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1.
Autophagy ; : 1-15, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34720021

RESUMO

Mitophagy is a selective autophagy mechanism for eliminating damaged mitochondria and plays a crucial role in the immune evasion of some viruses and bacteria. Here, we report that Mycobacterium bovis (M. bovis) utilizes host mitophagy to suppress host xenophagy to enhance its intracellular survival. M. bovis is the causative agent of animal tuberculosis and human tuberculosis. In the current study, we show that M. bovis induces mitophagy in macrophages, and the induction of mitophagy is impaired by PINK1 knockdown, indicating the PINK1-PRKN/Parkin pathway is involved in the mitophagy induced by M. bovis. Moreover, the survival of M. bovis in macrophages and the lung bacterial burden of mice are restricted by the inhibition of mitophagy and are enhanced by the induction of mitophagy. Confocal microscopy analysis reveals that induction of mitophagy suppresses host xenophagy by competitive utilization of p-TBK1. Overall, our results suggest that induction of mitophagy enhances M. bovis growth while inhibition of mitophagy improves growth restriction. The findings provide a new insight for understanding the intracellular survival mechanism of M. bovis in the host.

2.
Vet Microbiol ; 258: 109126, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34020176

RESUMO

Mycobacterium bovis (M. bovis) infection triggers cytokine production via pattern recognition receptors. These cytokines include type I interferons (IFNs) and interleukin-1ß (IL-1ß). Excessive type I IFN levels impair host resistance to M. bovis infection. Therefore, strict control of type I IFN production is helpful to reduce pathological damage and bacterial burden. Here, we found that a deficiency in caspase-1, which is the critical component of the inflammasome responsible for IL-1ß production, resulted in increased IFN-ß production upon M. bovis infection. Subsequent experiments demonstrated that caspase-1 activation reduced cyclic GMP-AMP synthase (cGAS) expression, thereby inhibiting downstream TANK-binding kinase 1 (TBK1)- interferon regulatory factor 3 (IRF3) signaling and ultimately reducing IFN production. A deficiency in caspase-1 activation enhanced the bacterial burden during M. bovis infection in vitro and in vivo and aggravated pathological lesion formation. Thus, caspase-1 activation reduced IFN-ß production upon M. bovis infection by dampening cGAS-TBK1-IRF3 signaling, suggesting that the inflammasome protects hosts by negatively regulating harmful cytokines.


Assuntos
Caspase 1/metabolismo , Animais , Inibidores de Caspase/farmacologia , Sobrevivência Celular , Dipeptídeos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamassomos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium bovis , Nucleotidiltransferases , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Distribuição Aleatória , para-Aminobenzoatos/farmacologia
3.
J Infect ; 83(1): 61-68, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33892015

RESUMO

Caspases are classified as inflammatory or apoptotic category. Inflammatory caspases participate in inflammasome activation, while apoptotic caspases mediate apoptotic activation. Previous studies have shown that apoptotic caspases prevent the production of IFN-ß during apoptosis or virus infection. However, the relationship between apoptotic caspases and IFN-ß production during intracellular bacterial infection is still unclear. Here, we investigated the role of apoptotic caspases in IFN-ß production induced by Mycobacterium bovis (M. bovis) infection. M. bovis is an intracellular bacterium and belongs to the Mycobacterium tuberculosis complex. M. bovis infection can cause tuberculosis in animals and human beings. In the current study, we found that M. bovis infection triggered mitochondrial stress, which caused the leakage of cytochrome c into the cytoplasm, and in turn, activated the downstream caspase-9 and-3. Furthermore, our results showed that activation of apoptotic caspases reduced IFN-ß production during M. bovis infection and vice versa. Confocal microscopy analysis revealed that apoptotic caspases prevented IFN-ß production by decreasing p-IRF3 nuclear translocation. Our findings demonstrate that apoptotic caspases negatively regulate the production of IFN-ß induced by an intracellular bacterial infection.


Assuntos
Apoptose , Caspases , Interferon beta/imunologia , Macrófagos/imunologia , Mycobacterium bovis , Animais , Caspases/genética , Macrófagos/microbiologia , Camundongos , Tuberculose
4.
Pharmaceutics ; 12(12)2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33271900

RESUMO

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex imposing a high zoonotic threat to human health. The limited efficacy of BCG (Bacillus Calmette-Guérin) and upsurges of drug-resistant tuberculosis require new effective vaccination approaches and anti-TB drugs. Poly (lactic-co-glycolic acid) (PLGA) is a preferential drug delivery system candidate. In this study, we formulated PLGA nanoparticles (NPs) encapsulating the recombinant protein bovine neutrophil ß-defensin-5 (B5), and investigated its role in immunomodulation and antimicrobial activity against M. bovis challenge. Using the classical water-oil-water solvent-evaporation method, B5-NPs were prepared, with encapsulation efficiency of 85.5% ± 2.5%. These spherical NPs were 206.6 ± 26.6 nm in diameter, with a negatively charged surface (ζ-potential -27.1 ± 1.5 mV). The encapsulated B5 protein from B5-NPs was released slowly under physiological conditions. B5 or B5-NPs efficiently enhanced the secretion of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-10 in J774A.1 macrophages. B5-NPs-immunized mice showed significant increases in the production of TNF-α and immunoglobulin A (IgA) in serum, and the proportion of CD4+ T cells in spleen compared with B5 alone. In immunoprotection studies, B5-NPs-immunized mice displayed significant reductions in pulmonary inflammatory area, bacterial burden in the lungs and spleen at 4-week after M. bovis challenge. In treatment studies, B5, but not B5-NPs, assisted rifampicin (RIF) with inhibition of bacterial replication in the lungs and spleen. Moreover, B5 alone also significantly reduced the bacterial load in the lungs and spleen. Altogether, our findings highlight the significance of the B5-PLGA NPs in terms of promoting the immune effect of BCG and the B5 in enhancing the therapeutic effect of RIF against M. bovis.

5.
Cell Commun Signal ; 18(1): 186, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239048

RESUMO

Mitochondria are important cellular organelles involved in many different functions, from energy generation and fatty acid oxidation to cell death regulation and immune responses. Accumulating evidence indicates that mitochondrial stress acts as a key trigger of innate immune responses. Critically, the dysfunctional mitochondria can be selectively eliminated by mitophagy. The elimination of dysfunctional mitochondria may function as an effective way employed by mitophagy to keep the immune system in check. In addition, mitophagy can be utilized by pathogens for immune evasion. In this review, we summarize how mitochondrial stress triggers innate immune responses and the roles of mitophagy in innate immunity and in infection, as well as the molecular mechanisms of mitophagy. Video Abstract.


Assuntos
Imunidade Inata , Mitocôndrias/patologia , Mitofagia/imunologia , Estresse Fisiológico , Humanos , Modelos Biológicos , Transdução de Sinais
6.
Front Microbiol ; 11: 433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265874

RESUMO

Mycobacterium bovis is the causative agent of bovine tuberculosis, has been identified a serious threat to human population. It has been found that sodium butyrate (NaB), the inhibitor of histone deacetylase, can promote the expression of cathelicidin (LL37) and help the body to resist a variety of injuries. In the current study, we investigate the therapeutic effect of NaB on the regulation of host defense mechanism against M. bovis infection. We found an increased expression of LL37 in M. bovis infected THP-1 cells after NaB treatment. In contrast, NaB treatment significantly down-regulated the expression of Class I HDAC in THP-1 cells infected with M. bovis. Additionally, NaB reduced the expression of phosphorylated P65 (p-P65) and p-IκBα, indicating the inhibition of nuclear factor-κB (NF-κB) signaling. Furthermore, we found that NaB treatment reduced the production of inflammatory cytokines (IL-1ß, TNF-α, and IL-10) and a key anti-apoptotic marker protein Bcl-2 in THP-1 cell infected with M. bovis. Notably, mice showed high resistance to M. bovis infection after NaB treatment. The reduction of viable M. bovis bacilli indicates that NaB-induced inhibition of M. bovis infection mediated by upregulation of LL37 and inhibition of NF-κB signaling pathway. These observations illustrate that NaB mediate protective immune responses against M. bovis infection. Overall, these results suggest that NaB can be exploited as a therapeutic strategy for the control of M. bovis in animals and human beings.

7.
J Infect Dis ; 221(3): 438-448, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31495880

RESUMO

BACKGROUND: Mycobacterium bovis persistently survives in macrophages by developing multiple strategies to evade host immune responses, and the early induction of interferon-ß (IFN-ß) is one of these critical strategies. The mitochondrial transcription factor A (TFAM) plays a vital role in mitochondrial DNA (mtDNA) metabolism and has been suggested to influence IFN-ß production in response to viral infection. However, its role in the production of IFN-ß by M. bovis has not been elucidated. METHODS: In the current study, we investigated the role of TFAM in the production of IFN-ß in M. bovis-infected macrophages. RESULTS: We found that knockdown of TFAM expression significantly reduced M. bovis-induced IFN-ß production, mtDNA copy numbers and cytosolic mtDNA were increased in murine macrophages with M. bovis infection, cytosolic mtDNA contributed to IFN-ß production, and TFAM was required for the increase in mtDNA copy numbers induced by M. bovis. We also observed that TFAM affected the intracellular survival of M. bovis. CONCLUSIONS: Our results suggest that TFAM plays an essential role in M. bovis-induced IFN-ß production by regulating mtDNA copy numbers. This might be a new strategy adopted by M. bovis for its intracellular survival.


Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Interferon beta/biossíntese , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Tuberculose/veterinária , Animais , Linhagem Celular Tumoral , Citosol/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mycobacterium bovis/metabolismo , Transdução de Sinais/genética , Tuberculose/microbiologia
8.
BMC Infect Dis ; 19(1): 1031, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801478

RESUMO

BACKGROUND: Mycobacterium bovis (M. bovis) is the principal causative agent of bovine tuberculosis; however, it may also cause serious infection in human being. Type I IFN is a key factor in reducing viral multiplication and modulating host immune response against viral infection. However, the regulatory pathways of Type I IFN signaling during M. bovis infection are not yet fully explored. Here, we investigate the role of Type I IFN signaling in the pathogenesis of M. bovis infection in mice. METHODS: C57BL/6 mice were treated with IFNAR1-blocking antibody or Isotype control 24 h before M. bovis infection. After 21 and 84 days of infection, mice were sacrificed and the role of Type I IFN signaling in the pathogenesis of M. bovis was investigated. ELISA and qRT-PCR were performed to detect the expression of Type I IFNs and related genes. Lung lesions induced by M. bovis were assessed by histopathological examination. Viable bacterial count was determined by CFU assay. RESULTS: We observed an abundant expression of Type I IFNs in the serum and lung tissues of M. bovis infected mice. In vivo blockade of Type I IFN signaling reduced the recruitment of neutrophils to the lung tissue, mediated the activation of macrophages leading to an increased pro-inflammatory profile and regulated the inflammatory cytokine production. However, no impact was observed on T cell activation and recruitment in the early acute phase of infection. Additionally, blocking of type I IFN signaling reduced bacterial burden in the infected mice as compared to untreated infected mice. CONCLUSIONS: Altogether, our results reveal that Type I IFN mediates a balance between M. bovis-mediated inflammatory reaction and host defense mechanism. Thus, modulating Type I IFN signaling could be exploited as a therapeutic strategy against a large repertoire of inflammatory disorders including tuberculosis.


Assuntos
Interferon Tipo I/metabolismo , Mycobacterium bovis/patogenicidade , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo , Animais , Anticorpos/farmacologia , Citocinas/metabolismo , Feminino , Humanos , Interferon Tipo I/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Mycobacterium bovis/imunologia , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/efeitos dos fármacos
9.
Int J Mol Sci ; 20(23)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795474

RESUMO

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.


Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Proteína Fosfatase 2/imunologia , Tuberculose/veterinária , Animais , Autofagia , Bovinos , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Camundongos , Mycobacterium bovis/fisiologia , Fagocitose , Células RAW 264.7 , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia
10.
Cells ; 8(5)2019 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060300

RESUMO

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.


Assuntos
Apoptose , Autofagia , Imunidade Inata , Calicreínas/metabolismo , Macrófagos/patologia , Mycobacterium bovis/fisiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Bovinos , Citocinas/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Células RAW 264.7 , Transdução de Sinais , Tuberculose Bovina/patologia
11.
Cells ; 8(5)2019 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-31130711

RESUMO

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne's disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.


Assuntos
Autofagia/efeitos dos fármacos , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Paratuberculose/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Tuberculose Bovina/tratamento farmacológico , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/microbiologia , Feminino , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Oncogênica v-akt/metabolismo , Paratuberculose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tuberculose Bovina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
12.
Front Immunol ; 10: 268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30846986

RESUMO

Mycobacterium bovis, the causative agent of tuberculosis in cattle and humans, infects host macrophages and induces endoplasmic reticulum stress (ERS), mitochondrial damage, and interleukin (IL)-1ß production. The relationship between these phenotypes is yet to be elucidated. In this study, we investigated the role of ERS in mitochondrial damage and IL-1ß production in macrophages during infection with a virulent M. bovis strain. We found that ERS activates the inflammasome via NOD-like receptor family, pyrin domain-containing 3 (NLRP3)-caspase-8 and that IFN-inducible protein absent in melanoma 2 (AIM2) triggered mitochondrial damage. ERS increased reactive oxygen species (ROS), which promoted translocation of the inflammasome to the mitochondria. NLRP3, but not AIM2, was involved in the ERS-induced cleavage of caspase-8 and Bid, leading to mitochondrial damage, which was required for the production of mature IL-1ß. Our data suggest that ERS induces macrophages to produce mature IL-1ß during infection with virulent M. bovis through a positive feedback loop between mitochondrial damage and inflammasome activation. To the best of our knowledge, this is the first evidence of the involvement of ERS and mitochondrial damage in inflammasome activation during M. bovis infection.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Infecções por Mycobacterium/metabolismo , Mycobacterium bovis/patogenicidade , Animais , Caspases/metabolismo , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-30042930

RESUMO

The mechanism by which microRNAs (miRNAs) modulate innate immunity and autophagy has not been fully elucidated in Mycobacterium bovis (M. bovis) infections. In this study, we identified that miR-199a inhibited key innate immune responses and autophagy in murine macrophages infected with M. bovis. Using ex vivo and in vitro approaches we show that the expression of miR-199a was significantly increased during M. bovis infection. Furthermore, miR-199a suppressed autophagy and interferon-ß (IFN-ß) production by directly targeting TANK-binding kinase 1 (TBK1) mRNA in both J774a.1 and BMDM cells. Upregulation of miR-199a or TBK1 silencing (siTBK1) inhibited maturation of autophagosomes and increased M. bovis survival. Our results demonstrate that, by targeting of TBK1, miR-199a modulates innate immune responses and promote the intracellular survival and growth of M. bovis.


Assuntos
Autofagia , Imunidade Inata , Interferon beta/biossíntese , Macrófagos/imunologia , MicroRNAs/metabolismo , Mycobacterium bovis/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana
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