The role of an individual's microbiota in maintaining homeostasis is recognized, with increasing frequency, as a worthwhile subject of study for a variety of conditions including irritable bowel syndrome, Crohn's disease, neuropsychiatric disorders, and substance use disorders [[1], [2], [3], [4]]. These homeostatic effects of the enteric microflora are primarily maintained through the host nervous, immune, and endocrine systems [5,6]. Recent studies have demonstrated that gut-brain communication utilizes the nervous system primarily via vagal nerve stimulation, and to some extent the endocrine system [5,[7], [8], [9]]. However, the mechanisms by which the immune system contributes to gut-brain axis communication and homeostasis appear extensive and condition-dependent.

At the epithelial interface of the gastrointestinal tract, macrophages, dendritic cells (DCs), Peyer's patches containing B and T cells, as well as other members of the immune system are constantly surveying the luminal surface for markers of inflammation: damaged epithelial cells, cytokine/chemokine release, or leakage of viral, protozoal or bacterial markers/cells [10,11]. As one of their many functions, tolerogenic macrophages (i.e., anti-inflammatory/M2) and DCs continuously sample the intestinal lumen for the types (commensal, pathogenic, pathobiontic) and relative compositions of microbial inhabitants presently harbored in order to maintain homeostasis through several mechanisms such as, instigating an inflammatory response via inflammatory cytokine release or presentation of antigen to other immune cells [11]. As DCs and macrophages interact with microbial antigens they are internalized and often trafficked and presented to immune cells in Peyer's patches or to naïve T cells and can thus facilitate differentiation to regulatory phenotypes (e.g., Treg or Th) [10]. Upon sensing commensal microbes, DCs and macrophages produce and excrete tolerogenic cytokines and signals such as TGFβ, IL-10, and GM-CSF [12]. A tolerogenic immune environment may be maintained under homeostatic microbial conditions using these and other mechanisms. Additionally, research indicates that bacteria play an active role in the establishment of a tolerogenic immune environment through vesicle and metabolite excretion [[13], [14], [15]]. Vesicles serve several functions for microbial population persistence; of particular interest is their role in the transmission of information between cells (microbe – host) and subsequent modulation of cellular activities, and reprogramming of recipient cell phenotypes negating the requirement for cell-cell contact [13,14].

There are many factors that can perturb microbe-host interactions and induce disease in the host. Dietary factors, for example, play a significant role in the selection of microbes residing in the gastrointestinal tract (GI tract) [[16], [17], [18]]. Diets consisting of high levels of carbohydrates, particularly sugars, have been shown to preferentially select for the saccharolytic organism, Fusobacterium nucleatum, which has been strongly associated with the formation of pre-gingival biofilms in the oral cavity as well as the development of colorectal cancer from populations residing in the oral cavities and proximal colon of humans, respectively [19,20]. Alternatively, a diet high in fiber will preferentially select for many fiber utilizing bacteria such as the Clostridia facilitating epithelial cell growth and regeneration through the production of short-chain fatty acids (SCFAs), specifically butyrate [21]. Drugs of abuse such as nicotine have also been shown to have a lasting and profound effect on the composition of the microbiome identifiable at nearly every taxonomic level [[22], [23], [24]].

These factors act as environmental stressors to the enteric microflora and can affect successful growth and colonization of the GI tract, bacterial metabolism, and vesiculation. Though these factors have been observed to modify the microbial composition and diversity of the gut, it tends to revert back to its original composition, if not more similar to, after the initial offending factor(s) have subsided [22]. However, chronic nicotine abuse has been correlated with lasting effects on microbial composition and diversity years after cessation [22,25,26].

The majority of studies on the effects of nicotine or tobacco on the enteric microflora have focused on compositional changes associated with smoking status (e.g., current, former, or never). However, there is little research on the mechanism(s) by which nicotine's dysbiotic effects on the microbiome alter microbe-host interactions [27,28]. Recent studies have shown that nicotine confers a degree of protection against Crohn's disease but exacerbates the severity of ulcerative colitis, both of which are inflammatory conditions of the GI tract and are increasingly linked to detrimental changes in the microbiota of these individuals [29,30]. Additionally, nicotine has been shown to place individuals at increased risk for more severe bacterial infections compared to non-smoking populations, which is believed to be a combined effect of the loss of commensal microbes, increased colonization of opportunistic pathogens and dampening of the anti-inflammatory cholinergic pathway via the vagovagal reflex [[31], [32], [33], [34]]. Together these studies suggest that nicotine potentially plays a role in the interaction of the immune system and the enteric microflora of the GI tract. While correlative studies have made clear the relationship between nicotine/tobacco administration, microbial composition changes, and a characteristically tolerogenic immune system, it is likely that nicotine's primary metabolite, cotinine, is responsible for at least a portion of these observations as it shares the anti-inflammatory properties of nicotine and is present for longer periods of time in the GI tract than nicotine [[35], [36], [37], [38], [39]]. Prior to clearance, nicotine and its various metabolites are subject to recirculation via enterohepatic recycling back into the GI tract from the liver [40], where cotinine concentrations in chronic smokers can compound over time (10–40 μM), in part due to cotinine's long half-life and slow removal from the body [36,37].

The objective of this study was to examine how commensal and pathobiontic microbes alter their interactions with the immune system, by measuring changes in vesiculation physiology and metabolism, and subsequently the immune system's response to microbial stimuli through cytokine production of tolerogenic macrophages following chronic exposure to nicotine and its primary metabolite, cotinine.