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-1β 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-1β. CONCLUSION: In conclusion, the NASH-induced brain pathology might be because of increased Lcn2-induced release of HMGB1 and accompanying neuroinflammation

Host 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 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\u27s 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 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

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 IL1β 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 IL1β, 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

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 , , and , 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 and known to be associated with gastrointestinal pathology. AG treatment significantly restored tight junction proteins that correlated well with decreased intestinal proinflammatory mediators IL-1β 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

Host microbiome associated low intestinal acetate correlates with progressive NLRP3-dependent hepatic-immunotoxicity in early life microcystin-LR exposure.

BACKGROUND: Microcystins (MCs), potent hepatotoxins pose a significant health risk to humans, particularly children, who are more vulnerable due to higher water intake and increased exposure during recreational activities. METHODS: Here, we investigated the role of host microbiome-linked acetate in modulating inflammation caused by early-life exposure to the cyanotoxin Microcystin-LR (MC-LR) in a juvenile mice model. RESULTS: Our study revealed that early-life MC-LR exposure disrupted the gut microbiome, leading to a depletion of key acetate-producing bacteria and decreased luminal acetate concentration. Consequently, the dysbiosis hindered the establishment of a gut homeostatic microenvironment and disrupted gut barrier function. The NOD-like receptor family pyrin domain - containing 3 (NLRP3) inflammasome, a key player in MC-induced hepatoxicity emerged as a central player in this process, with acetate supplementation effectively preventing NLRP3 inflammasome activation, attenuating hepatic inflammation, and decreasing pro-inflammatory cytokine production. To elucidate the mechanism underlying the association between early-life MC-LR exposure and the progression of metabolic dysfunction associated steatotic liver disease (MASLD), we investigated the role of acetate binding to its receptor -G-protein coupled receptor 43 (GPR43) on NLRP3 inflammasome activation. Our results demonstrated that acetate-GPR43 signaling was crucial for decreasing NLRP3 protein levels and inhibiting NLRP3 inflammasome assembly. Further, acetate-induced decrease in NLRP3 protein levels was likely mediated through proteasomal degradation rather than autophagy. Overall, our findings underscore the significance of a healthy gut microbiome and its metabolites, particularly acetate, in the progression of hepatotoxicity induced by early life toxin exposure, crucial for MASLD progression. CONCLUSIONS: This study highlights potential therapeutic targets in gut dysbiosis and NLRP3 inflammasome activation for mitigating toxin-associated inflammatory liver diseases

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-1β, 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 1β, 6, 8 (IL-1β, 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

Prolonged Antibiotic Use in a Preclinical Model of Gulf War Chronic Multisymptom-Illness Causes Renal Fibrosis-like Pathology via Increased micro-RNA 21-Induced PTEN Inhibition That Is Correlated with Low Host Lachnospiraceae Abundance.

Gulf War (GW) veterans show gastrointestinal disturbances and gut dysbiosis. Prolonged antibiotic treatments commonly employed in veterans, especially the use of fluoroquinolones and aminoglycosides, have also been associated with dysbiosis. This study investigates the effect of prolonged antibiotic exposure on risks of adverse renal pathology and its association with gut bacterial species abundance in underlying GWI and aims to uncover the molecular mechanisms leading to possible renal dysfunction with aging. Using a GWI mouse model, administration of a prolonged antibiotic regimen involving neomycin and enrofloxacin treatment for 5 months showed an exacerbated renal inflammation with increased NF-κB activation and pro-inflammatory cytokines levels. Involvement of the high mobility group 1 (HMGB1)-mediated receptor for advanced glycation end products (RAGE) activation triggered an inflammatory phenotype and increased transforming growth factor-β (TGF-β) production. Mechanistically, TGF-β- induced microRNA-21 upregulation in the renal tissue leads to decreased phosphatase and tensin homolog (PTEN) expression. The above event led to the activation of protein kinase-B (AKT) signaling, resulting in increased fibronectin production and fibrosis-like pathology. Importantly, the increased miR-21 was associated with low levels of Lachnospiraceae in the host gut which is also a key to heightened HMGB1-mediated inflammation. Overall, though correlative, the study highlights the complex interplay between GWI, host gut dysbiosis, prolonged antibiotics usage, and renal pathology via miR-21/PTEN/AKT signaling

Periodic heat waves-induced neuronal etiology in the elderly is mediated by gut-liver-brain axis: a transcriptome profiling approach.

Heat stress exposure in intermittent heat waves and subsequent exposure during war theaters pose a clinical challenge that can lead to multi-organ dysfunction and long-term complications in the elderly. Using an aged mouse model and high-throughput sequencing, this study investigated the molecular dynamics of the liver-brain connection during heat stress exposure. Distinctive gene expression patterns induced by periodic heat stress emerged in both brain and liver tissues. An altered transcriptome profile showed heat stress-induced altered acute phase response pathways, causing neural, hepatic, and systemic inflammation and impaired synaptic plasticity. Results also demonstrated that proinflammatory molecules such as S100B, IL-17, IL-33, and neurological disease signaling pathways were upregulated, while protective pathways like aryl hydrocarbon receptor signaling were downregulated. In parallel, Rantes, IRF7, NOD1/2, TREM1, and hepatic injury signaling pathways were upregulated. Furthermore, current research identified Orosomucoid 2 (ORM2) in the liver as one of the mediators of the liver-brain axis due to heat exposure. In conclusion, the transcriptome profiling in elderly heat-stressed mice revealed a coordinated network of liver-brain axis pathways with increased hepatic ORM2 secretion, possibly due to gut inflammation and dysbiosis. The above secretion of ORM2 may impact the brain through a leaky blood-brain barrier, thus emphasizing intricate multi-organ crosstalk