A non-invasive strategy for suppressing asthmatic airway inflammation and remodeling: Inhalation of nebulized hypoxic hUCMSC-derived extracellular vesicles

Abstract

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extremely promising nanoscale cell-free therapeutic agents. We previously identified that intravenous administration (IV) of human umbilical cord MSC-EVs (hUCMSC-EVs), especially hypoxic hUCMSC-EVs (Hypo-EVs), could suppress allergic airway inflammation and remodeling. Here, we further investigated the therapeutic effects of Hypo-EVs administration by atomizing inhalation (INH), which is a non-invasive and efficient drug delivery method for lung diseases. We found that nebulized Hypo-EVs produced by the atomization system (medical/household air compressor and nebulizer) maintained excellent structural integrity. Nebulized Dir-labeled Hypo-EVs inhaled by mice were mainly restricted to lungs. INH administration of Hypo-EVs significantly reduced the airway inflammatory infiltration, decreased the levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF), declined the content of OVA-specific IgE in serum, attenuated the goblet cell metaplasia, and the expressions of subepithelial collagen-1 and α-smooth muscle actin (α-SMA). Notably, Hypo-EV INH administration was generally more potent than Hypo-EV IV in suppressing IL-13 levels and collagen-1 and α-SMA expressions. RNA sequencing revealed that various biological processes, such as cell adhesion, innate immune response, B cell activation, and extracellular space, were associated with the activity of Hypo-EV INH against asthma mice. In addition, Hypo-EVs could load exogenous miR-146a-5p (miR-146a-5p-EVs). Furthermore, INH administration of miR-146a-5p-EVs resulted in a significantly increased expression of miR-146a-5p mostly in lungs, and offered greater protection against the OVA-induced increase in airway inflammation, subepithelial collagen accumulation and myofibroblast compared with nebulized Hypo-EVs. Overall, nebulized Hypo-EVs effectively attenuated allergic airway inflammation and remodeling, potentially creating a non-invasive route for the use of MSC-EVs in asthma treatment.

Keywords: asthma; extracellular vesicles; inhalation device; lung injury; mesenchymal stem cells; nebulized administration.

Figure 1

Construction of atomization inhalation system for mice. (A) atomization inhalation mask commonly used by human beings. (B) Six-way pipe inhalation chamber for mice depicting the flow pattern of the aerosol. This chamber consisted of three parts (inlet pipe, six-way pipe and base fixing device). (C) Holding chamber for mouse. (D) medical/household air compressor and nebulizer set-up. (E) Working diagram of mouse atomization inhalation system.

Figure 2

Characterization of Hypo-EVs. (A) The flowchart shows the process that nebulized Hypo-EVs ejected from nebulizer were liquefied by using an ice-cold centrifuge tube. (B) Western blot analysis of TSG101 and HSP70 expression in nebulized Hypo-EVs and fresh extracted Hypo-EVs. (C) EVs were observed under a transmission electron microscope (Scale bars = 200 nm), the images are shown at ×60000, and the size distributions were measured using the Nanoparticle Tracking Analysis.

Figure 3

Biodistribution of Dir-labeled Hypo-EVs in major organs (brain, heart, liver, spleen, lung, kidney, stomach, and intestines) was visualized at 1 and 7 day after nebulized inhalation administration.

Figure 4

Inhalation of nebulized Hypo-EVs attenuated OVA-induced chronic airway inflammation in mice. (A) Experimental protocol for the development of chronic allergic asthma and treatment with Hypo-EVs. (B) Representative photographs of HE stained lung sections from each group (black bar =100 μm), the images are shown at ×200 (up panel) and ×400 (down panel), and the inflammatory infiltration was quantified by inflammation score. (C) Statistical analysis of the total inflammatory cells and eosinophils in the BALF. (D–F) IL-4, IL-5 and IL-13 levels in the BALF were measured by using ELISA. (G) The levels of OVA-specific IgE levels in serum were analyzed using ELISA. EVs-INH, Hypo-EV nebulization inhalation; EVs-IV, Hypo-EV intravenous injection. Each dot represents data from one animal and n = 4-6 per group. One-way analysis of variance (Tukey Kramer post hoc tests): **P < 0.01, ***P < 0.001.

Figure 5

EVs-INH treatment prevented the airway remodeling in chronic asthma mice. (A) periodic acid-schiff (PAS) stained lung sections from each group (black bar = 100 μm), the images are shown at ×200 (up panel) and ×400 (down panel), and the goblet cell hyperplasia was quantified by PAS score. (B) Representative photomicrographs of airway stained with Masson trichrome (black bar = 100 μm), the images are shown at ×200 (up panel) and ×400 (down panel), and the percentage of collagen fiber content in airway was measured. Collagen-1 (C) and α-SMA (D) levels in airway were determined by immunohistochemistry staining, the images are shown at ×200 (up panel) and ×400 (down panel), and the percentage of immunostained area was quantified. Each dot represents data from one animal and n = 4-6 per group. One-way analysis of variance (Tukey Kramer post hoc tests): *P < 0.05, ***P < 0.001.

Figure 6

The differential expressed (DE) mRNAs in lungs between EVs-INH-treated mice and OVA-induced asthma mice. (A) Heat-map was constructed for DE miRNAs in EVs-INH vs OVA. (B) Real-time PCR verification of several of the lowlist differentially expressed genes. (C) GO analysis of the differentially up and down expressed genes in lungs between EVs-INH-treated mice and OVA-induced asthma mice. The top-ten statistically signifificant results identifified including biological process (BP), cellular component (CC), and molecular function (MF) are listed. Each dot represents data from one animal and n = 4-6 per group. Student’s t-test: *P < 0.05, **P < 0.01.

Figure 7

Inhalation of Hypo-EVs carrying miR-146a-5p (miR-146a-5p-EVs) more inhibited airway inflammation and remodeling in chronic asthma mice. (A) Relative miR-146a-5p levels in miR-146a-5p-EVs and NC-EVs were detected by real-time PCR (n=3). (B) Levels of miR-146a-5p in major organs (brain, heart, liver, spleen, lung, kidney, stomach, and intestines) following administration by aerosol miR-146a-5p-EVs and NC-EVs (n=3). Representative photographs of HE (C), PAS (D), and Masson trichrome (E) stained lung sections from each group (black bar = 100 μm), the images are shown at ×200 (up panel) and ×400 (down panel), and respectively inflammatory score, PAS score and and the percentage of collagen fiber content in airway were quantified (n=6). (F) The protein levels of TRAF6 and TIRAP in the lung samples were analyzed by western blot, and the GAPDH was as a loading control (n=3). Each dot represents data from one animal. Student’s t-test: *P < 0.05, **P < 0.01.

Figure 8

Biocompatibility evaluation of EVs-INH (n=6). (A) Schematic diagram for EVs-INH treatment. (B) HE staining, scale bar=100 μm, and the images are shown at ×200. (C) Mouse body weights. Indicators reflected the physiological function of the (D) liver (ALT and AST) and (E) kidney (CREA and UREA) were detected. ALT, glutamic pyruvic transaminase; AST, glutamic oxaloacetic transaminase; CREA, Creatinine. Each dot represents data from one animal and n = 6 per group. Student’s t-test.