Epithelial cell-derived microvesicles activate macrophages and promote inflammation via microvesicle-containing microRNAs

Abstract

Intercellular communications between lung epithelial cells and alveolar macrophages play an essential role in host defense against acute lung injury. Hyperoxia-induced oxidative stress is an established model to mimic human lung injury. We show that after hyperoxia-associated oxidative stress, a large amount of extracellular vesicles (EVs) are detectable in bronchoalveolar lavage fluid (BALF) and culture medium of lung epithelial cells. Microvesicles (MVs), but not exosomes (Exos) or apoptotic bodies (Abs), are the main type of EVs found in the early stages after hyperoxia. Among all the MV compositions, small RNAs are altered the most significantly after hyperoxia-associated oxidative stress. We further confirmed that hyperoxia up-regulates the levels of certain specific miRNAs in the epithelial cell-derived MVs, such as the miR-320a and miR-221. Functionally, the hyperoxia-induced epithelial MVs promote macrophage activation in vitro and facilitate the recruitment of immunomodulatory cells in vivo detected in BALF. Using MV as a cargo, delivery of the specific miRNA-enriched epithelial MVs (miR-221 and/or miR-320a) also triggers macrophage-mediated pro-inflammatory effects. Collectively, epithelial cell-derived MVs promote macrophage-regulated lung inflammatory responses via MV-shuttling miRNAs.

Figure 1. Characterization of the extracellular vesicles (EVs) generated from BALF and lung epithelial cells.

(A,B) Three types of EVs were isolated from mouse broncho-alveolar lavage fluid (BALF), including apoptotic bodies (ABs), microvesicles (MVs), and exosomes (Exos). The sizes of the isolated EVs were measured using Dynamic Light Scattering (DLS) (A). Pie graph indicates the percentages of each type of EVs derived from BALF (B). (C) Mice were exposed to hyperoxia for 3 days, followed by isolation of three types of EVs from BALF. Amount of EVs were shown in the bar graph. n = 4 mice per group. Bease2B cells (D) and primary epithelial cells (F) were exposed to hyperoxia for 2 days, followed by isolation of EVs. Left panels show pie graphs indicating the percentages of each type of EVs. Right panels show the protein amounts of EVs in bar graphs. (E) Three types of EVs were isolated from Beas2B cells, and visualized using transmission electron microscope (TEM). Data represent mean ± SD of three independent experiments with identical results.

Figure 2. RNAs are accumulated in MVs in response to hyperoxia.

(A) Heat map of the miRNA profiles in the MVs which were isolated from BALF. (B–E) Mice and Beas2B cells were exposed to hyperoxia for 2 days. MVs and Exos were isolated from BALF (B,C) and cultured medium of the Bease2B cells (D,E) followed by isolation of total RNAs from the MVs or Exos. Proteins, RNAs, and normalized RNA using protein amounts were shown. Data represent mean ± SD of three independent experiments with the similar results.

Figure 3. Quantification of RNA and protein amount in each MV.

(A) MVs were isolated from Beas2B cells, and labeled with carboxyfluorescein succinimidyl ester (CFSE). Indicated protein amount of MVs were fixed and visualized using fluorescence microscope. Vesicles in field (20x magnification) were counted using NIH ImageJ software (right panel). (B) A liner calibration standard curve (particle number vs. count). The counts of particles with the pre-set numbers were measured using DLS, followed by generation of a linear calibration curve (R² = 0.9978). For A and B, data representative of two independent experiments. (C–E) MVs were isolated from the Beas2B cells after hyperoxia. Total MV number (C) total MV protein amount (D) and protein amount per MV (total MV protein/MV counting numbers) (E) were shown. (F–H) Large and small RNAs were purified from the isolated MVs and subjected to electrophoresis using agarose gel, as illustrated in (F). Total RNA amount is shown in (G). RNA amount per MV (total MV RNA/MV counting numbers) are shown in (H). For C–H, data represent mean ± SD of three independent experiments.

Figure 4. Lung epithelial cell-derived MVs induce macrophage migration and cytokine release.

(A) Mice were exposed to hyperoxia for the designated time period. BALF cells were stained with hematoxylin and eosin (H&E), followed by cell counting. n = 4 mice per group. (B) BALF-derived MVs were isolated from mice 3 days after hyperoxia. BALF cells were stained with H&E and analyzed 24 h after exposed to the RA-MVs and hyperoxia-induced MVs (10 μg/50 μl per mice) intranasally. n = 4 mice per group. (C) MVs were isolated from the Beas2B cells 2 days after hyperoxia. THP1 macrophages were treated with the Beas2B cell-derived MVs (5 μg/500 μl per well). After 16 h, transwell migration assay of THP1 macrophages was performed, as described in Material and Methods. Representative images of migrated cells (left panel) and quantification graphs (right panel) are shown. (D) THP1 macrophage were treated with isolated MVs (5 μg/500 μl per well) described in (B). After 16 h, TNF-α, IL-1β, and IL-10 were analyzed using ELISA. For C and D, data represent mean ± SEM of three independent experiments.

Figure 5. BALF- and lung epithelial cell-derived MVs contain pro-inflammatory miRNAs.

(A) Elevated miRNAs derived from BALF MVs after hyperoxia. The table was generated from miRNA microarray data as shown in Fig. 2A. (B,C) MVs were isolated from Beas2B cells (B) and primary epithelial cells (C). RNA was isolated from the MVs and quantified using the real-time quantitative RCR (qPCR). Individual miRNA expression levels were shown in bar graphs (D). Transwell migration assay of THP1 macrophages was performed after treated with mir-221 and/or mir-320a mimics which were transfected using lipofectamine 2000, as described in Material and Methods. (E–G) THP1 macrophages were treated with mir-221 and/or mir-320a mimics. After 16 h, MMP-9 levels (E) and TNF-α levels (F) from the culture medium were analyzed using gelatin zymography and ELISA, respectively. Western blot analysis was performed using total cell lysates with the indicated antibodies (G). For (B–F) data represent mean ± SD of three (D–F) or four (B,C) independent experiments. For (G) data represent two independent experiments with the similar results. Unprocessed original scans of gel and blots are shown in Suppl. Fig. 1.

Figure 6. Synergistic effects of MV-containing mir-221 and 320a on the macrophage migration and cytokine secretion.

(A) Schematic illustration of the functional analysis of MV-containing miRNAs on the macrophage activation. MVs were isolated from the mouse primary lung epithelial cells and directly transfected with mir-221 and/or 320a as described in Material and Methods. Mouse BMDMs were treated with the transfected MVs and the macrophage migration and cytokine secretion were measured. (B) MV-containing RNAs derived from the primary epithelial cells were labeled with acridine orange and incubated with the mouse BMDMs. After 16 h, MV-RNAs were visualized using fluorescent microscope. The MV internalization was shown in the upper panels. Differential Interference Contrast (DIC) cell images were shown in the lower panels. (C–F) Mouse BMDMs were treated with the transfected MVs (5 μg/500 μl per well) described in (A). After 16 h, BMDM migration was measured using the transwell assay, as described in Material and Method (C). Representative images of migrated cells (upper panel) and quantification graphs (lower panel) are shown. MMP-9 levels (D) and TNF-α levels (E) from the culture medium were analyzed using gelatin zymography and ELISA, respectively. Western blot analysis was performed using total cell lysates (F) with the indicated antibodies. For (C–E) data represent mean ± SD of three independent experiments. For (B,F) data represent two independent experiments with the similar results. Unprocessed original scans of gel and blots are shown in Suppl. Fig. 1.