Hirsutella sinensis mycelium regulates autophagy of alveolar macrophages via TLR4/NF-κB signaling pathway

Abstract

Background: Hirsutella sinensis mycelium (HSM) has potent anti-pulmonary fibrotic activities and has been proposed as an effective treatment for idiopathic pulmonary fibrosis. Macrophages are the main innate immune cells in the lung tissue, playing key roles in pulmonary fibrosis repair and homeostasis. Excessive macrophage autophagy plays a vital role in pulmonary fibrosis. The protective effect of HSM on macrophages of bleomycin (BLM)-induced pulmonary fibrotic mice remain unclear. Methods: In this study, we collected lung tissue and bronchoalveolar lavage fluid (BALF) samples from pulmonary fibrotic mice. Meanwhile, alveolar macrophages were isolated and murine macrophage RAW264.7 cell line was cultured for further study of HSM autophagy. Results: First, we found that HSM decreased the number of autophagosomes, as well as the levels of LC3B and ATG5, and increased the protein level of P62 during the development of pulmonary fibrosis. Meanwhile, HSM reduced alveolar macrophages infiltration into the BALF and inhibited their accumulation in the fibrotic lung tissue. Flow cytometry analysis showed that HSM administration inhibited the autophagy marker LC3B expression in CD11b^loCD11c^hi alveolar macrophages in BLM-induced lung fibrosis without affecting CD11b^hiCD11c^lo interstitial macrophages. Transmission electron microscopy and JC-1 staining for mitochondrial membrane potential of alveolar macrophages also verified that the HSM significantly decreased autophagy in the alveolar macrophages of BLM-treated mice. In vitro, autophagosomes-lysosome fusion inhibitor chloroquine (CQ) was pre-incubated with RAW264.7 cells, and HSM reduced CQ-induced autophagosomes accumulation. TLR4 signaling inhibitor CLI095 reversed the above effects, suggesting HSM could reduce the cumulation of autophagosomes dependent on TLR4. Furthermore, lipopolysaccharide (LPS)-stimulated TLR4-related autophagy was significantly inhibited by HSM treatment. In addition, the protein expressions of TLR4 and phospho-NF-κB p65 were markedly inhibited in cells treated with HSM. Conclusions: These results indicated that HSM could inhibit the autophagy of alveolar macrophages through TLR4/NF-κB signaling pathway to achieve anti-fibrotic effect.

Keywords: Hirsutella sinensis mycelium; TLR4 signal pathway.; alveolar macrophage; autophagy; pulmonary fibrosis.

Figure 1

HSM down-regulates excessive autophagy in BLM-induced pulmonary fibrosis mice. (A) In vivo experimental design: Mice were intratracheally administered with BLM (3 mg/kg in 100 μl saline) on day 0, and HSM (200 mg/kg in 500 μl saline) was orally administered after the BLM administration from day 7 to day 27. On day 28, the mice were sacrificed and the corresponding samples were collected. (B) Transmission electron microscopy image of mouse lung tissue. Black arrow: autophagosome; yellow asterisk: cell nucleus; scale bar: 2 μm. The black bars represent the mean ± SEM values between the treated groups analyzed, n=4. (C) Analysis of the changes of LC3B, P62 and ATG5 proteins in mice of each group by Western blotting, n=3. Data are presented as means ± SEM of at least three separate experiments. *P < 0.05, **P < 0.01.

Figure 2

HSM reduces the number of alveolar macrophages in the fibrotic lung. (A) BALF was centrifuged and coated to count the changes of total cells, macrophages, lymphocytes, eosinophils and neutrophils. The CON, HSM, BLM and BLM+HSM mainly display representative morphological images of macrophages, lymphocytes, eosinophils and neutrophils. Scale: 10 μm, magnification: x200, n=4. (B) Gating strategy mainly used to identify macrophages in BALF and lung tissue of mice. The differential expression of CD11b and CD11c was used to distinguish pulmonary macrophages. (C) Effect of HSM on the number of total cells, CD45⁺ cells and alveolar macrophages in BALF, n=6. (D) Effect of HSM on the number of total cells, CD45⁺ cells, alveolar macrophages and interstitial macrophages in lung tissue, n=10. Data are presented as means ± SEM of at least three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

HSM inhibits autophagy of alveolar macrophages in fibrotic mice. (A) The effect of HSM on the count of LC3B positive cells (total cells, CD45⁺ leukocytes, alveolar macrophages and interstitial macrophages) in lung tissue, n=10. (B) The effect of HSM on LC3B MFI of total cells, CD45⁺ leukocytes, alveolar macrophages and interstitial macrophages in lung tissue, n=10. (C) The effect of HSM on the number of LC3B⁺ total cells, CD45⁺ leukocytes and alveolar macrophages in BALF, n=6. (D) The effect of HSM on LC3B MFI of alveolar macrophages in BALF, n=6. (E) Primary alveolar macrophages were extracted from BALF of each group of mice, and the changes of autophagosomes were observed by transmission electron microscopy, n=4. Black arrow: autophagosome; yellow asterisk: cell nucleus; scale bar: 2 μm. Data are presented as means ± SEM of at least three separate experiments. **P < 0.01, ***P < 0.001.

Figure 4

HSM regulates autophagosomes accumulation in macrophages dependent on TLR4 activity. (A) The effect of different concentrations HSM on the activity of RAW264.7 cells was analyzed by CCK-8 assay. (B) In vitro experiment to explore the effect of HSM on CQ-induced autophagy flux accumulation. RAW264.7 cells were pretreated with CQ (20 μM) for 2 h and then treated with or without HSM (16 μg/ml) for 16 h. Representative immunofluorescence image of LC3B in RAW264.7 cells treated with CQ and HSM. Scale bar: 40 μm. (C) The effect of HSM on the expression of LC3B mRNA was analyzed by q-PCR. (D) Western blotting to examine the effect of HSM on the expression of LC3B protein. (E) RAW264.7 cells were pretreated with CQ (20 μM) and CLI095 (100 μg/ml) for 2 h and then treated with or without HSM (16 μg/ml) for 16 h. Representative immunofluorescence image of LC3B in RAW264.7 cells treated with CLI095. Scale bar: 30 μm. Data are presented as means ± SEM of at least three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Decreased TLR4/NF-κB signaling in macrophages with HSM incubation. (A) RAW264.7 cells were pretreated with LPS (0.1 μg/ml) for 2 h and then treated with or without HSM (HSM1:16 μg/ml and HSM2: 160 μg/ml) for 16 h. Representative immunofluorescence image of LC3B in RAW264.7 cells treated with LPS. Scale bar: 40 μm. (B) Effect of HSM on TLR4 protein expression by flow cytometry. (C) Effect of HSM on the expression of TLR4 downstream proteins NF-κB P65, ERK and IRF 3 was analyzed by western blotting. Data are presented as means ± SEM of at least three separate experiments. *P < 0.05, ***P < 0.001.

Figure 6

Mechanism of HSM treatment of BLM-induced pulmonary fibrosis in mice.