Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria

Authors: LAKES, JOURDAN - Oak Ridge Institute For Science And Education (ORISE); FU, XU - University Of Kentucky; HARVEY, BROCK - University Of Kentucky; NEUPANE, KHAGA - University Of Kentucky; ARYAL, SURYA - University Of Kentucky; Ferrell, Jessica; Flythe, Michael; RICHARDS, CHRISTOPHER - University Of Kentucky

Submitted to: Anaerobe
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2023
Publication Date: 10/14/2023
Citation: Lakes, J.E., Fu, X., Harvey, B., Neupane, K.R., Aryal, S.P., Ferrell, J.L., Flythe, M.D., Richards, C.I. 2023. Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria. Anaerobe. 83. Article 102787. https://doi.org/10.1016/j.anaerobe.2023.102787.
DOI: https://doi.org/10.1016/j.anaerobe.2023.102787

Interpretive Summary: Previous research indicates that continued use of nicotine products (e.g., electronic cigarettes/vapes, cigarettes, cigars, nicotine gum, etc.) leads to negative (dysbiotic) changes in the bacterial composition of the gut. These dysbiotic changes have been associated with increased risk of severe bacterial infections, immune dysregulation, and the development of anxiety and depressive disorders. We sought to find mechanistic explanations for the immune dysregulation associated with negative compositional changes following continuous exposure to nicotine (the neuro- and immuno-active component of tobacco) and its break-down product, cotinine (also neuro- and immuno-active), which accumulates at much greater concentrations than nicotine in the gut as a function of use. We exposed bacterial representatives of the gut to increasing concentrations of nicotine or cotinine and measured the changes in bacterial metabolites produced and their vesicle (small cargo vessel) physiology following exposure. We chose to measure changes in vesicle physiology, as bacteria regularly use these small cargo vessels to acquire nutrients, evade antimicrobials, and aide in infection; due to the use of vesicles for multiple purposes, they regularly interact with immune cells at the gastrointestinal interface and beyond into the body cavity. We found that continuous exposure to nicotine and cotinine (separately) resulted in changes to bacterial metabolite production as well as vesicular protein cargo. We then introduced altered or unaltered vesicles to a highly common immune cell of the gut, the tolerogenic macrophage, to determine if the changes to the vesicles altered the way the immune cell responded to the bacterial stimulus. We found that, yes, despite cotinine or nicotine exposure of the macrophages themselves, if they were exposed to vesicles altered by nicotine or cotinine exposure that they produced fewer inflammatory messenger molecules (cytokines/chemokines) than when they were exposed to unaltered vesicles. Through these studies, we were able to determine that continuous nicotine use alters bacterial physiology and vesicle production, ultimately dampening the inflammatory response by first-responder (innate) immune cells (macrophages) to bacterial stimuli and this is a potential explanation for the higher rates of severe bacterial infection chronic smokers contract.

Technical Abstract: Previous research indicates continuous use of nicotine-containing products leads to alterations in the enteric microfloral composition. This study aimed to elucidate mechanistic explanation(s) for these compositional changes by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. We demonstrated that metabolites produced by Gram (-) Prevotella bryantii (B14) and Bacteroides fragilis (25285), were significantly reduced following nicotine exposure while metabolite production by the Gram (+) Acetoanaerobium sticklandii (SR) were increased. Cotinine exposure yielded concentration-dependent effects on metabolites where low (1 µM) concentrations increased metabolite production by all species, while high (10 µM) concentrations resulted in less metabolite production. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Total protein packaged in vesicles of the Gram (-) representatives were reduced following nicotine exposure and 10 µM cotinine exposure, while protein cargo of the Gram (+) vesicles were largely unaffected. Interestingly, we found that the physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Results indicated that tolerogenic macrophages regardless of nicotine/cotinine treatment, maintained an inflammatory response following stimulation by vesicles from unamended cultures. However, tolerogenic macrophages exposed to vesicles from 1 µM nicotine, and 5 or 10 µ' cotinine cultures produced significantly less IL-12p70, TNFa, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. These results indicate that nicotine/cotinine exposure alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.