A Double-Humanized Mouse Model for Studying Host Gut Microbiome-Immune Interactions in Gulf War Illness.

Unraveling the multisymptomatic Gulf War Illness (GWI) pathology and finding an effective cure have eluded researchers for decades. The chronic symptom persistence and limitations for studying the etiologies in mouse models that differ significantly from those in humans pose challenges for drug discovery and finding effective therapeutic regimens. The GWI exposome differs significantly in the study cohorts, and the above makes it difficult to recreate a model closely resembling the GWI symptom pathology. We have used a double engraftment strategy for reconstituting a human immune system coupled with human microbiome transfer to create a humanized-mouse model for GWI. Using whole-genome shotgun sequencing and blood immune cytokine enzyme linked immunosorbent assay (ELISA), we show that our double humanized mice treated with Gulf War (GW) chemicals show significantly altered gut microbiomes, similar to those reported in a Veteran cohort of GWI. The results also showed similar cytokine profiles, such as increased levels of IL-1ß, IL-6, and TNF R-1, in the double humanized model, as found previously in a human cohort. Further, a novel GWI Veteran fecal microbiota transfer was used to create a second alternative model that closely resembled the microbiome and immune-system-associated pathology of a GWI Veteran. A GWI Veteran microbiota transplant in humanized mice showed a human microbiome reconstitution and a systemic inflammatory pathology, as reflected by increases in interleukins 1ß, 6, 8 (IL-1ß, IL-6, IL-8), tumor necrosis factor receptor 1 (TNF R-1), and endotoxemia. In conclusion, though preliminary, we report a novel in vivo model with a human microbiome reconstitution and an engrafted human immune phenotype that may help to better understand gut-immune interactions in GWI.

Hepatic NLRP3-Derived Hsp70 Binding to TLR4 Mediates MASLD to MASH Progression upon Inhibition of PP2A by Harmful Algal Bloom Toxin Microcystin, a Second Hit.

Harmful algal bloom toxin microcystin has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) progression and hepatocellular carcinoma, though the mechanisms remain unclear. Using an established mouse model of MASLD, we show that the NLRP3-Hsp70-TLR4 axis drives in part the inflammation of the liver lobule that results in the progression of MASLD to metabolic dysfunction-associated steatohepatitis (MASH). Results showed that mice deficient in NLRP3 exhibited decreased MASH pathology, blocked Hsp70 expression, and co-binding with NLRP3, a crucial protein component of the liver inflammasome. Hsp70, both in the liver lobule and extracellularly released in the liver vasculature, acted as a ligand to TLR4 in the liver, primarily in hepatocytes to activate the NF-κB pathway, ultimately leading to hepatic cell death and necroptosis, a crucial pathology of MASH progression. The above studies show a novel insight into an inflammasome-triggered Hsp70-mediated inflammation that may have broader implications in MASLD pathology. MASLD to MASH progression often requires multiple hits. One of the mediators of progressive MASLD is environmental toxins. In this research report, we show for the first time a novel mechanism where microcystin-LR, an environmental toxin, advances MASLD to MASH by triggering the release of Hsp70 as a DAMP to activate TLR4-induced inflammation in the liver.

Microbiome Dysbiosis Shows Strong Association of Gut-Derived Altered Metabolomic Profile in Gulf War Chronic Multisymptom Illness Symptom Persistence Following Western Diet Feeding and Development of Obesity.

The pathophysiology of Gulf War Illness (GWI) remains elusive even after three decades. The persistence of multiple complex symptoms along with metabolic disorders such as obesity worsens the health of present Gulf War (GW) Veterans often by the interactions of the host gut microbiome and inflammatory mediators. In this study, we hypothesized that the administration of a Western diet might alter the host metabolomic profile, which is likely associated with the altered bacterial species. Using a five-month symptom persistence GWI model in mice and whole-genome sequencing, we characterized the species-level dysbiosis and global metabolomics, along with heterogenous co-occurrence network analysis, to study the bacteriome-metabolomic association. Microbial analysis at the species level showed a significant alteration of beneficial bacterial species. The beta diversity of the global metabolomic profile showed distinct clustering due to the Western diet, along with the alteration of metabolites associated with lipid, amino acid, nucleotide, vitamin, and xenobiotic metabolism pathways. Network analysis showed novel associations of gut bacterial species with metabolites and biochemical pathways that could be used as biomarkers or therapeutic targets to ameliorate symptom persistence in GW Veterans.

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.

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.

Health impacts of environmental contamination of micro- and nanoplastics: a review.

Plastics are extensively used in our daily life. However, a significant amount of plastic waste is discharged to the environment directly or via improper reuse or recycling. Degradation of plastic waste generates micro- or nano-sized plastic particles that are defined as micro- or nanoplastics (MNPs). Microplastics (MPs) are plastic particles with a diameter less than 5 mm, while nanoplastics (NPs) range in diameter from 1 to 100 or 1000 nm. In the current review, we first briefly summarized the environmental contamination of MNPs and then discussed their health impacts based on existing MNP research. Our review indicates that MNPs can be detected in both marine and terrestrial ecosystems worldwide and be ingested and accumulated by animals along the food chain. Evidence has suggested the harmful health impacts of MNPs on marine and freshwater animals. Recent studies found MPs in human stool samples, suggesting that humans are exposed to MPs through food and/or drinking water. However, the effect of MNPs on human health is scarcely researched. In addition to the MNPs themselves, these tiny plastic particles can release plastic additives and/or adsorb other environmental chemicals, many of which have been shown to exhibit endocrine disrupting and other toxic effects. In summary, we conclude that more studies are necessary to provide a comprehensive understanding of MNP pollution hazards and also provide a basis for the subsequent pollution management and control.

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.

Doxorubicin obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation.

Ovarian toxicity and infertility are major side effects of cancer therapy in young female cancer patients. We and others have previously demonstrated that doxorubicin (DOX), one of the most widely used chemotherapeutic chemicals, has a dose-dependent toxicity on growing follicles. However, it is not fully understood if the primordial follicles are the direct or indirect target of DOX. Using both prepubertal and young adult female mouse models, we comprehensively investigated the effect of DOX on all developmental stages of follicles, determined the impact of DOX on primordial follicle survival, activation, and development, as well as compared the impact of age on DOX-induced ovarian toxicity. Twenty-one-day-old CD-1 female mice were intraperitoneally injected with PBS or clinically relevant dose of DOX at 10 mg/kg once. Results indicated that DOX primarily damaged granulosa cells in growing follicles and oocytes in primordial follicles and DOX-induced growing follicle apoptosis was associated with the primordial follicle overactivation. Using the 5-day-old female mice with a more uniform primordial follicle population, our data revealed that DOX also directly promoted primordial follicle death and the DNA damage-TAp63α-C-CASP3 pathway was involved in DOX-induced primordial follicle oocyte apoptosis. Compared to 21-day- and 8-week-old female mice that were treated with the same dose of DOX, the 5-day-old mice had the most severe primordial follicle loss as well as the least degree of primordial follicle overactivation. Taken together, these results demonstrate that DOX obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation and the DOX-induced ovarian toxicity is age dependent.

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.

CD44 deletion leading to attenuation of experimental autoimmune encephalomyelitis results from alterations in gut microbiome in mice.

Dysbiosis in gut microbiome has been shown to be associated with inflammatory and autoimmune diseases. Previous studies from our laboratory demonstrated the pivotal role played by CD44 in the regulation of EAE, a murine model of multiple sclerosis. In the current study, we determined whether these effects resulted from an alteration in gut microbiota and the short-chain fatty acid (SCFA) production in CD44 knockout (CD44KO) mice. Fecal transfer from naïve CD44KO but not C57BL/6 wild type (CD44WT) mice, into EAE-induced CD44WT mice, led to significant amelioration of EAE. High-throughput bacterial 16S rRNA gene sequencing, followed by clustering sequences into operational taxonomic units (OTUs) and biochemical analysis, revealed that EAE-induced CD44KO mice showed significant diversity, richness, and evenness when compared to EAE-induced CD44WT mice at the phylum level, with dominant Bacteroidetes (68.5%) and low Firmicutes (26.8%). Further, data showed a significant change in the abundance of SCFAs, propionic acid, and i-butyric acid in EAE-CD44KO compared to EAE-CD44WT mice. In conclusion, our results demonstrate that the attenuation of EAE seen following CD44 gene deletion in mice may result from alterations in the gut microbiota and SCFAs. Furthermore, our studies also demonstrate that the phenotype of gene knock-out animals may be shaped by gut microbiota.

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.

Dietary Indoles Suppress Delayed-Type Hypersensitivity by Inducing a Switch from Proinflammatory Th17 Cells to Anti-Inflammatory Regulatory T Cells through Regulation of MicroRNA.

Aryl hydrocarbon receptor (AhR) has been shown to have profound influence on T cell differentiation, and use of distinct AhR ligands has shown that whereas some ligands induce regulatory T cells (Tregs), others induce Th17 cells. In the present study, we tested the ability of dietary AhR ligands (indole-3-carbinol [I3C] and 3,3'-diindolylmethane [DIM]) and an endogenous AhR ligand, 6-formylindolo(3,2-b)carbazole (FICZ), on the differentiation and functions of Tregs and Th17 cells. Treatment of C57BL/6 mice with indoles (I3C or DIM) attenuated delayed-type hypersensitivity (DTH) response to methylated BSA and generation of Th17 cells while promoting Tregs. In contrast, FICZ exacerbated the DTH response and promoted Th17 cells. Indoles decreased the induction of IL-17 but promoted IL-10 and Foxp3 expression. Also, indoles caused reciprocal induction of Tregs and Th17 cells only in wild-type (AhR(+/+)) but not in AhR knockout (AhR(-/-)) mice. Upon analysis of microRNA (miR) profile in draining lymph nodes of mice with DTH, treatment with I3C and DIM decreased the expression of several miRs (miR-31, miR-219, and miR-490) that targeted Foxp3, whereas it increased the expression of miR-495 and miR-1192 that were specific to IL-17. Interestingly, treatment with FICZ had precisely the opposite effects on these miRs. Transfection studies using mature miR mimics of miR-490 and miR-1192 that target Foxp3 and IL-17, respectively, or scrambled miR (mock) or inhibitors confirmed that these miRs specifically targeted Foxp3 and IL-17 genes. Our studies demonstrate, to our knowledge for the first time, that the ability of AhR ligands to regulate the differentiation of Tregs versus Th17 cells may depend on miR signature profile.

Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis.

Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused an increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression.

Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4+ T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

NKT cell modulates NAFLD potentiation of metabolic oxidative stress-induced mesangial cell activation and proximal tubular toxicity.

Obesity and nonalcoholic fatty liver disease (NAFLD) are associated with the development and progression of chronic kidney disease. We recently showed that NAFLD induces liver-specific cytochrome P-450 (CYP)2E1-mediated metabolic oxidative stress after administration of the CYP2E1 substrate bromodichloromethane (BDCM) (Seth RK, Das S, Kumar A, Chanda A, Kadiiska MB, Michelotti G, Manautou J, Diehl AM, Chatterjee S. Toxicol Appl Pharmacol 274: 42-54, 2014; Seth RK, Kumar A, Das S, Kadiiska MB, Michelotti G, Diehl AM, Chatterjee S. Toxicol Sci 134:291-303, 2013). The present study examined the effects of CYP2E1-mediated oxidative stress in NAFLD leading to kidney toxicity. Mice were fed a high-fat diet for 12 wk to induce NAFLD. NAFLD mice were exposed to BDCM, a CYP2E1 substrate, for 4 wk. NAFLD + BDCM increased CYP2E1-mediated lipid peroxidation in proximal tubular cells compared with mice with NAFLD alone or BDCM-treated lean mice, thus ruling out the exclusive role of BDCM. Lipid peroxidation increased IL-1ß, TNF-α, and interferon-γ. In parallel, mesangial cell activation was observed by increased α-smooth muscle actin and transforming growth factor-ß, which was blocked by the CYP2E1 inhibitor diallyl sulphide both in vivo and in vitro. Mice lacking natural killer T cells (CD1d knockout mice) showed elevated (>4-fold) proinflammatory mediator release, increased Toll-like receptor (TLR)4 and PDGF2 mRNA, and mesangial cell activation in the kidney. Finally, NAFLD CD1D knockout mice treated with BDCM exhibited increased high mobility group box 1 and Fas ligand levels and TUNEL-positive nuclei, indicating that higher cell death was attenuated in TLR4 knockout mice. Tubular cells showed increased cell death and cytokine release when incubated with activated mesangial cells. In summary, an underlying condition of progressive NAFLD causes renal immunotoxicity and aberrant glomerular function possibly through high mobility group box 1-dependent TLR4 signaling and mesangial cell activation, which, in turn, is modulated by intrinsic CD1D-dependent natural killer T cells.

Sparstolonin B attenuates early liver inflammation in experimental NASH by modulating TLR4 trafficking in lipid rafts via NADPH oxidase activation.

Although significant research data exist on the pathophysiology of nonalcoholic steatohepatitis (NASH), finding an efficient treatment regimen for it remains elusive. The present study used sparstolonin B (SsnB), a novel TLR4 antagonist derived from the Chinese herb Sparganium stoloniferum, as a possible drug to mitigate early inflammation in NASH. This study used an early steatohepatitic injury model in high-fat-fed mice with CYP2E1-mediated oxidative stress as a second hit. SsnB was administered for 1 wk along with bromodichloromethane (BDCM), an inducer of CYP2E1-mediated oxidative stress. Results showed that SsnB administration attenuated inflammatory morphology and decreased elevation of the liver enzyme alanine aminotransferase (ALT). Mice administered SsnB also showed decreased mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-1ß, and IL-23, while protein levels of both TNF-α and IL-1ß were significantly decreased. SsnB significantly decreased Kupffer cell activation as evidenced by reduction in CD68 and monocyte chemoattractant protein-1 (MCP1) mRNA and protein levels with concomitant inhibition of macrophage infiltration in the injured liver. Mechanistically, SsnB decreased TLR4 trafficking to the lipid rafts, a phenomenon described by the colocalization of TLR4 and lipid raft marker flotillin in tissues and immortalized Kupffer cells. Since we have shown previously that NADPH oxidase drives TLR4 trafficking in NASH, we studied the role of SsnB in modulating this pathway. SsnB prevented NADPH oxidase activation in vivo and in vitro as indicated by decreased peroxynitrite formation. In summary, the present study reports a novel use of the TLR4 antagonist SsnB in mitigating inflammation in NASH and in parallel shows a unique molecular mechanism of decreasing nitrative stress.

NADPH Oxidase-Derived Peroxynitrite Drives Inflammation in Mice and Human Nonalcoholic Steatohepatitis via TLR4-Lipid Raft Recruitment.

The molecular events that link NADPH oxidase activation and the induction of Toll-like receptor (TLR)-4 recruitment into hepatic lipid rafts in nonalcoholic steatohepatitis (NASH) are unclear. We hypothesized that in liver, NADPH oxidase activation is key in TLR4 recruitment into lipid rafts, which in turn up-regulates NF-κB translocation to the nucleus and subsequent DNA binding, leading to NASH progression. Results from confocal microscopy showed that liver from murine and human NASH had NADPH oxidase activation, which led to the formation of highly reactive peroxynitrite, as shown by 3-nitrotyrosine formation in diseased liver. Expression and recruitment of TLR4 into the lipid rafts were significantly greater in rodent and human NASH. The described phenomenon was NADPH oxidase, p47phox, and peroxynitrite dependent, as liver from p47phox-deficient mice and from mice treated with a peroxynitrite decomposition catalyst [iron(III) tetrakis(p-sulfonatophenyl)porphyrin] or a peroxynitrite scavenger (phenylboronic acid) had markedly less Tlr4 recruitment into lipid rafts. Mechanistically, peroxynitrite-induced TLR4 recruitment was linked to increased IL-1ß, sinusoidal injury, and Kupffer cell activation while blocking peroxynitrite-attenuated NASH symptoms. The results strongly suggest that NADPH oxidase-mediated peroxynitrite drove TLR4 recruitment into hepatic lipid rafts and inflammation, whereas the in vivo use of the peroxynitrite scavenger phenylboronic acid, a novel synthetic molecule having high reactivity with peroxynitrite, attenuates inflammatory pathogenesis in NASH.

Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals.

BACKGROUND: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-ß signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-ß, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-ß signaling and fibrogenesis in experimental and human NASH.

M1 polarization bias and subsequent nonalcoholic steatohepatitis progression is attenuated by nitric oxide donor DETA NONOate via inhibition of CYP2E1-induced oxidative stress in obese mice.

Activation of M1 macrophages in nonalcoholic steatohepatitis (NASH) is produced by several external or endogenous factors: inflammatory stimuli, oxidative stress, and cytokines are known. However, any direct role of oxidative stress in causing M1 polarization in NASH has been unclear. We hypothesized that CYP2E1-mediated oxidative stress causes M1 polarization in experimental NASH, and that nitric oxide (NO) donor administration inhibits CYP2E1-mediated inflammation with concomitant attenuation of M1 polarization. Because CYP2E1 takes center stage in these studies, we used a toxin model of NASH that uses a ligand and a substrate of CYP2E1 for inducing NASH. Subsequently, we used a methionine and choline-deficient diet-induced rodent NASH model where the role of CYP2E1 in disease progression has been shown. Our results show that CYP2E1 causes M1 polarization bias, which includes a significant increase in interleukin-1ß (IL-1ß) and IL-12 in both models of NASH, whereas CYP2E1-null mice or diallyl sulfide administration prevented it. Administration of gadolinium chloride (GdCl3), a macrophage toxin, attenuated both the initial M1 response and the subsequent M2 response, showing that the observed increase in cytokine levels is primarily from macrophages. Based on the evidence of an adaptive NO increase, the NO donor administration in vivo that mechanistically inhibited CYP2E1 catalyzed the oxidative stress during the entire study in NASH-abrogated M1 polarization and NASH progression. The results obtained show the association of CYP2E1 in M1 polarization, and that inhibition of CYP2E1 catalyzed oxidative stress by an NO donor (DETA NONOate [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate]) can be a promising therapeutic strategy in NASH.