**5. Conclusions**

The aim of this Review was to evaluate the involvement of nAChRs in the gut-brain axis by examining their role in different physiological and pathophysiological processes of the gastrointestinal tract. The extensive expression of nAChRs by neurons that innervate gastrointestinal organs influences numerous physiological processes including gu<sup>t</sup> motility, sensory detection of signaling molecules released by other neurons, immune cells, and bacteria. Importantly, control of gu<sup>t</sup> inflammation through α7 and α9 nAChRs, the vagus nerve, and the CAP is essential. We note that there is a surprising lack on information concerning several important areas of nAChR research on the gut-brain axis. First, sparse information is available detailing the functional nAChR subtypes expressed by ENS neurons and glial cells. Research on the role of glial cells in general in gu<sup>t</sup> function is also lacking. Determining the subtype composition of these receptors is important in the context of designing pharmacotherapeutics that treat IBD. Gene knock-out of CHRNA3 in mice produces gross ANS dysfunction, and certain human diseases of the gu<sup>t</sup> involve production of antibodies against α3 and β4 subunits. It is highly likely that ENS neurons express the α3β4\* subtype, but it is possible that multiple different subtypes containing α3 and β4 subunits are present, for example α3β2β4\*, α3β4α5\* and α3β2\*, which are highly expressed by other PNS neurons. Each of these nAChRs subtypes may show different sensitivities to ligands. Nevertheless, it is critical that potential drugs used to treat IBD be devoid of activity on α3β4 subtypes to avoid secondary side effects associated with excessive activation or inhibition of α3β4 receptors. Additionally, sparse information is available concerning the expression of the α7 subtype in the ENS and essentially nothing is known about the expression of subtypes containing α9 subunits. Secondly, information about the interaction of commensal bacteria and enterotoxins, produced by pathogenic strains, with nAChRs is lacking. It is known that bacteria from *Firmicutes* species and from *Bacteroides* and *Eubacteria* genera and other commensal bacteria produce SCFAs. These

fatty-acid molecules reach millimolar concentrations in the intestines and are involved in the esterification of choline. Choline and its various derivatives have been shown to modulate the release of cytokines from murine macrophages and human monocytes through α7- and/or α9-containing nAChRs as well as decrease chloride secretion from intestinal epithelia. Alterations in SCFA-producing populations of bacteria may therefore affect activities of nAChRs expressed by sensory neurons innervating the gut, ENS neurons, and immune cells and ultimately affect regulation of gu<sup>t</sup> homeostasis. Lastly, although numerous studies detailing the effects of nicotine on gu<sup>t</sup> microbiota have been reported, little information is available concerning the effects of other nAChR compounds including those listed in Table 2. In the context of pharmacotherapy of IBD with nAChR compounds, it is important to determine the potential effects these compounds might have on commensal bacterial populations. Clearly, more research is needed to elucidate the nAChR subtypes expressed in the gut-brain axis, their interactions with bacteria, and the effects of experimental nicotinic IBD therapeutics on commensal bacteria.

**Author Contributions:** Writing—original draft preparation, L.R.R., J.L.C., A.J.H.; writing—review and editing, L.R.R., J.L.C., A.J.H.; visualization, L.R.R., J.L.C., A.J.H.; project administration, A.J.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Published data for the x-ray crystallography structures of the SARS-CoV-2 spike protein and its peptide fragment are archived in the Protein Data Bank (PDB:6XR8 and PDB:7JJC) at rcsb.org.

**Conflicts of Interest:** The authors declare no conflict of interest.
