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

Diana Kimono,Patricia Janulewicz,Sutapa Sarkar,Stephen Lasley,Yuxi Li,Saurabh Chatterjee,Dipro Bose,Ayan Mondal,Amar N Kar,Ratanesh Seth,Muayad Albadrani,Mitzi Nagarkatti,Kimberly Sullivan,Prakash Nagarkatti,Ronnie Horner,Nancy Klimas

doi:10.3389/fphys.2019.01229

Abstract

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-S100β/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.

Highlights

Gastrointestinal disturbances are one of the most commonly reported chronic symptoms among veterans who returned from the Persian Gulf war of 1990–1991 (Murphy et al, 1999; Dunphy et al, 2003; Koch and Emory, 2005; White et al, 2016)
We test the hypothesis that, the altered microbiome which results in increased pathogen associated molecular patterns (PAMPS) (e.g., LPS, flagellin and other immunostimulatory bacterial parts), leaky gut and increase in circulatory damage associated molecular patterns (DAMPS) (e.g., High mobility group box 1 protein (HMGB-1)) in GWchemical (Permethrin and pyridostigmine bromide) treated mice results in a reactive EGC phenotype compared to mice treated with vehicle control treated mice and mice coexposed with Gulf war (GW) chemicals and antibiotics
We found that there was a significant increase (P < 0.05) in glial fibrillary acidic protein (GFAP) expression in the small intestine of mice treated with GW chemicals (PB + BER) compared to the control group, and mice coexposed to GW chemicals and antibiotics (Figures 2A,B)

Summary

Introduction

Gastrointestinal disturbances are one of the most commonly reported chronic symptoms among veterans who returned from the Persian Gulf war of 1990–1991 (Murphy et al, 1999; Dunphy et al, 2003; Koch and Emory, 2005; White et al, 2016). One of our own studies which reported metabolic reprogramming in liver as a result of leaky gut and endotoxemia did not find any biochemical markers of liver damage or altered metabolism in a mouse model of GWI. This was surprising because we had had previously shown that exposure to GW theater chemicals resulted in an alteration of gut microbiome and concomitant TLR4 mediated gastrointestinal and neuroinflammation with endotoxemia (Alhasson et al, 2017; Seth et al, 2018). This elusive nature of GWI is a strong reason for further studying underlying mechanisms of this condition in order to obtain effective therapies

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