Chronic alcohol induces M2 polarization enhancing pulmonary disease caused by exposure to particulate air pollution

Abstract

Background: Chronic alcohol consumption causes persistent oxidative stress in the lung, leading to impaired alveolar macrophage (AM) function and impaired immune responses. AMs play a critical role in protecting the lung from particulate matter (PM) inhalation by removing particulates from the airway and secreting factors which mediate airway repair. We hypothesized AM dysfunction caused by chronic alcohol consumption increases the severity of injury caused by PM inhalation.

Methods: Age- and sex-matched C57BL/6 mice were fed the Lieber-DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 8 weeks. Mice from both diet groups were exposed to combustion-derived PM (CDPM) for the final 2 weeks. AM number, maturation, and polarization status were assessed by flow cytometry. Noninvasive and invasive strategies were used to assess pulmonary function and correlated with histomorphological assessments of airway structure and matrix deposition.

Results: Co-exposure to alcohol and CDPM decreased AM number and maturation status (CD11c expression), while increasing markers of M2 activation (interleukin [IL]-4Rα, Ym1, Fizz1 expression, and IL-10 and transforming growth factor [TGF]-β production). Changes in AM function were accompanied by decreased airway compliance and increased elastance. Altered lung function was attributable to elevated collagen content localized to the small airways and loss of alveolar integrity. Intranasal administration of neutralizing antibody to TGF-β during the CDPM exposure period improved changes in airway compliance and elastance, while reducing collagen content caused by co-exposure.

Conclusions: Combustion-derived PM inhalation causes enhanced disease severity in the alcoholic lung by stimulating the release of latent TGF-β stores in AMs. The combinatorial effect of elevated TGF-β, M2 polarization of AMs, and increased oxidative stress impairs pulmonary function by increasing airway collagen content and compromising alveolar integrity.

Keywords: Alcohol; Environmentally Persistent Free Radicals; Macrophage; Particulate; Pollution.

Figure 1

Chronic alcohol ingestion impairs AM maturation and exposure to CDPM increases oxidative stress and suppresses pulmonary AM numbers. Animals were administered control/alcohol diet and exposed to air/DCB-230 as described in text. (A) Total isolated AMs (CD11c+ F4/80+) per lung as determined from the percentage of AMs multiplied by total live isolated cells. (B) Reduced:Oxidized glutathione ratio in the BAL fluid. (C) Mean fluorescent intensity (MFI) values for CD11c and (D) median fluorescent intensity for CD11b on gated AM populations. (E) Percentage of CD11b^hi AMs (F4/80+ CD11c+). Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad).

Figure 2

Chronic alcohol ingestion induces M2 polarization which is exacerbated following exposure to CDPM. (A) Mean fluorescent intensity and representative histogram of IL-10 and IL-4Rα in AMs. (B) M2 gene expression in BAL-isolated AMs. (C) M1 (Nos2) and M2 (Arg1) gene expression in whole lung RNA preps. Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad).

Figure 3

Chronic Alcohol ingestion during exposure to CDPM increases active TGF-β levels in the lung. (A) Total TGF-β in whole lung tissue homogenates. (B) Tgf-β gene expression in BAL-isolated AMs. (C) Percentage of LAP+ AMs in whole lung. Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad).

Figure 4

Exposure of chronic alcohol fed mice to CDPM reduces lung function in a manner consistent with fibrosis. Twenty-four hrs following the final exposure day, animals were assessed for baseline pulmonary function per methods section. (A) Quasi-static compliance (Cst) derived from pressure-volume loops. (B) Newtonian resistance (Rn) and (C) tissue elastance (H) derived from Primewave-8 perturbations. Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad).

Figure 5

Chronic alcohol and exposure to CDPM increases airway collagen. (A) Representative histological sections of mouse lungs with total collagen visualized by Masson’s trichrome staining. (B) Histomorphological assessment of airway collagen content normalized to airway length. Sections analyzed in blindfolded manner. (C) Hydroxyproline assay for total collagen content in whole lung preps. Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad). Scale bar = 100 μm.

Figure 6

CDPM exposure in chronic alcohol fed mice causes loss of alveolar integrity. (A) Representative histological sections of mouse lungs stained by H&E. (B) Alveolar space fraction as determined from total image area minus interstitial fraction. (C) Mean linear intercept measured across the alveoli (3 measurements taken from 6 random images of each replicate). Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column by the letters of the reference control group of the comparison. Control+Air (ca), Alcohol+Air (aa), Control+DCB-230 (cd), Alcohol+DCB-230 (ad). Scale bar = 100μm.

Figure 7

Neutralizing antibody to TGF-β improves pulmonary function and reduces collagen deposition sue to chronic alcohol ingestion and exposure to CDPM. (A) Static compliance (Cst) derived from pressure-volume loops. (B) Newtonian resistance (Rn) and (C) tissue elastance (H) derived from Primewave-8 perturbations. (D) Hydroxyproline assay for collagen content in whole lung preps. Significance (p<0.05, Two-way ANOVA, Bonferroni post-tests) indicated above sample column.