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.

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.

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.

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.

Mycoplasma ovipneumoniae induces sheep airway epithelial cell apoptosis through an ERK signalling-mediated mitochondria pathway.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovipneumoniae) is a species of Mycoplasma bacteria that specifically infects sheep and goat, causing ovine infectious pleuropneumonia. However, the mechanism underlying the pathogen-host interaction between M. ovipneumoniae and airway epithelial cells is unknown. METHODS: A primary air-liquid interface (ALI) epithelial culture model generated from the bronchial epithelial cells of Ningxia Tan sheep (ovis aries) was employed to explore the potential mechanism of M. ovipneumoniae-induced cell apoptosis by characterizing the production of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA) and anti-oxidative enzymes, as well as the mitochondrial membrane potentials, cytochrome C release, and activities of ERK and caspase signalling pathways. RESULTS: Increased ROS production and MDA concentration with mitochondrial membrane dysfunction and apoptotic cell death but decreased expression of the antioxidant enzymes catalase (CAT), glutathione synthetase (GSS), total superoxide dismutaes (T-SOD) and Mn-SOD were observed in sheep airway epithelial cells infected with M. ovipneumoniae. Mechanistically, the M. ovipneumoniae-induced cell apoptosis and disruption of mitochondrial integrity reflected mechanisms by which pathogen-activated mitogen-activated protein kinase (MAPK) signalling sequentially led to mitochondrial damage and release of Cyt-C into the cytoplasm, which in turn triggered the activation of caspase signalling cascade, resulting in the apoptosis of host cells. CONCLUSIONS: These results suggest that M. ovipneumoniae-induced ROS and MAPK signalling-mediated mitochondrial apoptotic pathways might play key roles in the pathogenesis of M. ovipneumoniae infection in sheep lungs.

Wnt/ß-catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway.

BACKGROUND: Necrosis of alveolar macrophages following Mycobacterium tuberculosis infection has been demonstrated to play a vital role in the pathogenesis of tuberculosis. Our previous study demonstrated that Wnt/ß-catenin signaling was able to promote mycobacteria-infected cell apoptosis by a caspase-dependent pathway. However, the functionality of this signaling in the necrosis of macrophage following mycobacterial infection remains largely unknown. METHODS: Murine macrophage RAW264.7 cells were infected with Bacillus Calmette-Guerin (BCG) in the presence of Wnt/ß-catenin signaling. The necrotic cell death was determined by cytometric assay and electronic microscopy; the productions of reactive oxygen species (ROS) and reduced glutathione (GSH) were measured by a cytometric analysis and an enzyme-linked immunosorbent assay, respectively; and the activity of poly (ADP-ribose) polymerase 1 (PARP-1)/apoptosis inhibition factor (AIF) signaling was examined by an immunoblotting assay. RESULTS: The BCG can induce RAW264.7 macrophage cells necrosis in a dose- and time-dependent manner along with an accumulation of reactive oxygen species (ROS). Intriguingly, an enhancement of Wnt/ß-catenin signaling shows an ability to reduce the mycobacteria-induced macrophage necrosis. Mechanistically, the activation of Wnt/ß-catenin signaling is capable of inhibiting the necrotic cell death in BCG-infected RAW264.7 cells through a mechanism by which the Wnt signaling scavenges intracellular ROS accumulation and increases cellular GSH concentration. In addition, immunoblotting analysis further reveals that Wnt/ß-catenin signaling is capable of inhibiting the ROS-mediated cell necrosis in part through a PARP-1/AIF- dependent pathway. CONCLUSIONS: An activation of Wnt/ß-catenin signaling can inhibit BCG-induced macrophage necrosis by increasing the production of GSH and scavenging ROS in part through a mechanism of repression of PARP-1/AIF signaling pathway. This finding may thus provide an insight into the underlying mechanism of alveolar macrophage cell death in response to mycobacterial infection.

A caspase-dependent pathway is involved in Wnt/ß-catenin signaling promoted apoptosis in Bacillus Calmette-Guerin infected RAW264.7 macrophages.

Apoptosis of alveolar macrophages following Mycobacterium tuberculosis infection have been demonstrated to play a central role in the pathogenesis of tuberculosis. In the present study, we found that Wnt/ß-catenin signaling possesses the potential to promote macrophage apoptosis in response to mycobacterial infection. In agreement with other findings, an activation Wnt/ß-catenin signaling was observed in murine macrophage RAW264.7 cells upon Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection at a multiple-of-infection of 10, which was accompanied with up-regulation of pro-inflammatory cytokines TNF-α and IL-6 production. However, the BCG-induced TNF-α and IL-6 secretion could be significantly reduced when the cells were exposed to a canonical Wnt signaling ligand, Wnt3a. Importantly, the activation of Wnt/ß-catenin signaling was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by a mitochondria-dependent apoptosis pathway. Immunoblotting analysis further demonstrated that Wnt/ß-catenin signaling-induced cell apoptosis partly through a caspase-dependent apoptosis mechanism by down-regulation of anti-apoptotic protein Mcl-1, and up-regulation of pro-apoptotic proteins Bax and cleaved-caspase-3, as well as enhancement of caspase-3 activity in BCG-infected RAW264.7 cells. These data may imply an underlying mechanism of alveolar macrophages in response to mycobacterial infection, by which the pathogen induces Wnt/ß-catenin signaling activation, which in turn represses mycobacterium-trigged inflammatory responses and promotes mycobacteria-infected cell apoptosis.

Mycobacterium manipulate glutaminase 1 mediated glutaminolysis to regulate macrophage autophagy for bacteria intracellular survival.

Autophagy plays a vital role in eliminating intracellular mycobacterium. It is regulated by multiple metabolic processes including glutaminolysis. Glutaminase 1 (GLS1) is the rate-limiting enzyme of glutaminolysis and has been reported to control intracellular Gln content. However, its function on regulating autophagy in mycobacterium infected macrophage is still obscure. Hence, the current study hired mycobacterium virulent strain H37Rv or attenuated strain BCG to infect macrophage and detected the changes in cell glutaminolysis. The function of GLS1 on regulating autophagy in mycobacterium infected macrophages was further investigated. The results showed that BCG infection promoted macrophage autophagy, enhanced glutaminolysis, reduced intracellular Gln content, accompanied with the up-regulation of GLS1. Conversely, H37Rv infection resulted in completely opposite effects. Meanwhile, knockdown of GLS1 increased Gln content and attenuated autophagy in BCG infected macrophages. In addition, the deprivation of Gln not only promoted the autophagy of H37Rv infected macrophages, but also abolished the effect of knockdown GLS1 on regulating BCG infection-induced mTOR activation or autophagy. To sum up, our study suggested that different virulent strains of mycobacterium infection have totally opposite effects on glutaminolysis and the expression of GLS1. Specifically, mycobacterium virulent strain reduced GLS1 expression and decreased Gln content but mycobacterium attenuated strain promoted GLS1 expression and enhanced Gln content. Furthermore, GLS1 inhibits the activation of the mTOR signaling pathway and promotes autophagy by decreasing Gln content.

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.

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.

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.

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.].

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.

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.

Acute respiratory distress syndrome induced by H9N2 virus in mice.

H9N2 avian influenza viruses have repeatedly caused infections in swine and humans in some countries. The purpose of the present study was to evaluate the pulmonary pathology caused by H9N2 viral infection in mice. Six- to eight-week-old BALB/c mice were infected intranasally with 1 x 10(4) MID(50) of A/Chicken/Hebei/4/2008(H9N2) virus. Clinical signs, pathological changes and viral replication in lungs, arterial blood gas, and cytokines in bronchoalveolar lavage fluid (BALF) were observed at different time points after infection. A control group was infected intranasally with noninfectious allantoic fluid. H9N2-infected mice exhibited severe respiratory syndrome, with a mortality rate of 60%. Gross observations showed that infected lungs were highly edematous. Major histopathological changes in infected lungs included diffuse pneumonia and alveolar damage, with neutrophil-dominant inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage, and severe bronchiolitis/peribronchiolitis. In addition, H9N2 viral infection resulted in severe progressive hypoxemia, lymphopenia, and a significant increase in neutrophils, tumor necrosis factor-alpha and interleukin-6 in BALF. The features described above satisfy the criteria for acute respiratory distress syndrome (ARDS). Our data show that H9N2 viral infection resulted in ARDS in mice, and this may facilitate studies of the pathogenesis of future potential H9N2 disease in humans.

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.

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.

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.

Pulmonary fibrosis induced by H5N1 viral infection in mice.

BACKGROUND: Inflammatory process results in lung injury that may lead to pulmonary fibrosis (PF). Here, we described PF in mice infected with H5N1 virus. METHODS: Eight-week-old BALB/c mice were inoculated intranasally with 1 x 101 MID50 of A/Chicken/Hebei/108/2002(H5N1) viruses. Lung injury/fibrosis was evaluated by observation of hydroxyproline concentrations, lung indexes, and histopathology on days 7, 14, and 30 postinoculation. RESULTS: H5N1-inoculated mice presented two stages of pulmonary disease over a 30-d period after infection. At acute stage, infected-mice showed typical diffuse pneumonia with inflammatory cellular infiltration, alveolar and interstitial edema and hemorrhage on day 7 postinoculation. At restoration stage, most infected-mice developed PF of different severities on day 30 postinoculation, and 18% of the survived mice underwent severe interstitial and intra-alveolar fibrosis with thickened alveolar walls, collapsed alveoli and large fibrotic areas. The dramatically elevated hydroxyproline levels in H5N1-infected mice showed deposition of collagen in lungs, and confirmed fibrosis of lungs. The dry lung-to-body weight ratio was significantly increased in infected group, which might be associated with the formation of PF in H5N1-infected mice. CONCLUSION: Our findings show that H5N1-infected mice develop the typical PF during restoration period, which will contribute to the investigation of fibrogenesis and potential therapeutic intervention in human H5N1 disease.