ABSTRACT Gulf War Illness (GWI) is a severe disease affecting over 25% of military personnel deployed in the Persian Gulf War. Because Gulf War veterans were exposed to several neurotoxicants, including anti-nerve gas pills Pyridostigmine Bromide (PB) and insecticide permethrin (PER), central nervous system dysfunction and microglia-mediated neuroinflammation are the most prominent symptoms of GWI. However, published observations demonstrated that periodontitis is linked to central system dysfunction and neurological manifestation in veterans with GWI, as a post-war factor. Notably, periodontitis is significantly elevated in senior 65+ age veterans compared to the general public population. Because the surviving cohort of the U.S. Gulf War Veterans (currently an average of 55 years old) is entering old age, it is plausible that periodontitis may be linked to the deteriorated neuroinflammation in veterans with GWI at a senior age of 65 and above. Importantly, our unpublished observation revealed that the key periodontal pathogen Porhyromonas gingivalis might worsen neuroinflammation in veterans with GWI. It was also demonstrated that ligation of lipopolysaccharide (LPS), a common bacterial-derived virulent factor, with its cognate Toll-Like Receptor 4 (TLR4), elevates GWI neuroinflammation in young mice exposed to a mixture of PR and PER toxins. In contrast, our group and others demonstrated that the TLR4 signaling axis decreases with aging in the general public population. In contrast, we also reported that P. gingivalis produces a unique isoC17:0-Dihydroceramide-1-phosphoglycerol (PG-isoC17:0- DHC-1-P) virulent factor, which induces pro-inflammatory activation of macrophages independently of TLR4 signaling. Furthermore, our preliminary data demonstrated that 1) diminished expression patterns of TLR4 mRNA in mid-age veterans with GWI; 2) concentration of anti-P. gingivalis IgG, but not other periodontal pathogens, is serum was significantly elevated in GWI veterans compared to healthy control veterans; 3) PG- isoC17:0-DHC-1-P promotes neuroinflammation and impairs learning behavior in an experimental mouse model of GWI induced by a mixture of PB and PER. Therefore, we hypothesize that a unique class of PG-isoC17:0- DHC-1-P dihydroceramide exacerbates the age-dependent severity of GWI neuroinflammation independently of TLR4 signaling. Aim 1 is designed to elucidate the age- and sex-dependent impacts of P. gingivalis-LPS, PG- isoC17:0-DHC-1-P on GWI microglia in vitro. To mediate the GWI response in microglia, cells will be isolated from young (3-month-old) and aged (20-month-old) male and female TLR4-knock out and corresponding wild- type mice and then pre-exposed to PB and PER alone or in an equivalent mixture. Aim 2 will evaluate whether periodontitis exacerbates GWI-like behavioral changes and neuroinflammation independently of TLR4 signaling in relation to age and sex. We will induce periodontitis using live transgenic PG-isoC17:0-DHC-1-P-null or parent P. gingivalis-W83 strains in young and aged TLR4-knock out and wild-type mice (both sexes) pre-treated with PB and PER alone or in the equivalent mixture. Then, we will compare learning and memory skills as well as levels of brain neuroinflammation. To further understand the molecular mechanisms of PG-isoC17:0-DHC-1-P- exacerbated GWI neuroinflammation, microglia collected from the cortical and hippocampal wild-type brain regions will be analyzed by single-cell RNA-seq assay. Altogether, proposed studies are crucial to establishing the age-associated pathophysiological role of unique PG-isoC17:0-DHC-1-P dihydroceramide in the exacerbated pathogenic hallmarks of neuroinflammation and microglia activation observed in veterans with GWI.

About one-fourth of the veterans who served in the Persian Gulf War (1990-1991) are affected by a multi- symptom condition known as Gulf War Illness (GWI). Currently, clinical practitioners take a palliative treatment approach to address such neurological symptoms of GWI as brain neuroinflammation, fatigue, headaches, and memory loss. Emerging evidence indicates that age-dependent chronic periodontitis, induced by oral bacterial dysbiosis and exaggerated by poor dental care, may be linked to neurological complications of GWI, as a post- war factor. Therefore, the proposed study will identify whether the key oral periodontal bacteria Porphyromonas gingivalis is responsible for exacerbating GWI neuroinflammation associated with aging, only evident in veterans with periodontitis who served in the Persian Gulf War. Once our hypothesis is tested, we will be well-placed to develop novel therapeutic approaches to improve oral health and reduce neuroinflammation in senior 65+ old age veterans living with GWI.