Genome-Wide CRISPR/Cas9 Screening Identifies That Mitochondrial Solute Carrier SLC25A23 Attenuates Type I IFN Antiviral Immunity via Interfering with MAVS Aggregation.

Activation of the mitochondrial antiviral signaling (MAVS) adaptor, also known as IPS-1, VISA, or Cardif, is crucial for antiviral immunity in retinoic acid-inducible gene I (RIG-I)-like receptor signaling. Upon interacting with RIG-I, MAVS undergoes K63-linked polyubiquitination by the E3 ligase Trim31, and subsequently aggregates to activate downstream signaling effectors. However, the molecular mechanisms that modulate MAVS activation are not yet fully understood. In this study, the mitochondrial solute carrier SLC25A23 was found to attenuate type I IFN antiviral immunity using genome-wide CRISPR/Cas9 screening. SLC25A23 interacts with Trim31, interfering with its binding of Trim31 to MAVS. Indeed, SLC25A23 downregulation was found to increase K63-linked polyubiquitination and subsequent aggregation of MAVS, which promoted type I IFN production upon RNA virus infection. Consistently, mice with SLC25A23 knockdown were more resistant to RNA virus infection in vivo. These findings establish SLC25A23 as a novel regulator of MAVS posttranslational modifications and of type I antiviral immunity.

The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy.

The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.

A cathelicidin antimicrobial peptide from Hydrophis cyanocinctus inhibits Zika virus infection by downregulating expression of a viral entry factor.

Zika virus (ZIKV) is a re-emerging flavivirus that causes conditions such as microcephaly and testis damage. The spread of ZIKV has become a major public health concern. Recent studies indicated that antimicrobial peptides are an ideal source for screening antiviral candidates with broad-spectrum antiviral activities, including against ZIKV. We herein found that Hc-CATH, a cathelicidin antimicrobial peptide identified from the sea snake Hydrophis cyanocinctus in our previous work, conferred protection against ZIKV infection in host cells and showed preventative efficacy and therapeutic efficacy in C57BL/6J mice, Ifnar1-/- mice, and pregnant mice. Intriguingly, we revealed that Hc-CATH decreased the susceptibility of host cells to ZIKV by downregulating expression of AXL, a TAM (TYRO3, AXL and MERTK) family kinase receptor that mediates ZIKV infection, and subsequently reversed the negative regulation of AXL on host's type I interferon response. Furthermore, we showed that the cyclo-oxygenase-2/prostaglandin E2/adenylyl cyclase/protein kinase A pathway was involved in Hc-CATH-mediated AXL downregulation, and Hc-CATH in addition directly inactivated ZIKV particles by disrupting viral membrane. Finally, while we found Hc-CATH did not act on the late stage of ZIKV infection, structure-function relationship studies revealed that α-helix and phenylalanine residues are key structural requirements for its protective efficacy against initial ZIKV infection. In summary, we demonstrate that Hc-CATH provides prophylactic and therapeutic efficacy against ZIKV infection via downregulation of AXL, as well as inactivating the virion. Our findings reveal a novel mechanism of cathelicidin against viral infection and highlight the potential of Hc-CATH to prevent and treat ZIKV infection.

Cardiomyocyte-derived HMGB1 takes a protective role in CVB3-induced viral myocarditis via inhibiting cardiac apoptosis.

Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) is characterized by immune cell infiltration and myocardial damage. High mobility group box 1 (HMGB1) is a highly conserved nuclear DNA-binding protein that participates in DNA replication, transcriptional regulation, repair response and inflammatory response in different disease models. To investigate the exact function of HMGB1 in CVB3-induced VMC, we crossed Hmgb1-floxed (Hmgb1f/f ) mice with mice carrying a suitable Cre recombinase transgenic strain to achieve conditional inactivation of the Hmgb1 gene in a cardiomyocyte-specific manner and to establish myocarditis. In this study, we found that cardiomyocyte-specific Hmgb1-deficient (Hmgb1f/f TgCre/+ ) mice exhibited exacerbated myocardial injury. Hmgb1-deficient cardiomyocytes may promote early apoptosis via the p53-mediated Bax mitochondrial pathway, as evidenced by the higher localization of p53 protein in the cytosol of Hmgb1-deficient cardiomyocytes upon CVB3 infection. Moreover, cardiomyocyte Hmgb1-deficient mice are more susceptible to cardiac dysfunction after infection. This study provides new insights into HMGB1 in VMC pathogenesis and a strategy for appropriate blocking of HMGB1 in the clinical treatment of VMC.

USP39 Serves as a Deubiquitinase to Stabilize STAT1 and Sustains Type I IFN-Induced Antiviral Immunity.

Deubiquitinating enzymes (DUBs) are cysteine proteases that reverse the ubiquitination by removing ubiquitins from the target protein. The human genome encodes ∼100 potential DUBs, which can be classified into six families, influencing multiple cellular processes, such as antiviral responses, inflammatory responses, apoptosis, etc. To systematically explore the role of DUBs involved in antiviral immunity, we performed an RNA interference-based screening that contains 97 human DUBs. We identified that ubiquitin-specific protease (USP) 39 expression modulates the antiviral activity, which is, to our knowledge, a previously unknown function of this enzyme. Small interfering RNA knockdown of USP39 significantly enhanced viral replication, whereas overexpression of USP39 had an opposite effect. Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK/STAT downstream of type I signaling by enhancing IFN-stimulated response elements promoter activity and expression of IFN-stimulated genes. Interestingly, USP39, previously considered not to have the deubiquitinase activity, in this study is proved to interact with STAT1 and sustain its protein level by deubiqutination. Furthermore, we found that through novel mechanism USP39 can significantly decrease K6-linked but not K48-linked ubiquitination of STAT1 for degradation. Taken together, these findings uncover that USP39 is, to our knowledge, a new deubiquitinase that positively regulates IFN-induced antiviral efficacy.

Retrospective analysis of HIV-1 drug resistance mutations in Suzhou, China from 2009 to 2014.

In this study, we investigated drug resistance levels in human immunodeficiency virus (HIV)-1-infected patients in Suzhou by retrospectively analyzing this property and the characteristics of circulating HIV-1 strains collected from 2009 to 2014. A total of 261 HIV-1-positive plasma samples, confirmed by the Suzhou CDC, were collected and evaluated to detect HIV-1 drug resistance genotypes using an in-house method. The pol gene fragment was amplified, and its nucleic acid sequence was determined by Sanger sequencing. Drug resistance mutations were then analyzed using the Stanford University HIV resistance database ( https://hivdb.stanford.edu ). A total of 216 pol gene fragments were amplified and sequenced with 16.7% (36/216) of sequences revealing these mutations. The drug resistance rates of protease, nucleoside reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were 4/36 (11.1%), 2/36 (5.6%), and 30/36 (83.3%), respectively. Five surveillance drug resistance mutations were found in 36 sequences, of which, three were found among specimens of men who have sex with men. Potential low-level resistance accounted for 33% of amino acid mutations associated with NNRTIs. Two of the mutations, M230L and L100I, which confer a high level of resistance efavirenz (EFV) and nevirapine (NVP) used as NNRTIs for first-line antiretroviral therapy (ART), were detected in this study. Therefore, when HIV-1 patients in Suzhou are administered fist-line ART, much attention should be paid to the status of these mutations that cause resistance to EVP, EFV, and NVP.

Semaphorin7A aggravates coxsackievirusB3-induced viral myocarditis by increasing α1ß1-integrin macrophages and subsequent enhanced inflammatory response.

Semaphorin7A (Sema7A) has been reported to play various roles in nerve axon growth, tumor suppression, and tissue remodeling, as well as regulation of intestinal inflammation diseases. Viral myocarditis (VMC) characterized by viral-myocardial-cell necrosis and inflammatory cell infiltration is a common clinical disease of the cardiovascular system. However, the role of Sema7A in coxsackievirus B3 (CVB3)-induced VMC has not been reported. In this study, we generated an acute VMC mouse model by CVB3 infection, and manipulated Sema7A expression by in vivo polyethyleneimine-mediated Sema7A down-regulation. Our results indicated that Sema7A was up-regulated in cardiomyocytes during VMC, and that Sema7A down-regulation following short hairpin RNA interference or mAb neutralization effectively protected mice from VMC. Additionally, reduced inflammatory responses were observed along with Sema7A down-regulation. Furthermore, adoptive transfer of α1ß1-integrin macrophages exacerbated CVB3-induced myocarditis, suggesting the significance of α1ß1-integrin macrophages in response to VMC. We observed that co-culture of neonatal myocardiocytes with macrophages increased the percentage of α1ß1-integrin macrophages, while Sema7A neutralization reduced α1ß1-integrin macrophages in heart tissue of VMC mice. These results demonstrated that Sema7A, as an inflammation regulator in CVB3-induced VMC, might interact with α1ß1-integrin in macrophages to enhance the inflammatory response and aggravate disease severity. Our findings provided insight into the potential role of Sema7A as a therapeutic treatment for VMC.

ODC1 inhibits the inflammatory response and ROS-induced apoptosis in macrophages.

Macrophage activation plays a critical role in the innate immune response. Ornithine decarboxylase (ODC1) metabolizes l-ornithine to polyamines and is the rate-limiting enzyme involved in the metabolism of polyamines, which are reportedly involved in cell differentiation, proliferation, and migration. However, the function of ODC1 in immune cells and especially in macrophages, as well as its underlying molecular mechanism, remains unclear. This study revealed the potential ODC1 function and mechanism associated with the lipopolysaccharide (LPS)-, Bacillus Calmette-Guerin (BCG)-, or carbon tetrachloride (CCl4)-induced inflammatory response in macrophages. We found significant upregulation of ODC1 in macrophages following LPS simulation and ODC1-specific suppression of proinflammatory cytokine secretion from macrophages upon stimulation with LPS, BCG and CCl4, respectively, suggesting a role as a common control element of the inflammatory response. Western blotting for nuclear factor-κB and mitogen-activated protein kinases revealed significant inhibition of phosphorylation of multiple transcription factors following ODC1 overexpression in macrophages. Moreover, ODC1 inhibited reactive oxygen species-induced and caspase-dependent apoptosis highlighted by decreased caspase-3 and -9 expression following ODC1 upregulation. These findings indicated that ODC1 was involved in attenuating the inflammatory response upon stimulation of macrophages, making it a potential therapeutic target for inflammatory diseases.

IL-33 enhances macrophage M2 polarization and protects mice from CVB3-induced viral myocarditis.

Viral myocarditis is the inflammation caused by myocardial virus infection, and the coxsackievirus group B3 virus (CVB3) is the most common pathogen. An efficient therapeutic agent against viral myocarditis is currently unavailable. IL-33, a new member of the IL-1 cytokine superfamily, exhibits potential immunotherapeutic effect against inflammatory and autoimmune diseases. However, the functional role of IL-33 in viral myocarditis has not been investigated. To examine the therapeutic role of IL-33 in viral myocarditis, an IL-33 overexpression plasmid (pDisplay-IL-33) and IL-33 knockdown plasmid (pLL3.7-IL-33) were packaged with polyethylenimine and delivered intravenously at the orbital area of BALB/c male mice after CVB3 infection. Then, myocarditis severity was assessed 7days after infection. Results showed that IL-33 up-regulation significantly alleviated the severity of viral myocarditis with an increased cardiac contractive function and survival rate. Mechanistic studies demonstrated that IL-33 can stimulate ST2L+F4/80+ macrophages and ST2L+CD4+T cells in cardiac tissue to express IL-4, which is a potent inducer for macrophage M2 polarization. Mice with adoptive transfer of M2 macrophages exhibited less cardiac inflammation and attenuated myocarditis, suggesting the protective role of M2 macrophage in viral myocarditis. Additionally, IL-4 neutralization abolished the IL-33-mediated cardiac functional improvement in myocarditis mice. Collectively, our findings provide a novel therapeutic role for IL-33 in CVB3-induced myocarditis.

Coxsackievirus B3 infection induces autophagic flux, and autophagosomes are critical for efficient viral replication.

Autophagy is an intrinsic cellular process that can degrade cytoplasmic components. It has been reported that several pathogens hijack this process to facilitate their replication. Coxsackievirus B3 (CVB3), a member of the family Picornaviridae, induces autophagy upon infection. However, the details of CVB3-induced autophagy remain a subject of debate. This study applied a combination of multiple assays for the measurement of autophagy and demonstrated that CVB3 induces a complete autophagic flux. Experiments with infected HEK293A cells revealed that autophagosomes were induced upon CVB3 infection. Most of these autophagosomes were mCherry positive in mCherry-GFP-LC3 cells. Conversely, mCherry-positive autophagosomes were rescued to green positive when treated with the acidification inhibitors chloroquine (CQ) and bafilomycin A1 (BAF), suggesting that autophagosomes fused with late endosomes or lysosomes. The co-localization of LC3-positive puncta with lysosome-associated membrane protein 1 (LAMP1) or LysoTracker confirmed that the autophagosomes fused primarily with lysosomes. Interestingly, the disruption of autophagosome formation by 3-methyladenine (3-MA) or ATG5 siRNA treatment during viral infection significantly decreased CVB3 replication. However, inhibitors of lysosomal acidification, fusion, or degradation did not affect viral replication. Therefore, autolysosomes may not be critical for viral replication in vitro.

Signaling lymphocyte-activation molecule SLAMF1 augments mycobacteria BCG-induced inflammatory response and facilitates bacterial clearance.

Tuberculosis, which is caused by intracellular mycobacterium Mycobacterium tuberculosis (Mtb), remains one of the most serious global public health concerns. The mechanisms by which innate immunity regulates the inflammatory responses and affects mycobacterial infection remain unclear. In this study, signaling lymphocyte-activation molecule family 1 (SLAMF1) was significantly upregulated in Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected RAW264.7 cells. Overexpression of SLAMF1 significantly increased the production of inflammatory factors TNF-α and IL-1ß, as well as chemokine MCP-1, both in vitro and in vivo upon mycobacteria BCG infection. By contrast, knockdown of SLAMF1 significantly decreased the production of TNF-α, IL-1ß, and MCP-1. Western blot analysis indicated that the NF-κB signaling pathway may contribute to the elevated inflammatory response promoted by SLAMF1, as evidenced by higher levels of phosphorylated p65 and IκBα detected with SLAMF1 overexpression. Furthermore, SLAMF1 upregulation facilitated bacterial clearance in infected RAW264.7 cells and in the lungs of infected mice. In conclusion, we demonstrated that BCG infection significantly upregulated SLAMF1, which enhanced inflammatory response by activating the NF-κB signaling pathway and facilitated bacterial clearance in BCG-infected RAW264.7 cells and mice.

The molecular mechanism of CRISPR/Cas9 system and its application in gene therapy of human diseases.

CRISPR/Cas system is an adaptive immune system that confers resistance to exogenous virus or plasmid in bacteria and archaea. In recent years, the booming CRISPR/Cas9 genome editing technology modified from type2 CRISPR/Cas adaptive immune system has been widely applied to various research fields of life science and led to revolutionary changes. In this review, we summarize the origin and development of CRISPR/Cas9 genome editing technology as well as its applications in life science research. We focus on the latest application of this system in gene therapy of human diseases and the associated side/off-target effects, which may provide references for researchers in related areas.

Blockade of macrophage autophagy ameliorates activated lymphocytes-derived DNA induced murine lupus possibly via inhibition of proinflammatory cytokine production.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a typical inflammatory autoimmune disease for its unknown pathogenesis and potential fatality. It has been reported that autophagy has a crosstalk with autoimmunity, but its impact on the pathogenesis of SLE remains unclear. Here, we investigated the role of autophagy in inflammatory response of macrophages under SLE conditions. METHODS: First, we detected the expression of autophagy-related genes (Atg5, Atg12 and Beclin 1) in the macrophages derived from activated lymphocytes-derived DNA (ALD-DNA) induced murine lupus as well as in the PBMC from SLE patients. And then through adoptive transfer of Beclin 1 knockdown macrophages, we further investigated the potential effect of macrophage autophagy on the SLE-associated inflammatory response and disease severity by evaluating serum anti-dsDNA antibodies and proteinuria levels, immune complex deposition as well as renal pathological changes. RESULTS: We found that autophagy related genes were significantly upregulated in the splenic and renal macrophages of lupus mice and in the PBMC of SLE patients. Adoptive transfer of Beclin 1 knockdown macrophages could significantly decrease the anti-dsDNA antibodies and proteinuria levels, robustly reduce renal immune complex deposition and remit glomerulonephritis, indicating the amelioration of murine lupus. This protective effect was associated with the obviously decreased production of proinflammatory cytokines IL-6 and TNF-α. CONCLUSIONS: Our results suggested that aberrant activated autophagy in macrophages contributed to the pathogenesis of murine lupus possibly via promoting the production of proinflammatory cytokines TNF-α and IL-6, and inhibition of autophagy might represent a novel regulation strategy for excessive activation of proinflammatory macrophages and a new therapeutic regime for SLE.

In vivo delivery of interleukin-35 relieves coxsackievirus-B3-induced viral myocarditis by inhibiting Th17 cells.

Interleukin (IL)-35 is a new member of the IL-12 cytokine family. The suppressive role of IL-35 in the immune response to parasitic and bacterial infections and in autoimmunity has been demonstrated in terms of its anti-inflammatory properties. However, the functional role of IL-35 in viral myocarditis has not been investigated. In this study, IL-35 expression was measured in heart tissues with coxsackievirus B3 (CVB3)-induced myocarditis. It was significantly reduced in the late stage of viral infection and correlated negatively with disease severity. To examine the therapeutic role of IL-35 in viral myocarditis, an IL-35-expressing plasmid (pIL-35-FC) was packaged with polyethyleneimine and delivered intraperitoneally to BALB/c male mice before and after CVB3 infection. The severity of myocarditis was assessed 7 days after infection. The in vivo delivery of IL-35 significantly ameliorated the severity of viral myocarditis, reflected in an increased survival rate and increased bodyweights, and reduced serum creatine kinase (CK) and CK-MB activities, cardiac pathological scores, and viral replication. We also show that the overexpression of IL-35 reduced splenic Th17 cells and Th17-related proinflammatory cytokines in heart tissues. In conclusion, our data indicate that IL-35 effectively protects the myocardium from the pathogenesis of CVB3-induced viral myocarditis, which may be attributable to reduced Th17 production. This suggests that supplementation with IL-35 could be a novel therapeutic treatment for viral myocarditis.

Mycobacterial SapM hampers host autophagy initiation for intracellular bacillary survival via dephosphorylating Raptor.

Secreted acid phosphatase (SapM) is an immunomodulator of Mycobacterium tuberculosis (Mtb) and consequently plays a crucial role in disease onset and development upon infection. Importantly, the virulence of SapM has rendered SapM an attractive target for drug development. However, the mechanism underlying the role of SapM in facilitating bacillary survival remains to be fully elucidated. In this context, the present study demonstrated that SapM hampered cellular autophagy to facilitate bacillary survival in mycobacterial-infected macrophages. Mechanically, SapM interacted with Raptor and was localized to the subcellular lysosomal organelle, causing the dephosphorylation of Raptor at the Ser792 position, resulting in mTORC1 hyperactivity and the subsequent autophagy inhibition. Consistent with this, SapM blocked the autophagy initiation and mitigated lung pathology in vivo. These findings highlighted the role of Raptor as a significant substrate of SapM for inhibiting autophagy, which is a novel clue for developing a treatment against tuberculosis.

Mycobacterial Rv1804c binds to the PEST domain of IκBα and activates macrophage-mediated proinflammatory responses.

Recognition of the components of Mycobacterium tuberculosis (Mtb) by macrophages is vital for initiating a cascade of host immune responses. However, the recognition of Mtb-secretory proteins by the receptor-independent pathways of the host remains unclear. Rv1804c is a highly conserved secretory protein in Mtb. However, its exact function and underlying mechanism in Mtb infection remain poorly understood. In the present study, we observed that Rv1804c activates macrophage-mediated proinflammatory responses in an IKKα-independent manner. Furthermore, we noted that Rv1804c inhibits mycobacterial survival. By elucidating the underlying mechanisms, we observed that Rv1804c activates IκBα by directly interacting with its PEST domain. Moreover, Rv1804c was enriched in attenuated but not in virulent mycobacteria and associated with the disease process of tuberculosis. Our findings provide an alternative pathway via which a mycobacterial secretory protein activates macrophage-mediated proinflammatory responses. Our study findings may shed light on the prevention and treatment of tuberculosis.

ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2-BECN1 interaction.

Growing evidences indicate that dysfunction of autophagy contributes to the disease pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative disorders. The GGGGCC·GGCCCC repeat RNA expansion in chromosome 9 open reading frame 72 (C9orf72) is the most genetic cause of both ALS and FTD. According to the previous studies, GGGGCC·GGCCCC repeat undergoes the unconventional repeat-associated non-ATG translation, which produces dipeptide repeat (DPR) proteins. Although there is a growing understanding that C9orf72 DPRs have a strong ability to harm neurons and induce C9orf72-linked ALS/FTD, whether these DPRs can affect autophagy remains unclear. In the present study, we find that poly-GR and poly-PR, two arginine-containing DPRs which display the most cytotoxic properties according to the previous studies, strongly inhibit starvation-induced autophagy. Moreover, our data indicate that arginine-rich DPRs enhance the interaction between BCL2 and BECN1/Beclin 1 by inhibiting BCL2 phosphorylation, therefore they can impair autophagic clearance of neurodegenerative disease-associated protein aggregates under starvation condition in cells. Importantly, our study not only highlights the role of C9orf72 DPR in autophagy dysfunction, but also provides novel insight that pharmacological intervention of autophagy using SW063058, a small molecule compound that can disrupt the interaction between BECN1 and BCL2, may reduce C9orf72 DPR-induced neurotoxicity.

MiR-21 confers resistance against CVB3-induced myocarditis by inhibiting PDCD4-mediated apoptosis.

PURPOSE: The participation of microRNAs (miRNAs) in cardiovascular diseases suggests them as potential targets for novel preventive and therapeutic strategies. In this study, the key myocardial miRNA, miR-21, was identified in the murine coxsackievirus B3 (CVB3)-induced myocarditis model and its contribution to disease progression was explored. METHODS: Myocardial microRNA expression changes in CVB3-infected mice were analyzed by real-time PCR and miR-21 was found to be the miRNA whose expression was significantly reduced. Mice were injected with plasmid encoding miR-21 (pMDH-miR-21) at day 1 post CVB3 infection and myocarditis severity was evaluated 7 days post-infection. The underlying mechanism of miR-21 in viral myocarditis was also investigated. RESULTS: Myocardial miR-21 expression was negatively related to viral myocarditis severity. Recovery of miR-21 expression, by injecting with pMDH-miR-21, significantly relieved CVB3-induced myocarditis as shown by increased body weight, reduced myocardial injury, lowered myocarditis score and increased survival rate. Further study showed that miR-21 could protect myocardial apoptosis by specifically inhibiting its target programmed cell death 4 (PDCD4) expression. CONCLUSION: miR-21 administration efficiently alleviated CVB3-induced myocarditis by repressing PDCD4-mediated apoptosis. Our study not only helps to better understand the pathogenesis of viral myocarditis, but also proves the potential of miR-21 as a novel therapeutic target for treatment of CVB3-induced myocarditis and other apoptosis-mediated cardiovascular diseases.

Human OTUD6B positively regulates type I IFN antiviral innate immune responses by deubiquitinating and stabilizing IRF3.

IMPORTANCE: Interferon (IFN) regulatory factor (IRF3) is one of the key factors for type I IFN transcription. To sophisticatedly regulate type I IFN antiviral immune response, IRF3 activity is closely controlled by a variety of post-translational modifications. However, the regulatory mechanisms are still not fully elucidated. In the present study, we found that human deubiquitinase OTUD6B positively regulates IRF3-mediated antiviral immune response. OTUD6B can stabilize the IRF3 protein level via hydrolyzing (Lys33)-linked polyubiquitin at Lys315. More importantly, mice with OTUD6B overexpression exhibited more resistance to RNA virus infection. Thus, unlike the previous report that zebrafish OTUD6B negatively regulates the antiviral response by suppressing K63-linked ubiquitination of IRF3 and IRF7, we demonstrate that human OTUD6B actually enhances type I IFN response and has the potential for antiviral therapy.

Chlorpromazine affects autophagy in association with altered Rag GTPase-mTORC1-TFEB signaling.

Autophagy is a critical protein and organelle quality control system, which regulates cellular homeostasis and survival. Growing pieces of evidence suggest that autophagic dysfunction is strongly associated with many human diseases, including neurological diseases and cancer. Among various autophagic regulators, microphthalmia (MiT)/TFE transcription factors, including transcription factor EB (TFEB), have been shown to act as the master regulators of autophagosome and lysosome biogenesis in both physiological and pathological conditions. According to the previous studies, chlorpromazine (CPZ), an FDA-approved antipsychotic drug, affects autophagy in diverse cell lines, but the underlying mechanism remains elusive. In our present study, we find that CPZ treatment induces TFEB nuclear translocation through Rag GTPases, the upstream regulators of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Meanwhile, CPZ treatment also blocks autophagosome-lysosome fusion. Notably, we find a significant accumulation of immature autophagosome vesicles in CPZ-treated cells, which may impede cellular homeostasis due to the dysfunction of the autophagy-lysosome pathway. Interestingly and importantly, our data suggest that the expression of the active form of Rag GTPase heterodimers helps in reducing the accumulation of autophagosomes in CPZ-treated cells, further suggesting a major contribution of the Rag GTPase-mTORC1-TFEB signaling axis in CPZ-induced autophagic impairment.