Cigarette smoke (CS) exacerbates symptoms in Crohn’s disease (CD) patients while protecting those with ulcerative colitis (UC). CD has been associated with immuno-dysregulation, mucosal dysfunction, and infection. Among the CD-debated pathogens are Mycobacterium avium subsp. paratuberculosis (MAP), adherent invasive Escherichia coli (AIEC), and Klebsiella pneumoniae. The mechanism of how CS modulates nicotinic acetylcholine receptor-α7 (α7nAChR) and elicits inflammatory response in CD-like macrophages is unknown. Here, we investigated the effect of CS/nicotine on macrophages infected with CD-associated pathogens. We measured apoptosis, bacterial viability, macrophage polarization, and gene expression/cytokine levels involved in macrophage response to nicotine/CS extracts from Havana-Leave extract (HLE-nicotine rich) and germplasm line of Maryland tobacco (LAMD-nicotine less). Nicotine (4 µg/mL) and HLE extracts (0.18%) significantly favored anti-inflammatory response in macrophages (increased CD-206 (M2) and IL-10, and decreased M1/M2 ratio; p < 0.05). While macrophages infected with MAP or treated with LPS promoted pro-inflammatory response. Further treatment of these macrophages with nicotine or HLE extracts caused higher inflammatory response (increased iNOS (M1), TNF-α, IL-6, and M1/M2 ratio, p < 0.05), increased MAP burden, and decreased apoptosis. Pre-conditioning macrophages with nicotine ahead of infection resulted in lower pro-inflammatory response. Blocking α7nAChR with an antagonist voided the effect of nicotine on macrophages. Overall, the study provides an insight toward understanding the contradictory effect of nicotine on Inflammatory Bowel Disease patients and about the mechanistic role of α7nAChR in modulation of macrophages in tobacco smokers.
Cigarette smoke (CS) has adverse effects in patients with Crohn’s disease (CD), an inflammatory bowel disease (IBD) that has been associated with microbial infection, immuno-dysregulation, and mucosal dysfunction. However, CS seems to provide relief and protection to patients with another IBD known as ulcerative colitis (UC). These two subsets are featured as M1- and M2-mediated responses, respectively. Nicotine is the most active, addictive, and studied ingredient in CS. The mechanism of how nicotine and/or other CS ingredients induce pro-inflammatory or anti-inflammatory phenotypes in IBD patients remains under investigation. Our most recent in vitro nicotine study provided significant insights toward understanding the contradictory effects of nicotine on IBD patients, and it elucidated the mechanistic role of α7nAChR in modulation of macrophages in tobacco smokers. Shifting the beneficial effect of nicotine to a harmful outcome in CD patients was linked to a nicotine-microbe interaction that supports a microbial etiology in CD pathogenesis. Among the most debated pathogens in CD etiology is Mycobacterium avium subspecies paratuberculosis (MAP). Other studies associated nicotine with upregulation of miR-124 expression in macrophages, which led to anti-inflammatory response. This review discusses published work on the role of nicotine in modulation of the innate immune response and subsequent signaling in macrophages in IBD subsets.
Recently, we reported that cigarette smoking, and especially nicotine, increases susceptibility to mycobacterial infection and exacerbates inflammation in patients with Crohn’s disease (CD). The macrophagic response to Mycobacterium avium subspecies paratuberculosis (MAP) in CD and Mycobacteria tuberculosis (MTB) continues to be under investigation. The role of toll-like-receptors (TLRs) and cytoplasmic adaptor protein (MyD88) in proinflammatory response during Mycobacterial infection has been suggested. However, the mechanism of how nicotine modulates macrophage response during infection in CD and exacerbates inflammatory response remain unclear. In this study, we elucidated the mechanistic role of nicotine in modulating MyD88-dependent/TLR pathway signaling in a macrophage system during mycobacterial infection. The data demonstrated that MAP infection in THP-1 derived macrophages was mediated through TLR2 and MyD88 leading to increase in IL-8 in expression and production. On the other hand, LPS-representing, Gram-negative bacteria mediated macrophage response through TLR4. Blocking TLR2 and TLR4 with antagonists voided the effect of MAP, and LPS, respectively in macrophages and reversed response with decrease in expression of iNOS, TNF-α and IL-8. Interestingly, nicotine in infected macrophages significantly (1) downregulated TLR2 and TLR4 expression, (2) activated MyD88, (3) increased M1/M2 ratio, and (4) increased expression and secretion of proinflammatory cytokines especially IL-8, as seen in CD smokers. We also discovered that blocking macrophages during MAP infection with MyD88 antagonist significantly decreased response which illustrates the key role for MyD88 during infection. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response and decreased MAP viability, which clearly validate the inflammatory role of nicotine in macrophages through TLR2/MyD88 pathway during infection. This is the first report to describe the mechanism by which nicotine modulates TLR2/MyDD88 and exacerbates inflammation in CD smokers associated with infection.
Recently, we reported that nicotine plays a role in the failure of the macrophage in the clearance of Mycobacterium avium subspecies paratuberculosis (MAP) during infection in Crohn’s disease smokers. We also demonstrated that nicotine enhances macrophages cellular survival during MAP infection. Blocking α7 nicotinic acetylcholine receptor (α7nAChR) with the pharmacological antagonist—mecamylamine—subverted the anti-inflammatory effect of nicotine in macrophages. Yet, it is still unknown how α7nAChR is involved in the modulation of the macrophage response during MAP infection. Here, we studied the mechanistic role of nicotine-α7nAChR interaction in modulating NF-ĸB survival pathway, autophagy, and effect on cathelicidin production in MAP-infected macrophages using THP-1 cell lines. Our results showed that nicotine upregulated α7nAChR expression by 5-folds during MAP infection compared to controls. Bcl-2 expression was also significantly increased after nicotine exposure. Moreover, Nicotine inhibited autophagosome formation whereas infection with MAP in absence of nicotine has significantly increased LC-3b in macrophages. Nicotine also further upregulated NF-ĸB subunits expression including Rel-B and p100, and increased nuclear translocation of p52 protein. We also discovered that cathelicidin production was significantly suppressed in MAP-infected macrophages, treatment with nicotine showed no effect. Overall, the study provides new insight toward understanding the cellular role of nicotine through α7nAChR/NF-ĸB p100/p52 signaling pathway in inducing anti-apoptosis and macrophage survival during MAP infection in Crohn’s disease smokers.
<p>Supplementary Figure S3: RAB27B is required for NSCLC tumorigenicity in vivo</p>
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