation caused by cannabinoids. THC + CBD treatment attenuated EAE and caused significant decrease in inflammatory cytokines such as IL-17 and IFN-γ while promoting the induction of anti-inflammatory cytokines such as IL-10 and TGF-ß. Use of 16S rRNA sequencing on bacterial DNA extracted from the gut revealed that EAE mice showed high abundance of mucin degrading bacterial species, such as Akkermansia muciniphila (A. muc), which was significantly reduced after THC + CBD treatment. Fecal Material Transfer (FMT) experiments confirmed that THC + CBD-mediated changes in the microbiome play a critical role in attenuating EAE. In silico computational metabolomics revealed that LPS biosynthesis, a key component in gram-negative bacteria such as A. muc, was found to be elevated in EAE mice which was confirmed by demonstrating higher levels of LPS in the brain, while treatment with THC + CBD reversed this trend. EAE mice treated with THC + CBD also had significantly higher levels of short chain fatty acids such as butyric, isovaleric, and valeric acids compared to naïve or disease controls. Collectively, our data suggest that cannabinoids may attenuate EAE and suppress neuroinflammation by preventing microbial dysbiosis seen during EAE and promoting healthy gut microbiota.

Environmental microcystin targets the microbiome and increases the risk of intestinal inflammatory pathology via NOX2 in underlying murine model of Nonalcoholic Fatty Liver Disease.

With increased climate change pressures likely to influence harmful algal blooms, exposure to microcystin, a known hepatotoxin and a byproduct of cyanobacterial blooms can be a risk factor for NAFLD associated comorbidities. Using both in vivo and in vitro experiments we show that microcystin exposure in NAFLD mice cause rapid alteration of gut microbiome, rise in bacterial genus known for mediating gut inflammation and lactate production. Changes in the microbiome were strongly associated with inflammatory pathology in the intestine, gut leaching, tight junction protein alterations and increased oxidative tyrosyl radicals. Increased lactate producing bacteria from the altered microbiome was associated with increased NOX-2, an NADPH oxidase isoform. Activationof NOX2 caused inflammasome activation as shown by NLRP3/ASCII and NLRP3/Casp-1 colocalizations in these cells while use of mice lacking a crucial NOX2 component attenuated inflammatory pathology and redox changes. Mechanistically, NOX2 mediated peroxynitrite species were primary to inflammasome activation and release of inflammatory mediators. Thus, in conclusion, microcystin exposure in NAFLD could significantly alter intestinal pathology especially by the effects on microbiome and resultant redox status thus advancing our understanding of the co-existence of NAFLD-linked inflammatory bowel disease phenotypes in the clinic.

Resveratrol protects mice against SEB-induced acute lung injury and mortality by miR-193a modulation that targets TGF-ß signalling.

Staphylococcal enterotoxin B (SEB) is a potent superantigen produced by Staphylococcus aureus that triggers a strong immune response, characterized by cytokine storm, multi-organ failure, and often death. When inhaled, SEB can cause acute lung injury (ALI) and respiratory failure. In this study, we investigated the effect of resveratrol (RES), a phytoallexin, on SEB-driven ALI and mortality in mice. We used a dual-exposure model of SEB in C3H/HeJ mice, which caused 100% mortality within the first 5 days of exposure, and treatment with RES resulted in 100% survival of these mice up to 10 days post-SEB exposure. RES reduced the inflammatory cytokines in the serum and lungs, as well as T cell infiltration into the lungs caused by SEB. Treatment with RES also caused increased production of transforming growth factor-beta (TGF-ß) in the blood and lungs. RES altered the miRNA profile in the immune cells isolated from the lungs. Of these, miR-193a was strongly induced by SEB and was down-regulated by RES treatment. Furthermore, transfection studies and pathway analyses revealed that miR-193a targeted several molecules involved in TGF-ß signalling (TGFß2, TGFßR3) and activation of apoptotic pathways death receptor-6 (DR6). Together, our studies suggest that RES can effectively neutralize SEB-mediated lung injury and mortality through potential regulation of miRNA that promote anti-inflammatory activities.

Resveratrol Attenuates Allergic Asthma and Associated Inflammation in the Lungs Through Regulation of miRNA-34a That Targets FoxP3 in Mice.

Asthma is a chronic inflammatory disease of airways mediated by T-helper 2 (Th2) cells involving complex signaling pathways. Although resveratrol has previously been shown to attenuate allergic asthma, the role of miRNA in this process has not been studied. We investigated the effect of resveratrol on ovalbumin-induced experimental allergic asthma in mice. To that end, BALB/c mice were immunized with ovalbumin (OVA) intraperitoneally followed by oral gavage of vehicle (OVA-veh) or resveratrol (100 mg/kg body) (OVA-res). On day 7, the experimental groups received intranasal challenge of OVA followed by 7 days of additional oral gavage of vehicle or resveratrol. At day 15, all mice were euthanized and bronchioalveolar fluid (BALF), serum and lung infiltrating cells were collected and analyzed. The data showed that resveratrol significantly reduced IL-5, IL-13, and TGF-ß in the serum and BALF in mice with OVA-induced asthma. Also, we saw a decrease in CD3+CD4+, CD3+CD8+, and CD4+IL-4+ cells with increase in CD4+CD25+FOXP3+ cells in pulmonary inflammatory cell infiltrate in OVA-res group when compared to OVA-veh. miRNA expression arrays using lung infiltrating cells showed that resveratrol caused significant alterations in miRNA expression, specifically downregulating the expression of miR-34a. Additionally, miR-34a was found to target FOXP3, as evidenced by enhanced expression of FOXP3 in the lung tissue. Also, transfection studies showed that miR-34a inhibitor upregulated FOXP3 expression while miR-34a-mimic downregulated FOXP3 expression. The current study suggests that resveratrol attenuates allergic asthma by downregulating miR-34a that induces increased expression of FOXP3, a master regulator of Treg development and functions.