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The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs, including two-dimensional cell culture models or animal models, have several limitations. Therefore, an appropriate model representative of the human organism is required. Here, we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hMφs) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably, M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore, a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus, the incorporation of hMφs and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies.
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Antituberculosos , Pulmão , Mycobacterium tuberculosis , Organoides , Humanos , Organoides/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Macrófagos/microbiologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Células Epiteliais/microbiologiaRESUMO
IMPORTANCE: Several studies have suggested that endoplasmic reticulum (ER) stress is important in the pathogenesis of infectious diseases; however, the precise function of ER stress regulation and the role of Herp as a regulator in Mtb H37Ra-induced ER stress remain elusive. Therefore, our study investigated ER stress and autophagy associated with Herp expression in Mycobacterium tuberculosis-infected macrophages to determine the role of Herp in the pathogenesis of tuberculosis.
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Mycobacterium tuberculosis , Espécies Reativas de Oxigênio , Macrófagos , Estresse do Retículo Endoplasmático , AutofagiaRESUMO
BACKGROUND: Mitophagy, mitochondrial selective autophagy, plays a pivotal role in the maintenance of cellular homeostasis in response to cellular stress. However, the role of mitophagy in macrophages during infection has not been elucidated. To determine whether mitophagy regulates intracellular pathogen survival, macrophages were infected with Mycobacterium tuberculosis (Mtb), an intracellular bacterium. RESULTS: We showed that Mtb-infected macrophages induced mitophagy through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) activation. In contrast, BNIP3-deficient macrophages failed to induce mitophagy, resulting in reduced mitochondrial membrane potential in response to Mtb infection. Moreover, the accumulation of damaged mitochondria due to BNIP3 deficiency generated higher levels of mitochondrial reactive oxygen species (mROS) compared to the control, suppressing the intracellular survival of Mtb. We observed that siBNIP3 suppressed intracellular Mtb in mice lungs. CONCLUSION: We found that BNIP3 plays a critical role in the regulation of mitophagy during Mtb infection. The inhibition of mitophagy suppresses Mtb growth in macrophages through increased mROS production. Therefore, BNIP3 might be a novel therapeutic target for tuberculosis treatment.
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Pulmonary nodular lymphoid hyperplasia (PNLH) is a rare, benign lymphoproliferative disease, which is characterized by nonclonal lymphoproliferation. PNLH is usually asymptomatic and usually detected incidentally on imaging studies. Common imaging findings include a solitary nodule, multiple nodules, or focal consolidation. Atypically, PNLH may present with persistent subsolid nodules, mimicking adenocarcinoma. Here, we report a rare case of PNLH presenting as multifocal subsolid nodules in both lower lobes. During follow-up, persistency and growth of the subsolid nodules suggested the possibility of malignancy. Wedge resection was performed bilaterally, and PNLH was confirmed on pathological examination.
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BACKGROUND: Iron has important roles as an essential nutrient for all life forms and as an effector of the host defense mechanism against pathogenic infection. Lipocalin 2 (LCN2), an innate immune protein, plays a crucial role in iron transport and inflammation. In the present study, we examined the role of LCN2 in immune cells during Mycobacterium tuberculosis (Mtb) infection. RESULTS: We found that infection with Mtb H37Ra induced LCN2 production in bone marrow-derived dendritic cells (BMDCs). Notably, expression of MHC class I molecules was significantly reduced in LCN2-/- BMDCs during Mtb infection. The reduced expression of MHC class I molecules was associated with the formation of a peptide loading complex through LCN2-mediated reactive oxygen species production. The reduced expression of MHC class I molecules affected CD8+ T-cell proliferation in LCN2-/- mice infected with Mtb. The difference in the population of CD8+ effector T cells might affect the survival of intracellular Mtb. We also found a reduction of the inflammation response, including serum inflammatory cytokines and lung inflammation in LCN2-/- mice, compared with wild-type mice, during Mtb infection. CONCLUSIONS: These data suggest that LCN2-mediated reactive oxygen species affects expression of MHC class I molecules in BMDCs, leading to lower levels of CD8+ effector T-cell proliferation during mycobacterial infection.
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It has been known that infection plays a role in the development of hypertension. However, the role of hypertension in the progression of infectious diseases remain unknown. Many countries with high rates of hypertension show geographical overlaps with those showing high incidence rates of tuberculosis (TB). To explore the role of hypertension in tuberculosis, we compared the effects of hypertension during mycobacterial infection, we infected both hypertensive Angiotensin II (Ang II) and control mice with Mycobacterium tuberculosis (Mtb) strain H37Ra by intratracheal injection. Ang II-induced hypertension promotes cell death through both apoptosis and necrosis in Mtb H37Ra infected mouse lungs. Interestingly, we found that lipid accumulation in pulmonary tissues was significantly increased in the hypertension group compared to the normal controls. Ang II-induced hypertension increases the formation of foamy macrophages during Mtb infection and it leads to cell death. Moreover, the hypertension group showed more severe granuloma formation and fibrotic lesions in comparison with the control group. Finally, we observed that the total number of mycobacteria was increased in the lungs in the hypertension group compared to the normal controls. Taken together, these results suggest that hypertension increases intracellular survival of Mtb through formation of foamy macrophages, resulting in severe pathogenesis of TB.
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Angiotensina II/toxicidade , Apoptose , Hipertensão/patologia , Pulmão/patologia , Macrófagos/patologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/patologia , Animais , Hipertensão/induzido quimicamente , Pulmão/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Tuberculose/microbiologia , Vasoconstritores/toxicidadeRESUMO
PURPOSE: To determine the intra- and extralesional factors that predict sclerotic degeneration of hepatic hemangiomas in the cirrhotic liver on long-term follow-up computed tomography (CT) examinations. MATERIALS AND METHODS: Fifty-seven hepatic hemangiomas (> 5 mm in diameter) in 41 cirrhotic patients, recruited over a 5-year period (January 2005-December 2009), were subjected to CT to determine which factors predict sclerotic contraction or degeneration in hemangiomas. Prior and follow-up CT examinations (from 2000 to 2018) were included to observe time-related changes. The patients' gender, age, cause of cirrhosis, progression of background liver cirrhosis, lesion size/location/contrast enhancement pattern, and serum aspartate transaminase to platelet ratio index were correlated with sclerotic changes of each lesion. RESULTS: According to the dynamic CT features, 36 of 57 (63%) hemangiomas were determined to have sclerotic changes during the follow-up period (1.1-14.4 years, median: 7.8 years), including 28 lesions (49%) reduced by ≥ 20% in diameter. In univariate analysis, age (p = 0.047) and morphological progression of background cirrhosis (p = 0.013) were significantly related to sclerotic change of hemangiomas. In the logistic regression analysis, only morphological progression of background liver cirrhosis independently predicted sclerotic change (odds ratio: 4.88, p = 0.007). With the exception of exophytic location free from size reduction (p = 0.023 in multivariate analysis), no other analyzed factors were significantly correlated with sclerotic changes. CONCLUSION: Overall, sclerotic changes of hepatic cavernous hemangioma followed the morphological progression of background liver cirrhosis, while exophytic lesions tended to be free of size reduction.
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Meios de Contraste , Hemangioma Cavernoso/diagnóstico por imagem , Cirrose Hepática/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Fígado/diagnóstico por imagem , Tomografia Computadorizada Multidetectores/métodos , Análise de Variância , Análise de Dados , Progressão da Doença , Feminino , Seguimentos , Hemangioma Cavernoso/patologia , Humanos , Fígado/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Intensificação de Imagem Radiográfica , Esclerose , Fatores de Tempo , Carga TumoralRESUMO
OBJECTIVES: Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-ß-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-ß-lactamase, and Guiana extended-spectrum ß-lactamase. METHODS: The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides. RESULTS: No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours. CONCLUSION: The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.
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Tumor suppressor p53 is not only affects immune responses but also contributes to antibacterial activity. However, its bactericidal function during mycobacterial infection remains unclear. In this study, we found that the p53-deficient macrophages failed to control Mycobacterium tuberculosis (Mtb), manifested as a lower apoptotic cell death rate and enhanced intracellular survival. The expression levels of p53 during Mtb infection were stronger in M1 macrophages than in M2 macrophages. The TLR2/JNK signaling pathway plays an essential role in the modulation of M1 macrophage polarization upon Mtb infection. It facilitates p53-mediated apoptosis through the production of reactive oxygen species, nitric oxide and inflammatory cytokines in Mtb-infected M1 macrophages. In addition, nutlin-3 effectively abrogated the intracellular survival of mycobacteria in both TB patients and healthy controls after H37Ra infection for 24 h, indicating that the enhancement of p53 production effectively suppressed the intracellular survival of Mtb in hosts. These results suggest that p53 can be a new therapeutic target for TB therapy.
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Macrófagos/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Animais , Apoptose , Feminino , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/fisiologia , Tuberculose/genética , Tuberculose/microbiologia , Tuberculose/fisiopatologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The original version of this article unfortunately contains an error in the acknowledgement section. The text "Brain Korea 21 PLUS Project for Medical Science, Chungnam National University" was omitted by mistake. The correct and complete acknowledgment is given below: Acknowledgments This work was supported by the research fund of Chungnam National University and the Brain Korea 21 PLUS Project for Medical Science, Chungnam National University. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.
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Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has been disseminating nationwide due to clonal spread and is taking a serious action at the national level in Korea. The mobilized colistin resistance (MCR1) gene confers plasmid-mediated resistance to colistin and is known to be capable of horizontal transfer between different strains of a bacterial species. We have experienced a fatal case of the patient who developed MCR1-possessing, ST307/Tn4401a[blaKPC2] K. pneumonia bacteremia in the community of non-capital region after being diagnosed as pancreatic cancer with multiple liver metastases and treated in the capital region. The ST307/Tn4401a[blaKPC2] K. pneumonia was the most commonly disseminated clone in Korea. Our strain is the first MCR1 and KPC2 co-producing K. pneumonia in Korea and our case is the critical example that the multi-drug resistant clone can cause inter-regional spread and the community-onset fatal infections. Fortunately, our patient was admitted to the intensive care unit on the day of visit, and the contact precaution was well maintained throughout and KPC-KP was not spread to other patients. The high risk patients for KPC-KP need to be screened actively, detected rapidly and preemptively isolated to prevent outbreak of KPC-KP. Inter-facility communications are essential and the nationwide epidemiologic data of KPC-KP should be analyzed and reported regularly to prevent spread of KPC-KP. The prompt identification of species and antimicrobial susceptibilities for successful treatment against KPC-KP should be emphasized as well.
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Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control.
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Apoptose , Estresse do Retículo Endoplasmático , GTP Fosfo-Hidrolases/deficiência , Macrófagos/microbiologia , Macrófagos/patologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/prevenção & controle , Animais , Células Cultivadas , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Mitocôndrias/patologia , Consumo de Oxigênio , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
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.
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Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Camundongos , Mycobacterium avium/patogenicidade , Células RAW 264.7 , Tuberculose Aviária/metabolismo , Tuberculose Aviária/microbiologiaRESUMO
Tuberculosis (TB) remains a global healthcare issue. Understanding the host-pathogen interactions in TB is vital to develop strategies and therapeutic tools for the control of Mycobacterium tuberculosis (Mtb). In this study, transcriptome analyses of macrophages infected with either the virulent Mtb strain H37Rv (Rv) or the avirulent Mtb strain H37Ra (Ra) were carried out and 750 differentially expressed genes (DEGs) were identified. As expected, the DEGs were mainly involved in the induction of innate immune responses against mycobacterial infections. Among the DEGs, solute carrier family 7 member 2 (Slc7a2) was more strongly expressed in Ra-infected macrophages. Induction of SLC7A2 was important for macrophages to control the intracellular survival of Mtb. Our results imply that SLC7A2 plays an important role in macrophages during Mtb infection. Our findings could prove useful for the development of new therapeutic strategies to control TB infection.
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Sistemas de Transporte de Aminoácidos Básicos/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis , Tuberculose , Animais , Células Cultivadas , Interações Hospedeiro-Patógeno , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , RNA-Seq/métodos , Tuberculose/imunologia , Tuberculose/microbiologia , VirulênciaRESUMO
The endoplasmic reticulum (ER) is the major organelle in the cell for protein folding and plays an important role in cellular functions. The unfolded protein response (UPR) is activated in response to misfolded or unfolded protein accumulation in the ER. However, the UPR successfully alleviates the ER stress. If UPR fails to restore ER homeostasis, apoptosis is induced. ER stress plays an important role in innate immune signaling in response to microorganisms. Dysregulation of UPR signaling contributes to the pathogenesis of a variety of infectious diseases. In this review, we summarize the contribution of ER stress to the innate immune response to invading microorganisms and its role in the pathogenesis of infectious diseases.
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Estresse do Retículo Endoplasmático/imunologia , Imunidade Inata/imunologia , Infecções/imunologia , Resposta a Proteínas não Dobradas/imunologia , Animais , HumanosRESUMO
BACKGROUND: Mycobacterium smegmatis, a rapidly growing non-tuberculosis mycobacterium, is a good model for studying the pathogenesis of tuberculosis because of its genetic similarity to Mycobacterium tuberculosis (Mtb). Macrophages remove mycobacteria during an infection. Macrophage apoptosis is a host defense mechanism against intracellular bacteria. We have reported that endoplasmic reticulum (ER) stress is an important host defense mechanism against Mtb infection. RESULTS: In this study, we found that M. smegmatis induced strong ER stress. M. smegmatis-induced reactive oxygen species (ROS) play a critical role in the induction of ER stress-mediated apoptosis. Pretreatment with an ROS scavenger suppressed M. smegmatis-induced ER stress. Elimination of ROS decreased the ER stress response and significantly increased the intracellular survival of M. smegmatis. Interestingly, inhibition of phagocytosis significantly decreased ROS synthesis, ER stress response induction, and cytokine production. CONCLUSIONS: Phagocytosis of M. smegmatis induces ROS production, leading to production of proinflammatory cytokines. Phagocytosis-induced ROS is associated with the M. smegmatis-mediated ER stress response in macrophages. Therefore, phagocytosis plays a critical role in the induction of ER stress-mediated apoptosis during mycobacterial infection.
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B-cell activating factor (BAFF) has been proposed to play a crucial role in the pathogenesis of chronic rhinosinusitis with nasal polyp (CRSwNP). The aim of this study was to evaluate the role of toll-like receptor (TLR) 9-mediated BAFF activation on the pathogenesis of CRSwNP. NP and uncinate tissue (UT) were obtained from patients with CRSwNP or CRS without NP, and control subjects. The expression of TLR9, high mobility group box-1 protein (HMGB1), type I interferon (IFN), BAFF, and anti-double stranded DNA (dsDNA) antibody were examined in the tissues and the cultured dispersed NP cells (DNPCs). The expression of TLR9, HMGB1, type I IFN, BAFF, and anti-dsDNA antibody were elevated in NP tissue compared to the UTs. Exposure to TLR9 agonist increased the type I IFN expression in vitro, which further increased BAFF production. In conclusion, we provided a novel therapeutic potential of TLR9 agonist in CRSwNP.
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Fator Ativador de Células B/genética , Proteína HMGB1/genética , Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator Ativador de Células B/efeitos dos fármacos , Fator Ativador de Células B/metabolismo , Doença Crônica , Feminino , Seio Frontal/metabolismo , Proteína HMGB1/metabolismo , Humanos , Técnicas In Vitro , Interferon-alfa/efeitos dos fármacos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/efeitos dos fármacos , Interferon beta/genética , Interferon beta/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/agonistasRESUMO
Mycobacterium fortuitum (MF), a rapidly growing nontuberculosis mycobacterium, is recognized as an important human pathogen. We investigated whether the endoplasmic reticulum (ER) stress response is associated with the apoptosis of MF-infected macrophages. The expression of ER molecular chaperones was significantly induced by MF infection. We found that MF-induced reactive oxygen species (ROS) generation plays a critical role in the induction of ER stress-mediated apoptosis. Excess TNF-α in the ER led to ER stress-mediated apoptosis during MF infection. The intracellular survival of MF was significantly increased by TNF-α knockdown compared with the control. This is the first report of MF-induced TNF-α as a cause of ER stress in macrophages. Furthermore, we found that TLR2-mediated ER stress response contributed to the elimination of intracellular MF in vivo. These results suggest that TNF-α-mediated ER stress during MF infection contributes to the suppression of intracellular survival of MF in macrophages. Our findings provide new insight into the importance of ER stress in mycobacterial infection.-Oh, S.-M., Lim, Y.-J., Choi, J.-A., Lee, J., Cho, S.-N., Go, D., Kim, S.-H., Song, C.-H. TNF-α-mediated ER stress causes elimination of Mycobacterium fortuitum reservoirs by macrophage apoptosis.
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Apoptose , Estresse do Retículo Endoplasmático , Macrófagos/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Mycobacterium fortuitum , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
Ajoene, a garlic-derived sulfur-containing compound, has broad-spectrum antimicrobial activity. To assess the potential of ajoene for treating tuberculosis (TB), we determined whether it induces the stress response of the endoplasmic reticulum (ER), which plays an important role in TB. We showed that ajoene stimulation induced the production of ER stress sensor molecules and reactive oxygen species (ROS) levels. Ajoene-induced ROS production was dependent on c-Jun N-terminal kinase (JNK) activation. Interestingly, the inhibition of JNK activity and suppression of ROS production reduced ajoene-induced CHOP production in macrophages. Because ER stress activates autophagy, the activation of which suppresses the growth of mycobacteria, we investigated the ajoene-induced production of autophagy-related factors, including LC3-II, P62 and Beclin-1. As expected, ajoene treatment increased the levels of these factors in RAW 264.7 cells. Remarkably, the total amount of Mycobacterium tuberculosis (Mtb) H37Rv was significantly reduced in ajoene-treated RAW 264.7 cells. The treatment of macrophages with ajoene resulted in the activation of JNK, induction of ROS synthesis and accumulation of ROS, possibly leading to the activation of ER stress and autophagy. These results reveal the mechanism of the antimycobacterial effects of ajoene against Mtb H37Rv. Our findings might facilitate the development of novel therapies for patients with TB.
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Antibacterianos/farmacologia , Dissulfetos/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mycobacterium tuberculosis/imunologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , SulfóxidosRESUMO
Endoplasmic reticulum (ER)-stress-mediated apoptosis is a host defense mechanism against Mycobacterium tuberculosis (Mtb) infection. Calreticulin (CRT) is the major calcium-binding chaperone protein. Previous reports have suggested a close relationship between the cell-surface expression of CRT and apoptosis. In this study, the role of CRT during Mtb infection was examined. The results showed that Mtb infection induces CRT production by macrophages and that CRT levels are correlated with the degree of apoptotic cell death. The enhanced production of CRT was associated with the ER stress induced by Mtb infection. A significant increase in CRT translocation from the cytosol to the plasma membrane after 24 h of infection suggested the importance of CRT localization in the induction of apoptosis during Mtb infection. An investigation of the factors associated with CRT translocation and the ability of ectopically expressed CRT to induce apoptosis showed that pretreatment with a reactive oxygen species scavenger decreased Mtb-induced CRT expression, leading to the reduction of CHOP and caspase-3 activation. The intracellular survival of Mtb was significantly higher in macrophages transfected with a CRT-specific small interfering RNA than in control cells. The key role of CRT in inducing apoptosis included its interaction with CXCR1 and TNFR1 in Mtb-infected macrophages. The CRT/CXCR1/TNFR1 complex was shown to induce the extrinsic apoptotic pathway during Mtb infection. Together, these results demonstrate that CRT is critical for the intracellular survival of Mtb, via ER-stress-induced apoptosis, as well as the importance of ER stress-mediated CRT localization in the pathogenesis of tuberculosis.