Reactive oxygen species-mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium-infected macrophages by activating regulated IRE1-dependent decay pathway

Abstract

Mycobacterium avium, a slow-growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress-mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1-dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol-requiring enzyme 1 (IRE1)/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD-associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium-infected macrophages. Interestingly, M. avium-induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N-acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC-pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin-, 4μ8c-, and NAC-treated macrophages compared with the control. The data indicate that the ROS-mediated ER stress response induces apoptosis of M. avium-infected macrophages by activating IRE1α-RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.

Keywords: ER stress; IRE1α; Mycobacterium avium; RIDD; apoptosis.

Figure 1

Mycobacterium avium‐induced apoptosis in macrophages. (a) RAW 264.7 cells were infected with M. avium at a multiplicity of infection (moi) of 5. Apoptosis of RAW 264.7 cells was assayed by flow cytometry with Annexin V/propidium iodide staining. RAW 264.7 cells were treated with staurosporine (STS, 1 μM) for 6 hr. (b) Percentage of apoptotic cells calculated from (a). (c) Activation of caspase‐12, caspase‐9, and caspase‐3 was determined in RAW 264.7 cells infected with M. avium in a moi‐dependent manner. RAW 264.7 cells were pretreated with (d) z‐ATAD‐fmk (20 μM), (e) z‐LEHD‐fmk (20 μM), or (f) z‐VAD‐fmk (20 μM) for 1 hr prior to M. avium infection. After 24 hr, induction of caspase‐12, caspase‐9, and caspase‐3 was analysed by Western blotting. Data are representative of at least three independent experiments with similar results (*p < .05, **p < .01, and ***p < .001)

Figure 2

Endoplasmic reticulum (ER) stress plays a crucial role in Mycobacterium avium‐induced apoptosis. (a) Polymerase chain reaction (PCR) amplification of CHOP and XBP‐1 for 0–24 hr. (b) RAW 264.7 cells were infected M. avium (multiplicity of infection [moi] = 5) and incubated for 6–24 hr. (c) RAW 264.7 cells were infected with M. avium at moi of 1, 5, and 10 and incubated for 24 hr. ER stress factors were analysed by Western blotting. (d) RAW 264.7 cells were pretreated with the ER stress inhibitor 4‐PBA (1–10 mM) and infected with M. avium for 24 hr. (e) RAW 264.7 cells were pretreated with 4‐PBA (10 mM) and infected with M. avium. (f) Apoptosis of 4‐PBA‐pretreated cells following infection with M. avium for 24 hr. Data are representative of three independent experiments

Figure 3

Inhibition of IRE1α reduces Mycobacterium avium‐mediated apoptosis of RAW 264.7 cells. (a) RAW 264.7 cells or (b) bone marrow‐derived macrophages (BMDMs) were infected with M. avium for 0–6 hr; and the inositol‐requiring enzyme 1 IRE1α, apoptosis signal‐regulating kinase 1 (ASK‐1), and (ASK1)/phospho‐c‐Jun N‐terminal kinase (JNK; p‐JNK) levels were determined by Western blotting. (c) RAW 264.7 cells or (d) BMDMs were pretreated with irestatin (10 mM) and infected with M. avium. (e) Cell lysates were subjected to Western blotting to determine the levels of C/EBP homologous protein (CHOP), cleaved caspase‐3, and β‐actin. (f) Apoptosis of irestatin‐pretreated cells following infection with M. avium for 24 hr. Data are representative of at least three independent experiments (*p < .05, **p < .01, and ***p < .001)

Figure 4

IRE1α‐dependent degradation of genes is involved in Mycobacterium avium‐mediated apoptosis. Polymerase chain reaction (PCR) amplification of Bloc1s1 and St3gal5 from M. avium‐infected (a) RAW 264.7 cells or (c) bone marrow‐derived macrophages (BMDMs). (b) Bloc1s1 and St3gal5 mRNA levels were identified by real‐time PCR. (d) RAW 264.7 cells or (f) BMDMs were pretreated with 4μ8c (100 μM) and infected with M. avium. (e) mRNA levels of St3gal5 and Bloc1s1 by real‐time PCR. (g) RAW 264.7 cells were pretreated with 4μ8c and infected with M. avium for 24 hr. (h) RAW 264.7 cells were incubated with 4μ8c prior to infection with M. avium. Data are representative of three independent experiments (*p < .05, **p < .01, and ***p < .001)

Figure 5

Mycobacterium avium‐induced reactive oxygen species (ROS) activates degradation of regulated IRE1‐dependent decay (RIDD) target genes via the endoplasmic reticulum (ER) stress‐IRE1α pathway. (a) RAW 264.7 cells were infected with M. avium at a moi of 5 for 0–60 min. Dihydroethidium (2 μM) assay of the intracellular ROS level. (b) RAW 264.7 cells were pretreated with N‐acetyl cysteine (NAC; 30 mM) and infected with M. avium for 5 min. (c) RAW 264.7 cells were pretreated with NAC and infected with M. avium for 24 hr. (d) RAW 264.7 cells or (e) bone marrow‐derived macrophages (BMDMs) were pretreated with NAC and infected with M. avium. IRE1α was determined by Western blotting. Bloc1s1 and St3gal5 mRNA levels were detected in NAC‐pretreated (f) RAW 264.7 cells or (g) BMDMs by PCR. (h) mRNA levels of RIDD target genes in NAC‐pretreated RAW 264.7 cells by real‐time PCR. (i) Apoptosis of NAC‐pretreated cells following infection with M. avium for 24 hr. Data are representative of at least three independent experiments (*p < .05, **p < .01, and ***p < .001)

Figure 6

The endoplasmic reticulum (ER) stress response regulates the intracellular survival of Mycobacterium avium. Surviving intracellular M. avium were counted at 24 hr after infection. (a–c) RAW 264.7 cells or (d–f) BMDMs were pretreated with irestatin, NAC, or 4μ8c and infected with M. avium for 24 hr. Data are representative of at least three independent experiments (*p < .05, **p < .01, and ***p < .001)