Lung epithelial cell-derived IL-25 negatively regulates LPS-induced exosome release from macrophages

Abstract

Background: Acute lung injury (ALI) is a major component of multiple organ dysfunction syndrome (MODS) following pulmonary and systemic infection. Alveolar macrophages (AMϕ) are at the center of ALI pathogenesis. Emerging evidence has shown that cell-cell interactions in the lungs play an important regulatory role in the development of acute lung inflammation. However, the underneath mechanisms remain poorly addressed. In this study, we explore a novel function of lung epithelial cells (LEPCs) in regulating the release of exosomes from AMϕ following LPS stimulation.

Methods: For the in vivo experiments, C57BL/6 wildtype (WT) mice were treated with lipopolysaccharide (LPS) (2 mg/kg B.W.) in 0.2 ml of saline via intratracheal aerosol administration. Bronchoalveolar lavage fluid was collected at 0-24 h after LPS treatment, and exosomes derived from AMϕ were measured. For the in vitro studies, LEPCs and bone marrow-derived Mϕ (BMDM) were isolated from WT or TLR4^-/- mice and were then cocultured in the Transwell™ system. After coculture for 0-24 h, the BMDM and supernatant were harvested for the measurement of exosomes and cytokines.

Results: We demonstrate that LPS induces macrophages (Mϕ) to release exosomes, which are then internalized by neighboring Mϕ to promote TNF-α expression. The secreted interleukin (IL)-25 from LEPCs downregulates Rab27a and Rab27b expression in Mϕ, resulting in suppressed exosome release and thereby attenuating exosome-induced TNF-α expression and secretion.

Conclusion: These findings reveal a previously unidentified crosstalk pathway between LEPCs and Mϕ that negatively regulates the inflammatory responses of Mϕ to LPS. Modulating IL-25 signaling and targeting exosome release may present a new therapeutic strategy for the treatment of ALI.

Keywords: Acute lung injury; Multiple organ failure; Rab27; Sepsis.

Fig. 1

LEPCs suppress LPS-induced exosome release from Mϕ. a Exosomes were isolated from BMDM cultured in serum-free medium, stained with PE-isotype and PE-CD63 antibodies and measured by flow cytometry. b Exosomes isolated from the culture media of control and LPS-treated BMDM were analyzed for the mean particle diameter and total number by NanoSight. c BMDM were treated with LPS (1 μg/ml) for 0, 6, 12, and 24 h. Exosomes were then isolated from the culture medium and detected by CD63 staining and flow cytometry. d BMDM were treated with LPS (1 μg/ml) in the presence or absence of dimethyl amiloride (DMA, 25 μM) for 24 h. Exosomes were then isolated from the culture medium and detected by CD63 staining and flow cytometry. e Lung cells were labeled with biotin-conjugated CD326 antibody and separated with streptavidin-conjugated immunomagnetic beads. Flow cytometric analysis shows a purity of greater than 95% for the yielded LEPCs. f BMDM were cultured alone or cocultured with LEPCs and were either treated with LPS (1 μg/ml) for 24 h or not treated. Exosomes were isolated from the culture medium and detected by CD63 staining and flow cytometry. All results are representative of at least three independent experiments. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups

Fig. 2

IL-25 mediates LEPC-mediated suppression of exosome release from Mϕ. a BMDM were treated with LPS (1 μg/ml) with or without recombinant IL-25 (200 ng/ml), IL-33 (200 ng/ml), or TSLP (200 ng/ml) for 24 h followed by isolation of exosomes from the cell culture supernatants, and CD63+ exosomes were identified by flow cytometry. b BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of anti-IL-25 neutralizing antibody (10 μg/ml) or control non-specific IgG (10 μg/ml). CD63+ exosomes were detected by flow cytometry. c and d LEPCs were treated with LPS (1 μg/ml) for 0, 6, 12, or 24 h, and the IL-25 mRNA expression in the LEPCs and IL-25 protein concentration in the supernatants were measured by RT-qPCR (c) and ELISA (d), respectively. e BMDM were treated with LPS (1 μg/ml) for 24 h, and the cell surface expression of IL-25R was measured by flow cytometry. All results are representative of three independent experiments. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups or with the control

Fig. 3

IL-25 downregulates LPS-induced Rab27a and Rab27b to suppress exosome release from macrophages. a BMDM were transfected with siRab27a, siRab27b, or siNC (control) for 36 h and were then treated with LPS (1 μg/ml) for 24 h. Exosomes were isolated from the culture media, and CD63 staining was detected by flow cytometry. b BMDM were treated with LPS (1 μg/ml) for 0, 6, 12, and 24 h. Rab27a and Rab27b expression were detected by Western blotting. c BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of recombinant IL-25 (200 ng/ml) for 24 h. The expression of Rab27a and Rab27b were detected by Western blotting. All results are representative of three independent experiments. The graphs show the mean ± SEM, n = 3; * or # P < 0.05, ** or ## P < 0.01, compared with the LPS groups. * and ** indicate Rab27a, # and ## indicate Rab27b

Fig. 4

Suppression of exosome release from Mϕ attenuates the secretion of TNFα from Mϕ. a and b BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) for 24 h. Then, TNF-α and IL-6 mRNA levels in the BMDM and protein levels in the supernatants were measured by RT-qPCR and ELISA, respectively. c BMDM were treated with LPS (1 μg/ml) with or without dimethyl amiloride (DMA, 25 μmol/L) for 24 h, and then the TNF-α and IL-6 levels in the supernatants were measured by ELISA. d BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of recombinant IL-25 (200 ng/ml), anti-IL-25 neutralizing antibody (10 μg/ml), or non-specific IgG (10 μg/ml) for 24 h. The level of TNF-α in the supernatants was measured by ELISA. e Immunofluorescence images show Mϕ internalization of exosomes. BMDM were incubated with DiI-labeled exosomes (red) that were isolated from the culture media of untreated or LPS-treated BMDM (1 μl/ml) for 2 h. Nuclei were counterstained with Hoechst (blue). The fold changes in DiI fluorescence intensity were calculated by ImageJ. f Exosomes were isolated from the culture media of untreated or LPS-treated BMDM (1 μl/ml) for 24 h and were then added to WT or TLR4^−/− BMDM and incubated for 6 h. The level of TNF-α in the supernatants was measured by ELISA. All results are representative of three independent experiments. Three random fields in the images were counted. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups or with the control. NS, no significant difference

Fig. 5

IL-25 suppresses exosome release from AMϕ in vivo. a and b WT mice were given intratracheal (i.t.) LPS (2 mg/kg BW in a volume of 100 μl/mouse) or sham (i.t. PBS, 100 μl/mouse) for 24 h, and BALF was collected. IL-25 was measured in the supernatant by ELISA (a) and the surface expression of IL-25R on AMϕ was detected by flow cytometry (b). c WT mice were treated with LPS (2 mg/kg BW in a volume of 100 μl/mouse, i.t.), sham (PBS, 100 μl/mouse, i.t.), LPS (2 mg/kg BW, i.t.) + IgG (1 mg/kg BW, i.t.); or LPS (2 mg/kg BW, i.t.) + anti-IL-25 antibody (1 mg/kg BW, i.t.) for 24 h. Exosomes were isolated from BALF and quantified by CD63 staining and flow cytometry. All results are representative of three independent experiments. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups or with the control