Alveolar macrophages from EVALI patients and e-cigarette users: a story of shifting phenotype

Abstract

Exposure to e-cigarette vapors alters important biologic processes including phagocytosis, lipid metabolism, and cytokine activity in the airways and alveolar spaces. Little is known about the biologic mechanisms underpinning the conversion to e-cigarette, or vaping, product use-associated lung injury (EVALI) from normal e-cigarette use in otherwise healthy individuals. We compared cell populations and inflammatory immune populations from bronchoalveolar lavage fluid in individuals with EVALI to e-cigarette users without respiratory disease and healthy controls and found that e-cigarette users with EVALI demonstrate a neutrophilic inflammation with alveolar macrophages skewed towards inflammatory (M1) phenotype and cytokine profile. Comparatively, e-cigarette users without EVALI demonstrate lower inflammatory cytokine production and express features associated with a reparative (M2) phenotype. These data indicate macrophage-specific changes are occurring in e-cigarette users who develop EVALI.

Keywords: Alveolar macrophages (AM); Bronchoalveolar lavage (BAL); E-cigarette; Or vaping; Product use-associated lung injury (EVALI).

Fig. 1

Increased neutrophils and MPO are detected in EVALI subjects. Healthy and e-cigarette controls were recruited as controls for EVALI subjects that had been admitted to the hospital for acute lung injury. Bronchoalveolar lavage fluids were collected following standard SPIROMICS procedures and cellular content was separated from supernatant. (A) Cytospins were prepared from the separate cells and eosinophils (EOS), lymphocytes (Lymphs), neutrophils (PMNs), Macrophages (MACs) and airway epithelial cells were quantified. At least 20–50 cells were counted field, 4–6 fields were counted per slide. Results as displayed as mean ± SEM. For healthy controls n = 7, e-cigarette controls (e-Cig) n = 13, and EVALI subjects n = 10. Statistical significance was determined by ordinary one-way ANOVA followed by Dunnett’s multiple comparison post-test to determine between groups differences

Fig. 2

Composition of immune lineage cells differ in EVALI compared to otherwise healthy e-cigarette controls. Flow cytometric analysis was completed using a macrophage specific flow panel that included a viability dye, and antibodies specific for CD45, CD11c, CD11b, CD14, CD16, CD206, CD64 and CD163. (A)-(C) Total immune cells were detected as CD45 + cells in three groups healthy participants (A; healthy), e-cigarette controls (B; e-Cig), and EVALI subjects (C; EVALI). (D) The counts of CD45 + cells were normalized per mL of returned lavage fluid and shown as count/mL. (G-I) gating schematic and counts of (E) dendritic cells and (F) lymphocytes per mL of returned BAL fluid. (M-O) quantitation for (M) CD16^lo and (N) CD16^high inflammatory monocytes and (O) neutrophils and (J-L) the representative flow gating for each group. (J-L) shows the representative gating for alveolar macrophages, populations are contained in the dark red box. The quantitation of (P) total alveolar macrophages (aMacs), (Q) CD163- M1 macrophages and (R) CD163 + M2 macrophages. The composition of total BAL of each of the above cell populations are displayed in S-U for each group. Statistical significance was determined using an ordinary One-Way ANOVA with Dunnett’s post-test to determine between groups differences; *indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. Sample size is n = 7 for healthy controls, n = 13 for e-Cig controls and n = 10 for EVALI subjects

Fig. 3

Markers of inflammation and airway leak are increased in EVALI subjects compared to e-cigarette and healthy controls. As above, BAL was precleared of cellular content and analyzed for markers of inflammation; (A) TNFα, (B) IL-6, (C) IL-8, (D) CCL2, (E) soluble ICAM and (F) RAGE by ELISA. Markers of airway leak were also detected; (G) C reactive protein (CRP), (H) serum amyloid A (SAA), and total protein (BCA; µg/mL). Statistical significance was determined using an ordinary One-Way ANOVA with Dunnett’s post-test to determine between groups differences; *indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Sample size is n = 7 for healthy controls, n = 13 for e-Cig controls and n = 10 for EVALI subjects