Pyridostigmine Bromide Exposure Impairs Enteric Neural Stem Cell Regeneration in Gulf War Illness

Introduction:: The enteric nervous system is the central controller of colonic motility (contractile patterns in peristalsis).
Nerves of the enteric nervous system are maintained and regenerated upon injury by a tissue-resident p75 + enteric neural
stem cell (ENSC) compartment. Our previous work on Gulf War Illness, a multi-symptom disorder occurring in combat
veterans of the Persian Gulf War, showed that colonic motility was significantly impaired due to toxic exposure during
combat. We linked the exposure agent (Pyridostigmine bromide; PB) taken prophylactically against nerve gas attacks to
acute neuro-inflammation in the enteric nervous system, leading to reduced contraction. Interestingly, Gulf War veterans
have impaired colonic motility that persists even decades after their initial exposure during combat. This led us to
investigate the hypothesis that the regenerative ENSC compartment is also altered upon toxic exposure to pyridostigmine
bromide (PB). The objective of our current work is to demonstrate that neuro-inflammation extends to the p75+ ENSC
compartment, that impairs the regeneration of the enteric nervous system in Gulf War Illness.

Materials and Methods:: C57BL/6 male mice were administered PB through oral administration (2mg/kg) for 7 days
and allowed 30 days to recover to mimic delayed-symptom onset following exposure in combat leading to GWI. Gene
expression changes were analyzed in cellular compartments that mediate neuro-inflammation and produce colonic
motility using qPCR (enteric nerves, smooth muscle, and macrophages). Control or PB exposed colons were also
histologically examined for changes in ENSCs (p75 NGFR neural stem cell marker), and mature neurons (βIII Tubulin).
Lastly, colon ring explants were tested for real time force generation (motility) through a custom-built organ bath
physiology system to assay for differences in colonic motility due to chronic PB exposure.

Results, Conclusions, and Discussions:: PB exposure significantly decreased the number of ENSCs and expression of enteric neural stem cell marker (p75 NGFR ) compared to control (12 ± 2.9% in control vs 3.9 ± 0.37% in PB; Fig 1A). PB exposed colons also demonstrated a 23% increase in enteric neurons, observed via βIII Tubulin immunofluorescence (Fig 1B). Gene expression analysis confirmed the loss of ENSC-related genes (Ngfr, Sox2, Nes; Fig 1C). The increased number of neurons observed via immunofluorescence translated to an increase in a specific subset of neurons (Chat, Fig 1C) that mediate the generation of contractile forces in the colon. This increased expression of Chat led to a hyper-contractile colon in PB-exposed animals compared to control animals (increase in forces by 36.87% compared to controls; Fig 1D). A fully functional colon maintains its motility via the balanced expression of contractile and inhibitory nerves, with a hyper-contractile phenotype often indicative of severe inflammation. Our gene expression data further supported the existence of chronic inflammation, since the ENSCs in PB-treated mice expressed Sox9 (2.9 fold; Fig 1C), along with an increased expression of inflammatory marker (2.6-fold). Sox9 is a marker of ENSCs that display a propensity to
differentiate into immuno-modulatory enteric glia that amplify inflammation in the colon. Our results show that even in delayed settings, i.e. 30 days after initial PB exposure, a significant loss of ENSCs is observed along with hallmarks of inflammatory insult in the colon. This loss of ENSCs translated to a lack of diverse subsets of enteric nerves required to maintain colonic motility. Instead, we observed hyper-contractility in colons, that typically result in chronic diarrhea and other functional gastrointestinal disorders often observed in Gulf War veterans. Our future work will focus on isolating macrophage-enteric nerve-enteric glia crosstalk, to identify
therapeutic targets to improve veteran quality of life.

Acknowledgements (Optional): : This work was supported by Department of Defense Gulf War Illness Research Program
W81XWH-21-1-0477 (SR). Claudia Collier is supported by the Dr. Dionel Avilés ’53 and Dr. James Johnson ’67
Fellowship Program.

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