TRAF6 triggers Mycobacterium-infected host autophagy through Rab7 ubiquitination.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase that is extensively involved in the autophagy process by interacting with diverse autophagy initiation and autophagosome maturation molecules. However, whether TRAF6 interacts with lysosomal proteins to regulate Mycobacterium-induced autophagy has not been completely characterized. Herein, the present study showed that TRAF6 interacted with lysosomal key proteins Rab7 through RING domain which caused Rab7 ubiquitination and subsequently ubiquitinated Rab7 binds to STX17 (syntaxin 17, a SNARE protein that is essential for mature autophagosome), and thus promoted the fusion of autophagosomes and lysosomes. Furthermore, TRAF6 enhanced the initiation and formation of autophagosomes in Mycobacterium-induced autophagy in both BMDMs and RAW264.7 cells, as evidenced by autophagic flux, colocalization of LC3 and BCG, autophagy rates, and autophagy-associated protein expression. Noteworthy to mention, TRAF6 deficiency exacerbated lung injury and promoted BCG survival. Taken together, these results identify novel molecular and cellular mechanisms by which TRAF6 positively regulates Mycobacterium-induced autophagy.

LncRNA NR_003508 Suppresses Mycobacterium tuberculosis-Induced Programmed Necrosis via Sponging miR-346-3p to Regulate RIPK1.

Emerging evidence suggests that long non-coding RNAs (LncRNAs) are involved in Mtb-induced programmed necrosis. Among these LncRNAs, LncRNA NR_003508 is associated with LPS-induced acute respiratory distress syndrome. However, whether LncRNA NR_003508 contributes to Mtb-induced programmed necrosis remains undocumented. Firstly, the expression of LncRNA NR_003508 was determined using RT-qPCR and FISH. The protein expression of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL was measured by Western blot in RAW264.7 and mouse lung tissues. Furthermore, luciferase reporter assays and bioinformatics were used to predict specific miRNA (miR-346-3p) and mRNA (RIPK1) regulated by LncRNA NR_003508. In addition, RT-qPCR was used to detect the RIPK1 expression in TB patients and healthy peripheral blood. The flow cytometry assay was performed to detect cell necrosis rates. Here we show that BCG infection-induced cell necrosis and increased LncRNA NR_003508 expression. si-NR_003508 inhibited BCG/H37Rv-induced programmed necrosis in vitro or in vivo. Functionally, LncRNA NR_003508 has been verified as a ceRNA for absorbing miR-346-3p, which targets RIPK1. Moreover, RIPK1 expression was elevated in the peripheral blood of TB patients compared with healthy people. Knockdown of LncRNA NR_003508 or miR-346-3p overexpression suppresses cell necrosis rate and ROS accumulation in RAW264.7 cells. In conclusion, LncRNA NR_003508 functions as a positive regulator of Mtb-induced programmed necrosis via sponging miR-346-3p to regulate RIPK1. Our findings may provide a promising therapeutic target for tuberculosis.

Wnt/ß-catenin signaling pathway-a versatile player in apoptosis and autophagy.

The Wnt/ß-catenin signaling pathway is a highly conserved pathway that is involved in cell development, proliferation, differentiation, apoptosis and autophagy. Among these processes, apoptosis and autophagy occur physiologically during host defense and the maintenance of intracellular homeostasis. Mounting evidence suggests that the crosstalk between Wnt/ß-catenin-regulated apoptosis and autophagy has broad functional significance in various diseases. Herein, we summarize the recent studies in understanding the role of the Wnt/ß-catenin signaling pathway in apoptosis and autophagy, and draw the following conclusions: a) For apoptosis, the regulation of Wnt/ß-catenin is generally positive. However, a small amount of evidence indicates the presence of a negatively regulated relationship between Wnt/ß-catenin and apoptosis; b) Wnt/ß-catenin influences the occurrence and development of autophagy by regulating autophagy-related factors, and these factors in turn affect Wnt/ß-catenin pathway; c) Wnt/ß-catenin always balances the molecular damage caused by the crosstalk between autophagy and apoptosis in a compensatory manner. Understanding the specific role of the Wnt/ß-catenin signaling pathway during different stages of autophagy and apoptosis may provide new insights into the progression of related diseases regulated by the Wnt/ß-catenin signaling pathway.

RIP3 impedes Mycobacterium tuberculosis survival and promotes p62-mediated autophagy.

Macrophage is believed to play a vital role in the fight against Mycobacterium tuberculosis (M.tb) infection by activating autophagy. Recently, receptor-interacting protein kinase-3 (RIP3), an essential kinase for necroptotic cell death signaling, has been demonstrated to be involved in autophagy. However, RIP3's role in fighting against M.tb infection remains elusive. Here we show that a substantial increase in inflammatory cell infiltration and higher bacterial burden are observed in the lungs of RIP3-/- mice with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection. Meanwhile, RIP3 ameliorates lung injury and promote autophagy via induce autophagosome and autophagolysosome formation which indicate that RIP3 is indispensable for host clearance of BCG via autophagy. Mechanically, RIP3 enhances p62 binding to ubiquitylated proteins and LC3 by interacting with p62, and RHIM domain is required for RIP3-p62 interaction. Hence, our results conclusively show that RIP3 impedes M.tb survival and promotes p62-mediated autophagy. The findings provide further insight into understanding the mechanism of M.tb immune escape and pathogenesis of tuberculosis.

Heme oxygenase-1 modulates ferroptosis by fine-tuning levels of intracellular iron and reactive oxygen species of macrophages in response to Bacillus Calmette-Guerin infection.

Macrophages are the host cells and the frontline defense against Mycobacterium tuberculosis (Mtb) infection, and the form of death of infected macrophages plays a pivotal role in the outcome of Mtb infections. Ferroptosis, a programmed necrotic cell death induced by overwhelming lipid peroxidation, was confirmed as one of the mechanisms of Mtb spread following infection and the pathogenesis of tuberculosis (TB). However, the mechanism underlying the macrophage ferroptosis induced by Mtb infection has not yet been fully understood. In the present study, transcriptome analysis revealed the upregulation of heme oxygenase-1 (HMOX1) and pro-ferroptosis cytokines, but downregulation of glutathione peroxidase 4 (GPX4) and other key anti-lipid peroxidation factors in the peripheral blood of both patients with extra-pulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB). This finding was further corroborated in mice and RAW264.7 murine macrophage-like cells infected with Bacillus Calmette-Guerin (BCG). A mechanistic study further demonstrated that heme oxygenase-1 protein (HO-1) regulated the production of reactive oxygen species (ROS) and iron metabolism, and ferroptosis in BCG-infected murine macrophages. The knockdown of Hmox1 by siRNA resulted in a significant increase of intracellular ROS, Fe2+, and iron autophagy-mediated factor Ncoa4, along with the reduction of antioxidant factors Gpx4 and Fsp1 in macrophages infected with BCG. The siRNA-mediated knockdown of Hmox1 also reduced cell survival rate and increased the release of intracellular bacteria in BCG-infected macrophages. By contrast, scavenging ROS by N-acetyl cysteine led to the reduction of intracellular ROS, Fe2+, and Hmox1 concentrations, and subsequently inhibited ferroptosis and the release of intracellular BCG in RAW264.7 cells infected with BCG. These findings suggest that HO-1 is an essential regulator of Mtb-induced ferroptosis, which regulates ROS production and iron accretion to alter macrophage death against Mtb infections.

Microarray Profiling and Co-Expression Network Analysis of LncRNAs and mRNAs in Acute Respiratory Distress Syndrome Mouse Model.

BACKGROUND: Long noncoding RNAs (LncRNAs) play critical roles in many respiratory diseases. Acute respiratory distress syndrome (ARDS) is a destructive clinical syndrome of respiratory diseases. However, the potential mechanism of LncRNAs on ARDS remains largely unknown. METHODS: To identify the profiles of LncRNAs and mRNAs in the LPS-induced ARDS mouse model, the microarray analyses were hired to detect the expression of LncRNAs and mRNAs in present study. Subsequently, microarray data were verified by quantitative qRT-PCR. Functional annotation on DE mRNAs and LncRNAs were carried out by bioinformatics analysis. Furthermore, the role of selected DE LncRNAs on correlated genes was confirmed by si-RNA and Western blot. RESULTS: The expression of 2110 LncRNAs and 2690 mRNAs were significantly changed, which were further confirmed by qRT-PCR. GO and KEGG analysis indicated that the up-regulated mRNAs were mainly related to a defense response and tumor necrosis factor (TNF) signaling pathway, respectively. LncRNA-mRNA co-expression analyses showed that LncRNAs NR_003508, ENSMUST00000131638, ENSMUST00000119467, and ENSMUST00000124853 may correlate to MLKL, RIPK3, RIPK1, Caspase1, and NLRP3, respectively, or cooperatively, which were highly involved in the cell necroptosis process. Furthermore, siRNA for NR_003508 confirmed the co-expression analyses results. CONCLUSION: To summarize, this study implied that the DE LncRNAs could be potent regulators and target genes of ARDS and will provide a novel insight into the regulation of the pathogenesis of ARDS.

Corrigendum: Heme oxygenase-1 modulates ferroptosis by fine-tuning levels of intracellular iron and reactive oxygen species of macrophages in response to Bacillus Calmette-Guerin infection.

[This corrects the article DOI: 10.3389/fcimb.2022.1004148.].

Impact of knockdown LincRNA-Cox2 on apoptosis of macrophage infected with Bacillus Calmette-Guérin.

Mycobacterium tuberculosis (Mtb)-induced apoptosis of alveolar macrophages plays an important role in the pathogenesis of tuberculosis. Previous studies indicated that massive LncRNAs could deteriorate MTB invasion or latent infection by regulating macrophage's apoptosis. However, whether LincRNA-Cox2 is involved in apoptosis of macrophage infected with Mtb is unclear. In this study, we found Bacillus Calmette-Guerin(BCG)infection induced cell apoptosis with a increasing LincRNA-Cox2 expression in RAW264.7 cells. Furthermore, the activation of TLR signal pathway elevated the expression of lincRNA-Cox2. In this regard, we used small interfering RNA to explore the role of LincRNA-Cox2 on regulating apoptosis of RAW264.7 cells infected with BCG. The results showed that si-LincRNA-Cox2 was capable of increased the expression of apoptosis-associated proteins and accumulation of ROS in BCG-infected RAW264.7 cells. Mechanically, si-LincRNA-Cox2 facilitated BCG-induced macrophage apoptosis by activating the intrinsic apoptotic pathway as well as increased the genes expression of PERK/eIF2α/CHOP. These results provide novel insights into host-pathogen interactions and highlight the potential role of LincRNA-Cox2 in regulating apoptosis induced by BCG-infection.

Autophagy plays a protective role during Pseudomonas aeruginosa-induced apoptosis via ROS-MAPK pathway.

Pseudomonas aeruginosa infection can induce alveolar macrophage apoptosis and autophagy, which play a vital role in eliminating pathogens. These two processes are usually not independent. Recently, autophagy has been found to interact with apoptosis during pathogen infections. Nevertheless, the role of autophagy in P. aeruginosa-infected cell apoptosis is unclear. In this study, we explored the impact of P. aeruginosa infection on autophagy and apoptosis in RAW264.7 cells. The autophagy activator rapamycin was used to stimulate autophagy and explore the role of autophagy on apoptosis in P. aeruginosa-infected RAW264.7 cells. The results indicated that P. aeruginosa infection induced autophagy and apoptosis in RAW264.7 cells, and that rapamycin could suppress P. aeruginosa-induced apoptosis by regulating the expression of apoptosis-related proteins. In addition, rapamycin scavenged the cellular reactive oxygen species (ROS) and diminished p-JNK, p-ERK1/2 and p-p38 expression of MAPK pathways in RAW264.7 cells infected with P. aeruginosa. In conclusion, the promotion of autophagy decreased P. aeruginosa-induced ROS accumulation and further attenuated the apoptosis of RAW264.7 cells through MAPK pathway. These results provide novel insights into host-pathogen interactions and highlight a potential role of autophagy in eliminating P. aeruginosa.

Knockdown of fatty acid binding protein 4 exacerbates Bacillus Calmette-Guerin infection-induced RAW264.7 cell apoptosis via the endoplasmic reticulum stress pathway.

Mycobacterial infection can induce alveolar macrophage apoptosis, which plays a vital role in the pathogenesis of tuberculosis. Accumulating evidence has demonstrated that fatty acid oxidation is involved in apoptosis during various pathological processes, including bacterial infection. However, whether fatty acid oxidation regulates mycobacterial infection-induced macrophage apoptosis remains unclear. Hence, the present study aimed to investigate the role of fatty acid binding protein 4 (FABP4) which is a carrier protein for fatty acids, in regulating apoptosis in RAW264.7 cells infected with Bacillus Calmette-Guerin (BCG). In our study, the impact of BCG infection on apoptosis and fatty acid oxidation in RAW264.7 cells was examined. Notably, we found that FABP4 was overexpressed during this process. Furthermore, small interfering RNAs targeting FABP4 were used to investigate the role of FABP4 in regulating apoptosis and fatty acid oxidation in BCG-infected RAW264.7 cells. The results indicated that mycobacterial infection promoted apoptosis and enhanced fatty acid oxidation in RAW264.7 cells. Moreover, FABP4 knockdown exacerbated BCG-induced apoptosis and upregulated the expression of p-PERK, p-eIF2α and chop, which are endoplasmic reticulum (ER) stress markers. In addition, FABP4 knockdown promoted fatty acid oxidation and ROS production, which result in the activation of ER stress. Our data suggested that FABP4 knockdown exacerbated BCG-induced apoptosis in RAW264.7 cells via the ER stress pathway.

Shifts in microbial community, pathogenicity-related genes and antibiotic resistance genes during dairy manure piled up.

The uncomposted faeces of dairy cow are usually stacked on cow breeding farms, dried under natural conditions and then used as cow bedding material or they may be continuously piled up. However, no information is available to evaluate variations in the human and animal pathogen genes and antibiotic resistance during the accumulation of fresh faeces of dairy cow to manure. Here, we present the metagenomic analysis of fresh faeces and manure from a dairy farm in Ning Xia, showing a unique enrichment of human and animal pathogen genes and antibiotic resistance genes (ARGs) in manure. We found that manure accumulation could significantly increase the diversity and abundance of the pathogenic constituents. Furthermore, pathogens from manure could spread to the plant environment and enphytotic pathogens could affect the yield and quality of crops during the use of manure as a fertilizer. Levels of virulence genes and ARGs increased with the enrichment of microbes and pathogens when faeces accumulated to manure. Accumulated manure was also the transfer station of ARGs to enrich the ARGs in the environment, indicating the ubiquitous presence of environmental antibiotic resistance genes. Our results demonstrate that manure accumulation and usage without effective manure management is an unreasonable approach that could enrich pathogenic microorganisms and ARGs in the environment. The manure metagenome structure allows us to appreciate the overall influence and interaction of animal waste on water, soil and other areas impacted by faecal accumulation and the factors that influence pathogen occurrence in products from dairy cows.

Magnesium sulfate attenuates lipopolysaccharides-induced acute lung injury in mice.

Acute lung injury (ALI) is a common and severe respiratory disease with high morbidity and mortality. Although some progress has been made in the past years, the pathogenesis of ALI is still poorly understood and the therapeutic outcome has still not been significantly improved. It is well-recognized that magnesium sulfate (MgSO4) possesses potent anti-inflammation capacity. The present study was designed to investigate the protective effects of MgSO4 in lipopolysaccharides (LPSs)-induced ALI taken into account that excessive inflammatory response plays critical role in the development of ALI. In this study, Kunming mice were intravenously injected with LPS through tail vein to establish the ALI model and in parallel, A549 cells were used to establish cell model. The lung wet-to-dry weight ratio, malondialdehyde (MDA) levels in lung tissue, lung permeability index, hematoxylin and eosin staining, cytokines in the serum and bronchoalveolar lavage fluid (BALF), neutrophil counts in BALF, LPS-induced A549 cell apoptosis as well as apoptosis-inducing factor (AIF), and Poly(ADP-ribose) polymerase-1 (PARP-1) expression in both mice and A549 cells were detected. Our results demonstrated that MgSO4 significantly attenuated the LPS-induced ALI, oxidative stress (decreased MDA levels), and lung inflammatory response. Moreover, MgSO4 exerted protective effects by mitigating LPS-induced A549 cell apoptosis. Furthermore, MgSO4 decreased the AIF and PARP-1 expression both in vivo and in vitro. Our results, taken together, demonstrated that MgSO4 is a potential therapeutic agent for ALI taken into consideration that MgSO4 is commonly used in clinical settings.

Characterization of Microbial Communities in a Dairy Farm Matrix in Ningxia, China, by 16S rDNA Analysis.

A large amount of dairy manure is produced annually in the Ningxia Hui Autonomous Region of China due to the increase in food-producing animal agriculture in this region. The presence of bovine-originated zoonotic, especially human, pathogenic bacteria in untreated manure poses a significant threat to the environment and to public health. However, little is known about the composition, diversity, and abundance of bacterial communities in untreated dairy manure in the Ningxia region. In this study, the microbial community structure of the dairy farm matrix was characterized through 16S rDNA sequencing. The impact of manure treatment methods on bacterial communities was also analyzed. The results showed that the microbial community in dairy manure contained both beneficial bacteria and pathogens, with Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, and Actinobacteria as dominant phyla. The results also showed the diversity and variety of abundance of zoonotic pathogens among different matrices. The number of pathogens was found to increase significantly in the accumulated but untreated manure, which appeared to be the main matrix of dairy farms that accumulated pathogens including zoonotic pathogens. Findings from this study suggested that farm management, particularly proper treatment of manure, is essential to achieve a shift in the bacterial community composition and a reduction in the environmental load of pathogens including zoonotic pathogens.

Oocyte-derived E-cadherin acts as a multiple functional factor maintaining the primordial follicle pool in mice.

In mammals, female fecundity is determined by the size of the primordial follicle (PF) pool, which is established during the perinatal period. As a non-renewable resource, the preservation of dormant PFs is crucial for sustaining female reproduction throughout life. Although studies have revealed that several oocyte-derived functional genes and pathways, such as newborn ovary homeobox (NOBOX) and 3-phosphoinositide-dependent protein kinase-1, participate in maintaining the PF pool, our understanding of the underlying molecular mechanisms is still incomplete. Here, we demonstrate that E-cadherin (E-cad) plays a crucial role in the maintenance of PFs in mice. E-cad is specifically localized to the cytomembrane of oocytes in PFs. Knockdown of E-cad in neonatal ovaries resulted in significant PF loss owing to oocyte apoptosis. In addition, the expression pattern of NOBOX is similar to that of E-cad. Knockdown of E-cad resulted in a decreased NOBOX level, whereas overexpression of Nobox partially rescued the follicle loss induced by silencing E-cad. Furthermore, E-cad governed NOBOX expression by regulating the shuttle protein, ß-catenin, which acts as a transcriptional co-activator. Notably, E-cad, which is a transmembrane protein expressed in the oocytes, was also responsible for maintaining the PF structure by facilitating cell-cell adhesive contacts with surrounding pregranulosa cells. In conclusion, E-cad in oocytes of PFs plays an indispensable role in the maintenance of the PF pool by facilitating follicular structural stability and regulating NOBOX expression. These findings shed light on the physiology of sustaining female reproduction.

A role for Wnt/ß-catenin signalling in suppressing Bacillus Calmette-Guerin-induced macrophage autophagy.

Mycobacterium tuberculosis (Mtb)-induced autophagy of alveolar macrophages has been confirmed to play a central role in the pathogenesis of tuberculosis. Growing evidence indicates that excessive or uncontrolled autophagic activity, which results in type II programmed cell death, can be regulated by many factors, including Wnt/ß-catenin signalling. Wnt/ß-catenin signalling has been demonstrated to be involved in multiple diseases through the regulation of autophagy; however, its exact role in regulating autophagy induced by Mtb remains unclear. Accordingly, this study examined the function of the Wnt/ß-catenin signalling pathway in regulating Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-induced autophagy in RAW264.7 macrophage cell line. In the present study, we found that BCG induced the autophagy of RAW264.7 cells in a time- and dose-dependent manner along with an accumulation of LC3 (Microtubule-associated protein 1 light chain 3) protein. Intriguingly, Wnt3a, a Wnt/ß-catenin signalling ligand, significantly inhibited autophagy, with decreased autophagy rates and autophagic flux. An immunoblot analysis further revealed that Wnt/ß-catenin signalling was capable of inhibiting the expression of the LC3 and autophagy-associated gene (Atg) cascade proteins in BCG-infected cells. Mechanistically, Wnt/ß-catenin signalling may inhibit autophagy in BCG-infected macrophages by activating mTOR-dependent pathways. Our findings reveal the mechanisms of Wnt/ß-catenin signalling regulates cellular autophagy induced by Mtb and provide novel insights into physiological and immune control of tuberculosis by modulating autophagy processes.

Involvement of PKCε in FSH-induced connexin43 phosphorylation and oocyte maturation in mouse.

Gap junctions (GJs) are indispensable for communication between cumulus cells (CCs) and oocytes in coordinating the gonadotropin-induced meiotic maturation of oocytes. Of all proteins that constitute GJs, phosphorylated connexin43 (pCx43) is vital for mediating the actions of gonadotropins. In this study, the mechanism of Cx43 phosphorylation in response to follicle stimulating hormone (FSH) stimulation was examined using an in vitro model of mouse cumulus-oocyte complexes (COCs). The results confirmed that Cx43 phosphorylation occurred twice during FSH treatment. Importantly, the second Cx43 phosphorylation was closely related to cAMP level reduction within oocytes, which initiated oocyte maturation. Exploration of the underlying mechanism revealed that the CC-specific protein kinase C ε (PKCε) level was upregulated by FSH stimulation. PKCε was a kinase downstream from mitogen-activated protein kinase (MAPK) and was responsible for Cx43 phosphorylation. Interestingly, MAPK was involved in both Cx43 phosphorylation processes, while PKCε was only involved in the second. In conclusion, PKCε-mediated MAPK signals might contribute to Cx43 phosphorylation in CCs during FSH-induced oocyte meiotic resumption. Our findings contribute to a better understanding of the molecular regulation mechanism of oocyte maturation in response to FSH in vitro.

A ROS-dependent and Caspase-3-mediated apoptosis in sheep bronchial epithelial cells in response to Mycoplasma Ovipneumoniae infections.

Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections.

Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1ß, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFß of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

Capsular Polysaccharide of Mycoplasma ovipneumoniae Induces Sheep Airway Epithelial Cell Apoptosis via ROS-Dependent JNK/P38 MAPK Pathways.

In an attempt to better understand the pathogen-host interaction between invading Mycoplasma ovipneumoniae (M. ovipneumoniae) and sheep airway epithelial cells, biological effects and possible molecular mechanism of capsular polysaccharide of M. ovipneumoniae (CPS) in the induction of cell apoptosis were explored using sheep bronchial epithelial cells cultured in air-liquid interface (ALI). The CPS of M. ovipneumoniae was first isolated and purified. Results showed that CPS had a cytotoxic effect by disrupting the integrity of mitochondrial membrane, accompanied with an increase of reactive oxygen species and decrease of mitochondrial membrane potential (ΔΨm). Of importance, the CPS exhibited an ability to induce caspase-dependent cell apoptosis via both intrinsic and extrinsic apoptotic pathways. Mechanistically, the CPS induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways. These findings provide the first evidence that CPS of M. ovipneumoniae induces a caspase-dependent apoptosis via both intrinsic and extrinsic apoptotic pathways in sheep bronchial epithelial cells, which may be mainly attributed by a ROS-dependent JNK and p38 MAPK signaling pathways.

The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin.

Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence. Results. A His-tagged rCPB2 with integrity and cytotoxicity of native CPB2 was purified from E. coli expressing system, which exhibited a moderate cytotoxicity on NCM460 human intestinal epithelial cells. The rCPB2 could induce apoptotic cell death rather than necrotic death in part through a pathway involved in caspase-3 signaling. Mechanistically, rCPB2 was able to first bind to cell membrane and dynamically translocate into cytoplasm for its cytotoxic activity. Three McAbs 1E23, 2G7 and 2H7 were characterized to be able to immunologically react with CPB2 and neutralize rCPB2 cytotoxicity on NCM460 cells. Conclusion. These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future, which warrants for further investigations.