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.].

Prior exposure to microcystin alters host gut resistome and is associated with dysregulated immune homeostasis in translatable mouse models.

A strong association between exposure to the common harmful algal bloom toxin microcystin and the altered host gut microbiome has been shown. We tested the hypothesis that prior exposure to the cyanotoxin microcystin-LR may alter the host resistome. We show that the mice exposed to microcystin-LR had an altered microbiome signature that harbored antibiotic resistance genes. Host resistome genotypes such as mefA, msrD, mel, ant6, and tet40 increased in diversity and relative abundance following microcystin-LR exposure. Interestingly, the increased abundance of these genes was traced to resistance to common antibiotics such as tetracycline, macrolides, glycopeptide, and aminoglycosides, crucial for modern-day treatment of several diseases. Increased abundance of these genes was positively associated with increased expression of PD1, a T-cell homeostasis marker, and pleiotropic inflammatory cytokine IL-6 with a concomitant negative association with immunosurveillance markers IL-7 and TLR2. Microcystin-LR exposure also caused decreased TLR2, TLR4, and REG3G expressions, increased immunosenescence, and higher systemic levels of IL-6 in both wild-type and humanized mice. In conclusion, the results show a first-ever characterization of the host resistome following microcystin-LR exposure and its connection to host immune status and antimicrobial resistance that can be crucial to understand treatment options with antibiotics in microcystin-exposed subjects in clinical settings.

Host gut resistome in Gulf War chronic multisymptom illness correlates with persistent inflammation.

Chronic multisymptom illness (CMI) affects a subsection of elderly and war Veterans and is associated with systemic inflammation. Here, using a mouse model of CMI and a group of Gulf War (GW) Veterans' with CMI we show the presence of an altered host resistome. Results show that antibiotic resistance genes (ARGs) are significantly altered in the CMI group in both mice and GW Veterans when compared to control. Fecal samples from GW Veterans with persistent CMI show a significant increase of resistance to a wide class of antibiotics and exhibited an array of mobile genetic elements (MGEs) distinct from normal healthy controls. The altered resistome and gene signature is correlated with mouse serum IL-6 levels. Altered resistome in mice also is correlated strongly with intestinal inflammation, decreased synaptic plasticity, reversible with fecal microbiota transplant (FMT). The results reported might help in understanding the risks to treating hospital acquired infections in this population.

Andrographolide Attenuates Gut-Brain-Axis Associated Pathology in Gulf War Illness by Modulating Bacteriome-Virome Associated Inflammation and Microglia-Neuron Proinflammatory Crosstalk.

Gulf War Illness (GWI) is a chronic multi-symptomatic illness that is associated with fatigue, pain, cognitive deficits, and gastrointestinal disturbances and presents a significant challenge to treat in clinics. Our previous studies show a role of an altered Gut-Brain axis pathology in disease development and symptom persistence in GWI. The present study utilizes a mouse model of GWI to study the role of a labdane diterpenoid andrographolide (AG) to attenuate the Gut-Brain axis-linked pathology. Results showed that AG treatment in mice (100 mg/kg) via oral gavage restored bacteriome alterations, significantly increased probiotic bacteria Akkermansia, Lachnospiraceae, and Bifidobacterium, the genera that are known to aid in preserving gut and immune health. AG also corrected an altered virome with significant decreases in virome families Siphoviridae and Myoviridae known to be associated with gastrointestinal pathology. AG treatment significantly restored tight junction proteins that correlated well with decreased intestinal proinflammatory mediators IL-1ß and IL-6 release. AG treatment could restore Claudin-5 levels, crucial for maintaining the BBB integrity. Notably, AG could decrease microglial activation and increase neurotrophic factor BDNF, the key to neurogenesis. Mechanistically, microglial conditioned medium generated from IL-6 stimulation with or without AG in a concentration similar to circulating levels found in the GWI mouse model and co-incubated with neuronal cells in vitro, decreased Tau phosphorylation and neuronal apoptosis. In conclusion, we show that AG treatment mitigated the Gut-Brain-Axis associated pathology in GWI and may be considered as a potential therapeutic avenue for the much-needed bench to bedside strategies in GWI.

Environmental Microcystin exposure in underlying NAFLD-induced exacerbation of neuroinflammation, blood-brain barrier dysfunction, and neurodegeneration are NLRP3 and S100B dependent.

Nonalcoholic fatty liver disease (NAFLD) has been shown to be associated with extrahepatic comorbidities including neuronal inflammation and Alzheimer's-like pathology. Environmental and genetic factors also act as a second hit to modulate severity and are expected to enhance the NAFLD-linked neuropathology. We hypothezied that environmental microcystin-LR (MC-LR), a toxin produced by harmful algal blooms of cyanobacteria, exacerbates the neuroinflammation and degeneration of neurons associated with NAFLD. Using a mouse model of NAFLD, exposed to MC-LR subsequent to the onset of fatty liver, we show that the cyanotoxin could significantly increase proinflammatory cytokine expression in the frontal cortex and cause increased expression of Lcn2 and HMGB1. The above effects were NLRP3 inflammasome activation-dependent since the use of NLRP3 knockout mice abrogated the increase in inflammation. NLRP3 was also responsible for decreased expression of the blood-brain barrier (BBB) tight junction proteins Occludin and Claudin 5 suggesting BBB dysfunction was parallel to neuroinflammation following microcystin exposure. An increased circulatory S100B release, a hallmark of astrocyte activation in MC-LR exposed NAFLD mice also confirmed BBB integrity loss, but the astrocyte activation observed in vivo was NLRP3 independent suggesting an important role of a secondary S100B mediated crosstalk. Mechanistically, conditioned medium from reactive astrocytes and parallel S100B incubation in neuronal cells caused increased inducible NOS, COX-2, and higher BAX/ Bcl2 protein expression suggesting oxidative stress-mediated neuronal cell apoptosis crucial for neurodegeneration. Taken together, MC-LR exacerbated neuronal NAFLD-linked comorbidities leading to cortical inflammation, BBB dysfunction, and neuronal apoptosis.

Host gut microbiome and potential therapeutics in Gulf War Illness: A short review.

AIMS: Since our troops had returned from the first Persian Gulf War in 1990-91, the veterans have reported chronic multisymptomatic illness widely referred to as Gulf War Illness (GWI). We aim to review the current directions of GWI pathology research in the context of chronic multisymptomatic illness and its possible gut microbiome targeted therapies. The veterans of Gulf War show symptoms of chronic fatigue, cognitive deficits, and a subsection report of gastrointestinal complications. METHOD: Efforts of finding a suitable treatment regimen and clinical management remain a challenge. More recently, we have shown that the pathology is connected to alterations in the gut microbiome, and efforts of finding a suitable regimen for gut-directed therapeutics are underway. We discuss the various clinical interventions and summarize the possible effectiveness of gut-directed therapies such as the use of short-chain fatty acids (SCFA), phenolic compounds, and their metabolites, use of probiotics, and fecal microbiota transfer. SIGNIFICANCE: The short review will be helpful to GWI researchers to expand their studies to the gut and find an effective treatment strategy for chronic multisymptomatic illness.

Obesity Worsens Gulf War Illness Symptom Persistence Pathology by Linking Altered Gut Microbiome Species to Long-Term Gastrointestinal, Hepatic, and Neuronal Inflammation in a Mouse Model.

Persistence of Gulf War illness (GWI) pathology among deployed veterans is a clinical challenge even after almost three decades. Recent studies show a higher prevalence of obesity and metabolic disturbances among Gulf War veterans primarily due to the existence of post-traumatic stress disorder (PTSD), chronic fatigue, sedentary lifestyle, and consumption of a high-carbohydrate/high-fat diet. We test the hypothesis that obesity from a Western-style diet alters host gut microbial species and worsens gastrointestinal and neuroinflammatory symptom persistence. We used a 5 month Western diet feeding in mice that received prior Gulf War (GW) chemical exposure to mimic the home phase obese phenotype of the deployed GW veterans. The host microbial profile in the Western diet-fed GWI mice showed a significant decrease in butyrogenic and immune health-restoring bacteria. The altered microbiome was associated with increased levels of IL6 in the serum, Claudin-2, IL6, and IL1ß in the distal intestine with concurrent inflammatory lesions in the liver and hyperinsulinemia. Microbial dysbiosis was also associated with frontal cortex levels of increased IL6 and IL1ß, activated microglia, decreased levels of brain derived neurotrophic factor (BDNF), and higher accumulation of phosphorylated Tau, an indicator of neuroinflammation-led increased risk of cognitive deficiencies. Mechanistically, serum from Western diet-fed mice with GWI significantly increased microglial activation in transformed microglial cells, increased tyrosyl radicals, and secreted IL6. Collectively, the results suggest that an existing obese phenotype in GWI worsens persistent gastrointestinal and neuronal inflammation, which may contribute to poor outcomes in restoring cognitive function and resolving fatigue, leading to the deterioration of quality of life.

TLR Antagonism by Sparstolonin B Alters Microbial Signature and Modulates Gastrointestinal and Neuronal Inflammation in Gulf War Illness Preclinical Model.

The 1991 Persian Gulf War veterans presented a myriad of symptoms that ranged from chronic pain, fatigue, gastrointestinal disturbances, and cognitive deficits. Currently, no therapeutic regimen exists to treat the plethora of chronic symptoms though newer pharmacological targets such as microbiome have been identified recently. Toll-like receptor 4 (TLR4) antagonism in systemic inflammatory diseases have been tried before with limited success, but strategies with broad-spectrum TLR4 antagonists and their ability to modulate the host-microbiome have been elusive. Using a mouse model of Gulf War Illness, we show that a nutraceutical, derived from a Chinese herb Sparstolonin B (SsnB) presented a unique microbiome signature with an increased abundance of butyrogenic bacteria. SsnB administration restored a normal tight junction protein profile with an increase in Occludin and a parallel decrease in Claudin 2 and inflammatory mediators high mobility group box 1 (HMGB1), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in the distal intestine. SsnB also decreased neuronal inflammation by decreasing IL-1ß and HMGB1, while increasing brain-derived neurotrophic factor (BDNF), with a parallel decrease in astrocyte activation in vitro. Mechanistically, SsnB inhibited the binding of HMGB1 and myeloid differentiation primary response protein (MyD88) to TLR4 in the intestine, thus attenuating TLR4 downstream signaling. Studies also showed that SsnB was effective in suppressing TLR4-induced nod-like receptor protein 3 (NLRP3) inflammasome activation, a prominent inflammatory disease pathway. SsnB significantly decreased astrocyte activation by decreasing colocalization of glial fibrillary acid protein (GFAP) and S100 calcium-binding protein B (S100B), a crucial event in neuronal inflammation. Inactivation of SsnB by treating the parent molecule by acetate reversed the deactivation of NLRP3 inflammasome and astrocytes in vitro, suggesting that SsnB molecular motifs may be responsible for its anti-inflammatory activity.

Higher intestinal and circulatory lactate associated NOX2 activation leads to an ectopic fibrotic pathology following microcystin co-exposure in murine fatty liver disease.

Clinical studies implicated an increased risk of intestinal fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Our previous studies have shown that microcystin-LR (MC-LR) exposure led to altered gut microbiome and increased abundance of lactate producing bacteria and intestinal inflammation in underlying NAFLD. This led us to further investigate the effects of the MC-LR, a PP2A inhibitor in activating the TGF-ß fibrotic pathway in the intestines that might be mediated by increased lactate induced redox enzyme NOX2. Exposure to MC-LR led to higher lactate levels in circulation and in the intestinal content. The higher lactate levels were associated with NOX2 activation in vivo that led to increased Smad2/3-Smad4 co-localization and high alpha-smooth muscle actin (α-SMA) immunoreactivity in the intestines. Mechanistically, primary mouse intestinal epithelial cells treated with lactate and MC-LR separately led to higher NOX2 activation, phosphorylation of TGFßR1 receptor and subsequent Smad 2/3-Smad4 co-localization inhibitable by apocynin (NOX2 inhibitor), FBA (a peroxynitrite scavenger) and DMPO (a nitrone spin trap), catalase and superoxide dismutase. Inhibition of NOX2-induced redox signaling also showed a significant decrease in collagen protein thus suggesting a strong redox signaling induced activation of an ectopic fibrotic manifestation in the intestines. In conclusion, the present study provides mechanistic insight into the role of microcystin in dysbiosis-linked lactate production and subsequently advances our knowledge in lactate-induced NOX2 exacerbation of the cell differentiation and fibrosis in the NAFLD intestines.

Host Akkermansia muciniphila Abundance Correlates With Gulf War Illness Symptom Persistence via NLRP3-Mediated Neuroinflammation and Decreased Brain-Derived Neurotrophic Factor.

Neurological disorders are commonly reported among veterans who returned from the Gulf war. Veterans who suffer from Gulf War illness (GWI) complain of continued symptom persistence that includes neurological disorders, muscle weakness, headaches, and memory loss, that developed during or shortly after the war. Our recent research showed that chemical exposure associated microbial dysbiosis accompanied by a leaky gut connected the pathologies in the intestine, liver, and brain. However, the mechanisms that caused the symptoms to persist even 30 years after the war remained elusive to investigators. In this study, we used a rodent model of GWI to investigate the persistence of microbiome alterations, resultant chronic inflammation, and its effect on neurotrophic and synaptic plasticity marker BDNF. The results showed that exposure to GW chemicals (the pesticide permethrin and prophylactic drug pyridostigmine bromide) resulted in persistent pathology characterized by the low relative abundance of the probiotic bacteria Akkermansia muciniphila in the gut, which correlated with high circulatory HMGB1 levels, blood-brain barrier dysfunction, neuroinflammation and lowered neurotrophin BDNF levels. Mechanistically, we used mice lacking the NLRP3 gene to investigate this inflammasome's role in observed pathology. These mice had significantly decreased inflammation and a subsequent increase in BDNF in the frontal cortex. This suggests that a persistently low species abundance of Akkermansia muciniphila and associated chronic inflammation due to inflammasome activation might be playing a significant role in contributing to chronic neurological problems in GWI. A therapeutic approach with various small molecules that can target both the restoration of a healthy microbiome and decreasing inflammasome activation might have better outcomes in treating GWI symptom persistence.

Lipocalin 2 induces neuroinflammation and blood-brain barrier dysfunction through liver-brain axis in murine model of nonalcoholic steatohepatitis.

BACKGROUND: Recent clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with ectopic manifestations including neuroinflammation and neurodegeneration, but the mediators and critical pathways involved are not well understood. Lipocalin 2 (Lcn2) is one of the important mediators exclusively produced in the liver and circulation during NASH pathology. METHODS: Using murine model of NASH, we studied the role of Lcn2 as a potent mediator of neuroinflammation and neurodegeneration in NASH pathology via the liver-brain axis. RESULTS: Results showed that high circulatory Lcn2 activated 24p3R (Lipocalin2 receptor) in the brain and induced the release of high mobility group box 1 (HMGB1) preferably from brain cells. Released HMGB1 acted as a preferential ligand to toll-like receptor 4 (TLR4) and induced oxidative stress by activation of NOX-2 signaling involving activated p65 protein of the NF-κB complex. Further, the HMGB1-derived downstream signaling cascade activated NLRP3 inflammasome and release of proinflammatory cytokines IL-6 and IL-1ß from brain cells. In addition, to advance our present understanding, in vitro studies were performed in primary brain endothelial cells where results showed high circulatory Lcn2 influenced HMGB1 secretion. Mechanistically, we also showed that elevated Lcn2 level in underlying NASH might be a likely cause for induction of blood-brain barrier dysfunction since the adipokine decreased the expression of tight junction protein Claudin 5 and caused subsequent elevation of pro-inflammatory cytokines IL-6 and IL-1ß. CONCLUSION: In conclusion, the NASH-induced brain pathology might be because of increased Lcn2-induced release of HMGB1 and accompanying neuroinflammation.

Early microcystin-LR exposure-linked inflammasome activation in mice causes development of fatty liver disease and insulin resistance.

Evidence from pediatric studies show that infants and children are at risk for early exposure to microcystin. The present report tests the hypothesis that early life exposure to microcystin (MC), a principal component of harmful algal blooms followed by a juvenile exposure to high-fat diet feeding potentiate the development of nonalcoholic fatty liver disease phenotype in adulthood. Results showed classical symptoms of early NAFLD linked inflammation. Cytokines and chemokines such as CD68, IL-1ß, MCP-1, and TNF-α, as well as α-SMA were increased in the groups that were exposed to MC-LR with the high-fat diet compared to the vehicle group. Also, mechanistically, NLRP3 KO mice showed a significant decrease in the inflammation and NAFLD phenotype and resisted the metabolic changes such as insulin resistance and glucose metabolism in the liver. The data suggested that MC-LR exposure and subsequent NLRP3 inflammasome activation in childhood could impact liver health in juveniles.

Microcystin exposure worsens nonalcoholic fatty liver disease associated ectopic glomerular toxicity via NOX-2-MIR21 axis.

NAFLD often results in cardiovascular, intestinal and renal complications. Previous reports from our laboratory highlighted NAFLD induced ectopic inflammatory manifestations in the kidney that gave rise to glomerular inflammation. Extending our studies, we hypothesized that existing inflammatory conditions in NAFLD could make the kidneys more susceptible to environmental toxicity. Our results showed that exposure of Microcystin-LR (MC) in NAFLD mice caused a marked increase in cellular scarring with a concomitant increase in mesangial cell activation as observed by increased α-SMA in the extracellular matrix surrounding the glomeruli. Renal tissue surrounding the glomeruli also showed increased NOX2 activation as shown by greater co-localization of p47 Phox and its membrane component gp91Phox both in the mesangial cell and surrounding tissue. Mechanistically, mesangial cells incubated with apocynin, nitrone spin trap DMPO and miR21 inhibitor showed significantly decreased α-SMA, miR21 levels and proinflammatory cytokine release in the supernatant. In parallel, mice lacking miR21, known to be activated by NOX2, when exposed to MC in NAFLD showed decreased mesangial cell activation. Strikingly, phenyl boronic acid incubated cells that were exposed to MC showed significantly decreased mesangial cell activation showing that peroxynitrite might be the major reactive species involved in mediation of the activation process, release of proinflammatory micro RNAs and cytokines that are crucial for renal toxicity. Thus, in conclusion, MC exposure causes NOX2 activation that leads to mesangial cell activation and toxicity via release of peroxynitrite that also represses PTEN by the upregulation of miR21 thus amplifying the toxicity.

Dysbiosis-Associated Enteric Glial Cell Immune-Activation and Redox Imbalance Modulate Tight Junction Protein Expression in Gulf War Illness Pathology.

About 14% of veterans who suffer from Gulf war illness (GWI) complain of some form of gastrointestinal disorder but with no significant markers of clinical pathology. Our previous studies have shown that exposure to GW chemicals resulted in altered microbiome which was associated with damage associated molecular pattern (DAMP) release followed by neuro and gastrointestinal inflammation with loss of gut barrier integrity. Enteric glial cells (EGC) are emerging as important regulators of the gastrointestinal tract and have been observed to change to a reactive phenotype in several functional gastrointestinal disorders such as IBS and IBD. This study is aimed at investigating the role of dysbiosis associated EGC immune-activation and redox instability in contributing to observed gastrointestinal barrier integrity loss in GWI via altered tight junction protein expression. Using a mouse model of GWI and in vitro studies with cultured EGC and use of antibiotics to ensure gut decontamination we show that exposure to GW chemicals caused dysbiosis associated change in EGCs. EGCs changed to a reactive phenotype characterized by activation of TLR4-S100ß/RAGE-iNOS pathway causing release of nitric oxide and activation of NOX2 since gut sterility with antibiotics prevented this change. The resulting peroxynitrite generation led to increased oxidative stress that triggered inflammation as shown by increased NLRP-3 inflammasome activation and increased cell death. Activated EGCs in vivo and in vitro were associated with decrease in tight junction protein occludin and selective water channel aquaporin-3 with a concomitant increase in Claudin-2. The tight junction protein levels were restored following a parallel treatment of GWI mice with a TLR4 inhibitor SsnB and butyric acid that are known to decrease the immunoactivation of EGCs. Our study demonstrates that immune-redox mechanisms in EGC are important players in the pathology in GWI and may be possible therapeutic targets for improving outcomes in GWI symptom persistence.

The Gut-Microbiome in Gulf War Veterans: A Preliminary Report.

Gulf War Illness (GWI) is a chronic multi-symptom disorder affecting the central nervous system (CNS), immune and gastrointestinal (GI) systems of Gulf War veterans (GWV). We assessed the relationships between GWI, GI symptoms, gut microbiome and inflammatory markers in GWV from the Boston Gulf War Illness Consortium (GWIC). Three groups of GWIC veterans were recruited in this pilot study; GWV without GWI and no gastrointestinal symptoms (controls), GWV with GWI and no gastrointestinal symptoms (GWI-GI), GWV with GWI who reported gastrointestinal symptoms (GW+GI). Here we report on a subset of the first thirteen stool samples analyzed. Results showed significantly different gut microbiome patterns among the three groups and within the GWI +/-GI groups. Specifically, GW controls had a greater abundance of firmicutes and the GWI+GI group had a greater abundance of the phyla bacteroidetes, actinobacteria, euryarchaeota, and proteobacteria as well as higher abundances of the families Bacteroidaceae, Erysipelotrichaceae, and Bifidobacteriaceae. The GWI+GI group also showed greater plasma levels of the inflammatory cytokine TNF-RI and they endorsed significantly more chemical weapons exposure during the war and reported significantly greater chronic pain, fatigue and sleep difficulties than the other groups. Studies with larger samples sizes are needed to confirm these initial findings.

Gut DNA Virome Diversity and Its Association with Host Bacteria Regulate Inflammatory Phenotype and Neuronal Immunotoxicity in Experimental Gulf War Illness.

Gulf War illness (GWI) is characterized by the persistence of inflammatory bowel disease, chronic fatigue, neuroinflammation, headache, cognitive impairment, and other medically unexplained conditions. Results using a murine model show that enteric viral populations especially bacteriophages were altered in GWI. The increased viral richness and alpha diversity correlated positively with gut bacterial dysbiosis and proinflammatory cytokines. Altered virome signature in GWI mice also had a concomitant weakening of intestinal epithelial tight junctions with a significant increase in Claudin-2 protein expression and decrease in ZO1 and Occludin mRNA expression. The altered virome signature in GWI, decreased tight junction protein level was followed by the presence an activation of innate immune responses such as increased Toll-like receptor (TLR) signaling pathways. The altered virome diversity had a positive correlation with serum IL-6, IL-1ß, and IFN-γ, intestinal inflammation (IFN-γ), and decreased Brain-Derived Neurotrophic Factor (BDNF), a neurogenesis marker. The co-exposure of Gulf War chemical and antibiotic (for gut sterility) or Gulf War chemical and Ribavirin, an antiviral compound to suppress virus alteration in the gut showed significant improvement in epithelial tight junction protein, decreased intestinal-, systemic-, and neuroinflammation. These results showed that the observed enteric viral dysbiosis could activate enteric viral particle-induced innate immune response in GWI and could be a novel therapeutic target in GWI.

Exogenous PP2A inhibitor exacerbates the progression of nonalcoholic fatty liver disease via NOX2-dependent activation of miR21.

Nonalcoholic fatty liver disease (NAFLD) is an emerging global pandemic. Though significant progress has been made in unraveling the pathophysiology of the disease, the role of protein phosphatase 2A (PP2A) and its subsequent inhibition by environmental and genetic factors in NAFLD pathophysiology remains unclear. The present report tests the hypothesis that an exogenous PP2A inhibitor leads to hepatic inflammation and fibrogenesis via an NADPH oxidase 2 (NOX2)-dependent pathway in NAFLD. Results showed that microcystin (MC) administration, a potent PP2A inhibitor found in environmental exposure, led to an exacerbation of NAFLD pathology with increased CD68 immunoreactivity, the release of proinflammatory cytokines, and stellate cell activation, a process that was attenuated in mice that lacked the p47phox gene and miR21 knockout mice. Mechanistically, leptin-primed immortalized Kupffer cells (a mimicked model for an NAFLD condition) treated with apocynin or nitrone spin trap 5,5 dimethyl-1- pyrroline N-oxide (DMPO) had significantly decreased CD68 and decreased miR21 and α-smooth muscle actin levels, suggesting the role of NOX2-dependent reactive oxygen species in miR21-induced Kupffer cell activation and stellate cell pathology. Furthermore, NOX2-dependent peroxynitrite generation was primarily responsible for cellular events observed following MC exposure since incubation with phenylboronic acid attenuated miR21 levels, Kupffer cell activation, and inflammatory cytokine release. Furthermore, blocking of the AKT pathway attenuated PP2A inhibitor-induced NOX2 activation and miR21 upregulation. Taken together, we show that PP2A may have protective roles, and its inhibition exacerbates NAFLD pathology via activating NOX2-dependent peroxynitrite generation, thus increasing miR21-induced pathology.NEW & NOTEWORTHY Protein phosphatase 2A inhibition causes nonalcoholic steatohepatitis (NASH) progression via NADPH oxidase 2. In addition to a novel emchanism of action, we describe a new tool to describe NASH histopathology.

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.

Sparstolonin B (SsnB) attenuates liver fibrosis via a parallel conjugate pathway involving P53-P21 axis, TGF-beta signaling and focal adhesion that is TLR4 dependent.

SsnB previously showed a promising role to lessen liver inflammation observed in a mouse model of NAFLD. Since NAFLD can progress to fibrosis, studies were designed to unravel its role in attenuating NAFLD associated fibrosis. Using both in vivo and in vitro approaches, the study probed the possible mechanisms that underlined the role of SsnB in mitigating fibrosis. Mechanistically, SsnB, a TLR4 antagonist, decreased TLR4-PI3k akt signaling by upregulating PTEN protein expression. It also decreased MDM2 protein activation and increased p53 and p21 gene and protein expression. SsnB also downregulated pro-fibrogenic hedgehog signaling pathway, inhibited hepatic stellate cell proliferation and induced apoptosis in hepatic stellate cells, a mechanism that was LPS dependent. Further, SsnB decreased fibrosis by antagonizing TLR4 induced TGFß signaling pathway. Alternatively, SsnB augmented BAMBI (a TGFß pseudo-receptor) expression in mice liver by inhibiting TLR4 signaling pathway and thus reduced TGFß signaling, resulting in decreased hepatic stellate cell activation and extracellular matrix deposition. In vitro experiments on human hepatic stellate cell line showed that SsnB increased gene and protein expression of BAMBI. It also decreased nuclear co-localization of phospho SMAD2/3 and SMAD4 protein and thus attenuated TGFß signaling in vitro. We also observed a significant decrease in phosphorylation of SMAD2/3 protein, decreased STAT3 activation, alteration of focal adhesion protein and stress fiber disassembly upon SsnB administration in hepatic stellate cells which further confirmed the antagonistic effect of SsnB on TLR4-induced fibrogenesis.

Increased butyrate priming in the gut stalls microbiome associated-gastrointestinal inflammation and hepatic metabolic reprogramming in a mouse model of Gulf War Illness.

Most of the associated pathologies in Gulf War Illness (GWI) have been ascribed to chemical and pharmaceutical exposures during the war. Since an increased number of veterans complain of gastrointestinal (GI), neuroinflammatory and metabolic complications as they age and there are limited options for a cure, the present study was focused to assess the role of butyrate, a short chain fatty acid for attenuating GWI-associated GI and metabolic complications. Results in a GWI-mouse model of permethrin and pyridostigmine bromide (PB) exposure showed that oral butyrate restored gut homeostasis and increased GPR109A receptor copies in the small intestine (SI). Claudin-2, a protein shown to be upregulated in conditions of leaky gut was significantly decreased following butyrate administration. Butyrate decreased TLR4 and TLR5 expressions in the liver concomitant to a decrease in TLR4 activation. GW-chemical exposure showed no clinical signs of liver disease but a significant alteration of metabolic markers such as SREBP1c, PPAR-α, and PFK was evident. Liver markers for lipogenesis and carbohydrate metabolism that were significantly upregulated following GW chemical exposure were attenuated by butyrate priming in vivo and in human primary hepatocytes. Further, Glucose transporter Glut-4 that was shown to be elevated following liver complications were significantly decreased in these mice after butyrate administration. Finally, use of TLR4 KO mice completely attenuated the liver metabolic changes suggesting the central role of these receptors in the GWI pathology. In conclusion, we report a butyrate specific mechanistic approach to identify and treat increased metabolic abnormalities in GWI veterans with systemic inflammation, chronic fatigue, GI disturbances, metabolic complications and weight gain.