E-cigarette-induced pulmonary inflammation and dysregulated repair are mediated by nAChR α7 receptor: role of nAChR α7 in SARS-CoV-2 Covid-19 ACE2 receptor regulation

Abstract

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

Keywords: Dysregulated repair; E-cig exposure; Extracellular matrix; Inflammation; nAChRα7.

Fig. 1

Sub-chronic e-cig exposure augments inflammatory cell influx in BALF. Differential inflammatory cell counts were measured in BALF from mice exposed to air, PG or PG with nicotine (PG + Nic) for 30 days (2 h/day). (a) Total inflammatory cell counts were analyzed by Cellometer using AO/PI staining. Differential inflammatory cell counts were determined as percentages by flow cytometry. Absolute cell counts for (b) F4/80+ macrophages, (c) Ly6B.2+ neutrophils, (d) CD4a + T-lymphocytes, and (e) CD8a + T-lymphocytes were normalized to the total cell counts. Data are shown as mean ± SEM (n = 4–6/group; equal number of male and female mice). * P < 0.05, ** P < 0.01 between groups; & P < 0.05, compared to PG exposed WT group; # P < 0.05, compared to PG + Nic exposed WT group

Fig. 2

Sub-chronic e-cig exposure-induced pro-inflammatory mediators in BALF. Bio-Plex Pro mouse cytokine 23-plex assay kit (Bio-Rad) was used to determined levels of pro-inflammatory cytokines/chemokines in BALF from mice exposed to e-cig with or without nicotine for 30 days (2 h/day). Significant changes were found in cytokines related to macrophages (IL-1α, MCP-1, TNF-α, GM-CSF, and MIP-1β) and T-lymphocytes (IL-2, IL-5, IL-9, RANTES, and IFN-γ). Data are shown as mean ± SEM (n = 6–10/group; equal number of male and female mice). * P < 0.05, ** P < 0.01, *** P < 0.001, compared to air control; # P < 0.05, compared to PG + Nic exposed WT mice

Fig. 3

Normalized gene distribution and unique/common mRNAs identified among different multiple comparisons. Mice were exposed to e-cig aerosol with or without nicotine for 30 days, and sacrificed 24 hrs later. Lungs were snap-frozen and RNA was isolated for Nanostring analysis. The data set from Nanostring was analyzed using R. (a) Boxplot representing the distribution of genes among multiple groups/comparisons (Air, PG and PG with nicotine in WT and nAChRα7 global KO mice). (b) Venn diagram showing unique or common mRNAs among different exposure groups and mouse genotypes as described in a

Fig. 4

Sub-chronic e-cig exposure affects mRNA expression of myeloid and innate immune response target genes analyzed by Nanostring analysis. Mice were exposed to e-cig aerosol with or without nicotine for 30 days (2 hrs/day), and sacrificed 24 hrs after final exposure. RNA was isolated from lungs and screened via nCounter Mouse Myeloid Innate Immunity Panel using Nanostring analysis. Targets were selected based on significant differences, especially (a). between) PG+Nic exposed WT and nAChRα7 KO mice, or (b) ECM remodeling focused. RNA counts were normalized to multiple housekeeping genes and quantified using nSolver. Data are shown as mean ± SEM (n=6/group; equal number of male and female mice). * P < 0.05, ** P < 0.01, *** P < 0.001 between groups; # P < 0.05, # # P < 0.01 compared with PG+Nic exposed WT group

Fig. 5

Sub-chronic e-cig exposure lead to dysregulateddifferentially affects the protein abundance of NF-κB subunits (p50/p105) and Angiotensin-converting enzyme 2 (ACE2) in mouse lungs with sex differences. The protein abundance of (a) p50/p105 and (b) Angiotensin-converting enzyme 2 (ACE2) were measured in whole lung homogenates via Western blot. Representative blot images for female and male mice are shown. Densitometry analysis of individual blots was performed for p50/p105 and ACE2 for both female and male, and β-actin was used as an endogenous control. Data are shown as mean ± SEM. (n=4-5/group). * P < 0.05 significant compared between groups; P < 0.05, compared with air exposed WT group; # P < 0.05, compared with PG+Nic exposed WT group

Fig. 6

Sub-chronic e-cig exposure affects protein abundance of matrix metalloproteinases (MMPs) in mouse lungs. Protein levels of several MMPs (MMP9, MMP2, MMP12, and MMP8) were measured in whole mouse lung homogenates via Western blot. Representative blot images and densitometry analyses for female (a) and male (b) mice are shown. β-actin was used as an endogenous control. Data are shown as mean ± SEM. (n = 4–5/group). * P < 0.05 between groups; $ P < 0.05, compared with air exposed WT group; & P < 0.05, compared with PG exposed WT group. # P < 0.05, compared with PG + Nic exposed WT group

Fig. 7

Sub-chronic e-cig exposure affects protein abundance of ECM-related markers in mouse lungs. The abundance of ECM proteins (PAI-1, COL1A1, COL1A2, and fibronectin) were measured in whole mouse lung homogenates via Western blot. Representative blot images and densitometry analyses for female (a) and male (b) mice are shown. β-actin was used as an endogenous control. Data are shown as mean ± SEM. (n = 4–5/group). (* P < 0.05 between groups; $ P < 0.05, compared with air exposed WT group; & P < 0.05, compared with PG exposed WT group. # P < 0.05, compared with PG + Nic exposed WT group)