Cigarette Smoke-Induced Pulmonary Inflammation Becomes Systemic by Circulating Extracellular Vesicles Containing Wnt5a and Inflammatory Cytokines

Abstract

Chronic obstructive pulmonary disease (COPD) is a devastating, irreversible pathology affecting millions of people worldwide. Clinical studies show that currently available therapies are insufficient, have no or little effect on elevated comorbidities and on systemic inflammation. To develop alternative therapeutic options, a better understanding of the molecular background of COPD is essential. In the present study, we show that non-canonical and pro-inflammatory Wnt5a is up-regulated by cigarette smoking with parallel up-regulation of pro-inflammatory cytokines in both mouse and human model systems. Wnt5a is not only a pro-inflammatory Wnt ligand but can also inhibit the anti-inflammatory peroxisome proliferator-activated receptor gamma transcription and affect M1/M2 macrophage polarization. Both Wnt5a and pro-inflammatory cytokines can be transported in lipid bilayer sealed extracellular vesicles that reach and deliver their contents to every organ measured in the blood of COPD patients, therefore, demonstrating a potential mechanism for the systemic nature of this crippling disease.

Keywords: Wnt5a; chronic obstructive pulmonary disease; extracellular vesicles; inflammatory cytokines; peroxisome proliferator-activated receptor gamma.

Figure 1

Effects of cigarette smoke (CS) on C57BL/6 mouse lungs. (A) Hematoxylin-eosin staining of paraffin embedded sections of healthy control and CS exposed lungs. Mice were exposed to CS for 1 h/day for 2 months. (Scale bars, 100 µm.) (B) Changes in EpCAM⁺ and CD45⁺ cell populations in control and CS exposed lungs determined by flow cytometry. (C) Wnt5a mRNA expression changes in sorted EpCAM⁺ and CD45⁺cells following 1 and 2 months of CS exposure (n = 3). (D) Western blot analysis of Wnt5a protein expression in control and CS exposed lung tissue extracts. (E) Detection of Wnt5a protein expression in control and CS exposed lung tissue sections [EpCAM⁺ (green)/Wnt5a (red)/Nucleus (blue), Scale bars, 200 µm].

Figure 2

Effects of cigarette smoke extracts (CSE) on primary human 3D lung aggregate cultures. (A) Wnt5a mRNA was measured by qRT-PCR in control and CSE treated (48 h) aggregate cultures containing and not containing macrophages (M). (B) Fluorescence intensity of Wnt5a protein was analyzed by ImageJ. (C) Immunofluorescence staining of Wnt5a (green) and Cytokeratin7 (red) proteins in frozen sections of control aggregates and CSE (48 h) exposed aggregates not containing M. (D) Immunofluorescence staining of Wnt5a (green) and Cytokeratin7 (red) proteins in frozen sections of control aggregates and CSE (48 h) exposed aggregates containing M. Nuclei were stained by TO-PRO-3 Iodide (1:1,000, pseudo-blue). [Scale bars: 100 µm (magnification, 20×), 20 µm (magnification, 63×)].

Figure 3

Inflammatory cytokine and Wnt mRNA levels in primary human 3D lung aggregate cultures and human macrophages (M) after cigarette smoke extracts (CSE) exposure. (A) IL-6 and IL-8 inflammatory cytokine mRNA levels in control and CSE exposed (48 h) lung aggregate cultures, containing and not containing M. (B) IL-6 and IL-8 inflammatory cytokine mRNA levels in control and rWnt5a (1 µg/ml) treated lung aggregate cultures, containing and not containing M. (C) Taqman array heat map analysis of pooled (n = 4) cDNA of human M compared with control. M were treated with CSE for 3 h. (D) Changes in mRNA levels of Wnt signaling pathway genes measured by Taqman Array analysis using pooled (n = 4) cDNA samples of human M compared with control.

Figure 4

Changes of peroxisome proliferator-activated receptor (PPAR) gamma mRNA levels in M1 and M2 M type differentiation in primary human 3D lung aggregate cultures and M monocultures. (A) qRT-PCR analysis of M1 (differentiation marker IL-23) and M2 (differentiation marker IL-10) after human rWnt5a (1 µg/ml) treatment for 48 h. (B) Effects of cigarette smoke extracts (CSE), PPAR gamma agonist (RSG), and antagonist (GW9662) treatment on expression of M1 (IL-23) and M2 (IL-10) differentiation markers. CSE treatment (48 h) and also CSE + RSG (10 µM) and CSE + GW9662 (10 µM) treatment on human M. (C) PPAR gamma mRNA levels in CSE treated human macrophage cultures after 3 and 48 h of CSE treatment compared with controls.

Figure 5

Wnt5a and proinflammatory cytokine levels in extracellular vesicles (EV-s). (A) Wnt5a protein levels were measured in EV-s in sera of healthy donors and chronic obstructive pulmonary disease (COPD) patients as well as EV-s of A549 and Wnt5a-A549 cell lines as controls using a Wnt5a-specific enzyme-linked immunosorbent assay kit. (B) Inflammatory cytokine levels were measured in pooled megavesicles/oncosome samples of five healthy and five COPD patients isolated from human sera measured by cytometric bead array Flex kit using flow cytometry. (C) Inflammatory cytokine proteins were detected in pooled microvesicle/exosome samples of five healthy and five COPD patients isolated from human sera. (D) Fluorescent staining (Dil) labeled Wnt5a-containing EV-s are detectable in various organs (representative image of n = 3). (E) Fluorescence intensity of Wnt5a containing EV-s in various organs (representative data of n = 3). (F) Electron microscopic image of purified EV-s (microvesicle/exosome population) purified from the Wnt5a-A549 cell line. Scale bar is 100 nm.

Figure 6

Summary figure of cigarette smoke induced local inflammation becoming systemic by the contribution of Wnt5a and inflammatory cytokines.