Lipopolysaccharides may aggravate apoptosis through accumulation of autophagosomes in alveolar macrophages of human silicosis

Abstract

Silica dust mainly attacks alveolar macrophages (AMs) and increases the apoptosis of AMs in silicosis patients. However, it is still unclear whether autophagy is affected. Autophagy mainly has defensive functions in response to stress, contributing to cell survival in adverse conditions, and conversely it has also been implicated in cell death. Lipopolysaccharide (LPS) induces autophagy and apoptosis in macrophages. The role of LPS in autophagy and apoptosis in AMs of silicosis patients is unknown. In this study, we collected AMs from 53 male workers exposed to silica and divided them into an observer (control) group, and stage I, II and III patient groups. We found increased levels of LC3B, SQSTM1/p62 and BECN1，whereas the phosphorylation of MTOR，and levels of LAMP2, TLR4, MYD88, TICAM1, as well as the number of lysosomes decreased with the development of silicosis. LPS stimulation triggered autophagy and increased levels of SQSTM1 in AMs. The autophagy inhibitor, 3-methyladenine (3MA), inhibited LPS-induced apoptosis in the AMs of silicosis patients. Moreover, 3MA reversed the LPS-induced decrease in BCL2 and the increase in BAX and CASP3 levels in AMs. These results suggest that autophagosomes accumulate in AMs during silicosis progression. LPS can induce the formation of autophagosomes through a TLR4-dependent pathway, and LPS may exacerbate the apoptosis in AMs. Blockade of the formation of autophagosomes may inhibit LPS-induced apoptosis via the intrinsic apoptotic pathway in AMs. These findings describe novel mechanisms that may lead to new preventive and therapeutic strategies for pulmonary fibrosis.

Keywords: LPS; TLR4; alveolar macrophages; apoptosis; autophagic degradation; autophagosome; autophagy; lysosome; silicosis.

Figure 1.

Autophagosomes increase in AMs with the development of silicosis. (A) A representative image of fixed alveolar macrophages from different groups stained for LC3B (original magnification ×600). Green, LC3B protein; Red, PI-labeled nuclei; PI was used for nuclear staining. Scale bar: 40 μm. (B) AMs from observers and different silicosis stages were analyzed for LC3B by western blot. ACTB/beta-actin protein was used as a loading control. (C) LC3-II/LC3-I ratios for each patient group. Significance was determined using one-way ANOVA (n = 11 for the observer group; n = 14 for stages I, II and III. ^*, P < 0.05 compared to the observer group，^#, P < 0.05 vs. stage I patient group. O, observer group; I, stage I patient group; II, stage II patient group; III, stage III patient group).

Figure 2.

Autophagosomes increase in AMs of silicosis patients. (A) Representative transmission electron microscopic images. Upward arrows indicate autophagic vacuoles and rightward arrows indicate lysosomes in the AMs of different groups. Images i to iv show a macrophage of each patient group, and images v to viii show the same macrophage at a higher magnification. The observer (O) group displayed numerous lysosomes in the cytoplasm of AMs, and also fewer autophagosomes, than those of the silicosis patient groups (I, II, III). Groups I, II, III exhibited increased autophagic vacuoles and reduced lysosomes relative to the observer group. Top images (i to iv): ×7000; Bottom images (v to viii): ×15000. Scale bar: 1 μm (B) A graph of the number of autophagosomes per 25 AMs for each group. (C) A graph of the mean number of lysosomes per 15 AMs for each group. n = 6 for observer and stages I, II and III groups. ^*, P < 0.05 vs. observer group，^#, P < 0.05 vs. stage I patient group. ^▴, P < 0.05 vs. stage II patient group. O, observer group; I, stage I patient group; II, stage II patient group; III, stage III patient group.

Figure 3.

Delivery of autophagosomes to lysosomes was blocked in AMs of silicosis patients. (A) The expression of LAMP2 and SQSTM1 proteins was analyzed by western blot. ACTB was used as a loading control. (B and C) LAMP2/ACTB and SQSTM1/ACTB ratios of each patient group. n = 11 for the observer group, n = 14 for stage I, II and III groups. ^*, P < 0.05 vs. observer group，^#, P < 0.05 vs. stage I patient group, ^▴, P < 0.05 vs. stage II patient group. O, observer group; I, stage I patient group; II, stage II patients group, III; stage III patient group.

Figure 4.

Decreased TLR4 in AMs of silicosis patients. (A) TLR4, MYD88 and TICAM1 proteins were analyzed by western blot. ACTB was used as a loading control. (B to D) Ratios of the indicated proteins to ACTB (n = 8 for observer, and stage I, II and III groups. ^*, P < 0.05 vs. observer group, ^#, P < 0.05 vs. stage I patient group. O, observer group; I, stage I patient group; II, stage II patient group; III, stage III patient group).

Figure 5.

LPS can increase the formation of autophagosomes in AMs at different stages of silicosis. (A) Lysates of LPS- and anti-HTA-treated AMs were separated by SDS-PAGE and western blot. C indicates AMs from lavage fluids of silicosis patients without intervention. LPS and HTA indicate AMs were untreated or treated with anti-human TLR4 antibody HTA125 prior to 10 μg/ml LPS exposure for 24 h, respectively. ACTB protein was used as a loading control. (B to G) Ratios of the indicated proteins to ACTB, or LC3-II/LC3-I, at different stages of human silicosis. n = 8 for observer, stage I, II and III groups. ^*, P < 0.05 vs. C; ^#, P < 0.05 vs. LPS.

Figure 6.

LPS increases the SQSTM1 levels in AMs at different stages of silicosis. (A) AMs were treated without (C) or with 10 or 100 μg/ml LPS exposure for 24 h, respectively. SQSTM1 protein was separated by SDS-PAGE and analyzed by western blot. ACTB protein was used as a loading control. (B) Ratios of SQSTM1 to ACTB at different stages of human silicosis (n = 8 for observer, stage I, II and III groups. ^*, P < 0.05 vs. C).

Figure 7.

LPS can induce apoptosis in AMs of silicosis subjects. (A) AMs in the observer group were treated without or with LPS (100, 150, 200 μg/ml), and cell apoptosis determined after 24 h using TUNEL. Scale bar: 30 μm. (B) Quantification of TUNEL-positive apoptotic cells in AMs treated with different LPS concentrations. Six fields per sample were analyzed for each cell population. The bar graph shows the percentage of TUNEL-positive nuclei relative to DAPI-positive total nuclei (n = 5 each group. ^*, P < 0.05 vs. CON; ^#, P < 0.05 vs. 100 μg/ml LPS group). (C) Representative photographs of TUNEL staining in AMs at different stages of silicosis. Scale bar: 30 μm. (D) Quantification of TUNEL-positive apoptotic cells in AMs at different stages of silicosis. n = 5 for observer, stage I, II and III groups. ^*, P < 0.05 vs. observer group; ^#, P < 0.05 vs. stage I patient group).

Figure 8.

Inhibition of the formation of autophagosomes may reduce LPS-induced apoptosis in AMs at different stages of silicosis. (A–D) Representative photographs of TUNEL staining in AMs treated without or with 3MA (7.5 mM), or cotreated with or without E64d (10 μg/ml) and pepstatin A (10 μg/ml), and stimulated with LPS (150 μg/ml) for 24 h. Scale bar: 30 μm. (E) Ratios of TUNEL-positive apoptotic cells. 6 fields were analyzed for each sample from each cell population. The bar graph shows the percentage of TUNEL-positive nuclei relative to DAPI-positive total nuclei. n = 5 for observer, stage I, II and III groups. ^*, P < 0.05 vs. CON; ^#, P < 0.05 vs. LPS+3MA.

Figure 9.

Effects of LPS and 3MA on the expression of BCL2, BAX and cleaved CASP3 in AMs at different stages of silicosis. (A) C indicates AMs from lavage fluids of silicosis patients without intervention. AMs treated without or with 3MA (7.5 mM), and stimulated with LPS (150 μg/ml) for 24 h. BCL2, BAX and cleaved CASP3 proteins were analyzed by western blot. (B–D) Ratios of the indicated proteins to ACTB. n = 8 for observer, and stage I, II and III groups. ^*, P < 0.05 vs. C; ^#, P < 0.05 vs. LPS.

Figure 10.

A hypothetical diagram illustrates potential mechanisms in silicosis. Silica may lead to apoptosis via increasing the formation of autophagosomes in AMs. LPS regulates the formation of autophagosomes through a blockade of the MTOR signaling pathway and activation of MYD88, TICAM1 and BECN1 signaling pathways. However, silica engulfment results in lysosomal rupture, which may lead to the accumulation of autophagosomes. The excessive accumulation of autophagosomes may lead to apoptosis in AMs. Thus LPS or other substances that can activate autophagy may be risk factors for silicosis.