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.

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.

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.