*3.2. Gut Dysbiosis*

Gastrointestinal dysbiosis is associated with a number of pathophysiological conditions including neurodegenerative diseases, psychiatric conditions, diabetes, obesity, autism, and IBD [146]. Alterations in the normal populations of intestinal microbiota can allow the proliferation of harmful bacterial strains and the toxins they produce. Celiac disease and IBS are associated with a decrease in intestinal microbial diversity in general, with alterations in *Firmicutes/Bacteroidetes* ratio and in members of the *Proteobacteria* phylum [147,148]. For instance, elevated levels of endotoxins in the bloodstream such as LPS, derived from the outer membrane of gram-negative bacteria, is a common alteration that can cause a severe immune system response that leads to systemic inflammation and sepsis. Peripheral blood mononuclear cells from patients with IBS show elevated levels of pro-inflammatory cytokine release when challenged with LPS from *Escherichia coli* [149]. Similarly, *Clostridium difficile*, the bacteria responsible for diarrhea associated with overuse of certain antibiotics and the etiology of pseudomembranous colitis, attacks the lining of the intestine through the release of toxins A and B. Both toxins induce damage to the intestinal epithelium, increase permeability of the mucosal barrier, and generate an inflammatory response [150,151].

#### *3.3. Effects of nAChR Stimulation by Nicotine on Intestinal Microbiota Populations*

As mentioned above, the composition of commensal intestinal microbiota is essential for proper gastrointestinal function. Alterations in the proportion of certain bacterial strains produce negative impacts that lead to the onset, progression, and/or maintenance of IBDs. In relation to nAChRs, results from several studies have shown a disruptive effect from nicotine on the composition of intestinal microbiota populations in mice [108,152]. During a 9-week smoking cessation period, an increase in *Firmicutes* and *Actinobacteria* and a decrease in *Bacteroidetes* and *Proteobacteria* was found in human fecal samples [153]. In mice, chronic oral administration of nicotine increased bacterial alpha-diversity including members of the *Lactobacillus* and *Lachnospiraceae* genera and *Firmicutes* phylum [108]. Interestingly, administration of nicotine in the drinking water of mice showed a sex-dependent effect on the bacterial composition of the intestinal microbiome [152]. The relative abundance of bacteria from the *Christensenellaceae* and *Anaeroplasmataceae* families showed significant reductions in female mice after a 13-week exposure to nicotine whereas males showed decreased *Dehalobacteriaceae* bacteria. Similarly, daily exposure to tobacco smoke increased cecal *Clostridium clostridiforme* and decreased *Lactoccoci*, *Ruminococcus albus*, *Enterobacteriaceae* and *Bifidobacterium* compared to controls in mice and rats [154,155]. In addition, SCFAs such as butyrate, propionate, and acetate were reduced by the effect of smoke exposure [154]. Activation of the free fatty acid receptor 3 (FFA3) by SCFAs has been shown to reduce colonic motility and abolish chloride secretion involving nAChRs via G protein-coupled receptors in rats [156,157]. Thus, the composition of gu<sup>t</sup> microbiota is essential for maintaining the ability of the host organism to regulate intestinal inflammation and respond to pathogenic organisms that target the intestinal tract. Table 4 lists the effects of nicotine on the bacterial composition of gu<sup>t</sup> microbiota.

**Table 4.** Effects of nicotine on gu<sup>t</sup> microbiota and their function.


#### **4. Potential Involvement of nAChRs in COVID-19 and Associated Dysbiosis**

*The Pathophysiology of COVID-19 May Involve α7 nAChRs and Inhibition of the CAP*

In late December of 2019, a novel strain of coronavirus was reported in Hubei province, China in patients with viral pneumonia and was determined to be similar to other coronaviruses that causes severe acute respiratory syndrome (SARS) [158]. The sequence of this virus, SARS-CoV-2, was quickly determined and showed high similarity to other members of the coronavirus family including SARS-CoV-1 and RaTG13 but with one notable difference [158]. Unlike SARS-CoV-1 and RaTG13, SARS-CoV-2 contains additional residues (681-PRRA-684) between the S1 and S2 domains of the spike protein [159,160]. These residues serve as a cleavage site for the furin enzyme and have been proposed to impart increased infectiousness of SARS-CoV-2 relative to other members of the SARS-CoV family. This hypothesis is controversial, however, and requires further investigation [161,162].

Researchers at the Pasteur Institute and the Sorbonne in Paris, France observed that the sequence of the furin cleavage site along with seven residues (674-YQTQTNS-680) upstream and one arginine-685 residue downstream were similar to a motif found in neurotoxins from *Elapidea* serpents [163] (Figure 2). This motif allows serpent neurotoxins to bind to and inhibit nAChRs, most notably α7 nAChRs, which led Changeux and his colleagues to hypothesize that inhibition of α7 receptors by the SARS-CoV-2 spike protein may contribute to the pathophysiology of COVID-19 and specifically to elevated levels of cytokines. Computational modeling experiments later suggested that the spike protein may potentially interact with receptors that contain α7 subunits and/or α9-containing subtypes [164]. Given the possibility that the spike protein interacts with α7 nAChRs, inhibition of this receptor has been proposed as a contributor to the so-called 'cytokine storm' through inhibition the CAP [163–165].

**Figure 2.** Cartoon representation of the SARS-CoV-2 spike protein trimer (green) showing the proposed domains that interact with α7 and α9α10 nAChRs. Note that residues 675-QTNSPRRARSVA-686 are unresolved in this structure. Residues highlighted in yellow are those that show homology with sequences of the three-finger neurotoxins from *Elapidea* serpents including α-bungarotoxin from *Bungurus multicintus* and α-cobratoxin from *Naja naja* species [163]. Residues highlighted in red have also been proposed to interact with α7 and α9α10 nAChRs [164]. Rendition of the spike protein was accomplished using PyMOL [166] and adapted from Cai et al., 2020 (PDB:6XR8) [167]; rendition of the NSPRRAR sequence was adapted from Daly et al., 2020 (PDB: 7JJC) [168].

SARS-CoV-2 not only produces acute respiratory distress but has shown a propensity for inducing severe dysfunction of neurological, pulmonary, cardiovascular, and gastrointestinal systems. Some patients develop acute gastrointestinal distress including diarrhea and vomiting which initially led to the assumption that patients with IBD would experience more severe gastrointestinal symptoms than those without due to the presence of significant angiotensin-converting enzyme-2 receptor expression in the ileum and colon as suggested by analysis of transcriptomics data [169]. In addition, immunosuppressive therapies are often first-line treatments for IBD. However, analysis of clinical data has, in fact, suggested the contrary leading to speculation that immunotherapies with biologics

and other immune system modulators may actually reduce COVID-19-related symptoms by suppressing the cytokine storm [170–173]. Similarly, pharmacological stimulation of α7 receptors and the CAP has been proposed as a mechanism to 'calm the storm' [174]. As discussed above, α7 is highly involved in inflammatory conditions of the gastrointestinal tract and low expression levels of α7 receptors are associated with worse outcomes in Crohn's, other IBDs, and sepsis [82,92]. The systemic presence of an antagonist of α7 receptors would almost certainly worsen the gastrointestinal symptoms associated with COVID-19 by inhibiting the anti-inflammatory actions of the CAP. Therefore, treatment with an agonist such as nicotine might be beneficial and do two things: (1) bind to the ligand-binding site of α7 receptors and compete with or inhibit spike protein binding while simultaneously activating the receptor, and (2) stimulate the CAP to inhibit the cytokine storm. Indeed, such a treatment has been proposed by several authors [165,175,176].

The gastrointestinal symptoms associated with COVID-19, as experienced by some patients, including increased prevalence of diarrhea and vomiting may cause alterations in the gu<sup>t</sup> microbiome and influence the severity of the disease [177,178]. COVID-19 has been shown to be associated with reduced bacterial diversity in the gu<sup>t</sup> and increased prevalence of harmful strains of bacteria [179]. Analysis of fecal samples from patients with COVID-19 found differences in the gu<sup>t</sup> microbiome in those with high fecal levels of SARS-CoV-2 mRNA compared to those with low levels of mRNA [180]. Specifically, patients with high levels of viral mRNA showed increased prevalence, relative to those with low or no fecal viral mRNA, of *Collinsella aerofaciens* and *Morganella morganii*, bacteria that are associated with opportunistic infections in humans. By contrast, patients with low (or no) detectable levels of viral mRNA showed higher levels of bacteria known to produce SCFAs including members of *Parabacteroides*, *Bacteroides*, and *Lachnospiraceae* families. Therefore, alterations in the gu<sup>t</sup> microbiome in patients with COVID-19 may influence the course and severity of the disease. Treatment of COVID-19 with probiotics to combat such alterations has been suggested as a way to ameliorate COVID-19 symptoms [178,181].
