Selective autophagy of the adaptor TRIF regulates innate inflammatory signaling.

Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-ß and IL-1ß. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-ß and IL-1ß. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.

UBX Domain Protein 6 Positively Regulates JAK-STAT1/2 Signaling.

Type I/III IFNs induce expression of hundreds of IFN-stimulated genes through the JAK/STAT pathway to combat viral infections. Although JAK/STAT signaling is seemingly straightforward, it is nevertheless subjected to complex cellular regulation. In this study, we show that an ubiquitination regulatory X (UBX) domain-containing protein, UBXN6, positively regulates JAK-STAT1/2 signaling. Overexpression of UBXN6 enhanced type I/III IFNs-induced expression of IFN-stimulated genes, whereas deletion of UBXN6 inhibited their expression. RNA viral replication was increased in human UBXN6-deficient cells, accompanied by a reduction in both type I/III IFN expression, when compared with UBXN6-sufficient cells. Mechanistically, UBXN6 interacted with tyrosine kinase 2 (TYK2) and inhibited IFN-ß-induced degradation of both TYK2 and type I IFNR. These results suggest that UBXN6 maintains normal JAK-STAT1/2 signaling by stabilizing key signaling components during viral infection.

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice.

BACKGROUND & AIMS: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Distinct Tissue-Specific Roles for the Disease-Associated Autophagy Genes ATG16L2 and ATG16L1.

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.

Noncanonical autophagy in dermal dendritic cells mediates immunosuppressive effects of UV exposure.

BACKGROUND: Control of the inflammatory response is critical to maintaining homeostasis, and failure to do so contributes to the burden of chronic inflammation associated with several disease states. The mechanisms that underlie immunosuppression, however, remain largely unknown. Although defects in autophagy machinery have been associated with inflammatory pathologic conditions, we now appreciate that autophagic components participate in noncanonical pathways distinct from classical autophagy. We have previously demonstrated that LC3-associated phagocytosis (LAP), a noncanonical autophagic process dependent on Rubicon (rubicon autophagy regulator [RUBCN]), contributes to immunosuppression. OBJECTIVE: We used Rubcn-/- mice to examine the role of the LAP pathway in mediating the UV-induced immunotolerant program in a model of contact hypersensitivity (CHS). METHODS: Flow cytometry and transcriptional analysis were used to measure immune cell infiltration and activation in the skin of Rubcn+/+ and Rubcn-/- mice during the CHS response. RESULTS: Here, we demonstrate that LAP is required for UV-induced immunosuppression and that UV exposure induces a broadly anti-inflammatory transcriptional program dependent on Rubicon. Rubcn-/- mice are resistant to UV-induced immunosuppression and instead display exaggerated inflammation in a model of CHS. Specifically, RUBCN deficiency in CD301b+ dermal dendritic cells results in their increased antigen presentation capacity and subsequent hyperactivation of the CD8+ T-cell response. CONCLUSIONS: LAP functions to limit the immune response and is critical in maintaining the balance between homeostasis and inflammation.

Silencing Autophagy-Related Gene 2 (ATG2) Results in Accelerated Senescence and Enhanced Immunity in Soybean.

Autophagy plays a critical role in nutrient recycling and stress adaptations. However, the role of autophagy has not been extensively investigated in crop plants. In this study, soybean autophagy-related gene 2 (GmATG2) was silenced, using virus-induced silencing (VIGS) mediated by Bean pod mottle virus (BPMV). An accelerated senescence phenotype was exclusively observed for the GmATG2-silenced plants under dark conditions. In addition, significantly increased accumulation of both ROS and SA as well as a significantly induced expression of the pathogenesis-related gene 1 (PR1) were also observed on the leaves of the GmATG2-silenced plants, indicating an activated immune response. Consistent with this, GmATG2-silenced plants exhibited a significantly enhanced resistance to Pseudomonas syringae pv. glycinea (Psg) relative to empty vector control plants (BPMV-0). Notably, the activated immunity of the GmATG2-silenced plants was independent of the MAPK signaling pathway. The fact that the accumulation levels of ATG8 protein and poly-ubiquitinated proteins were significantly increased in the dark-treated GmATG2-silenced plants relative to the BPMV-0 plants indicated that the autophagic degradation is compromised in the GmATG2-silenced plants. Together, our results indicated that silencing GmATG2 compromises the autophagy pathway, and the autophagy pathway is conserved in different plant species.

ATG10 (autophagy-related 10) regulates the formation of autophagosome in the anti-virus immune response of pacific oyster (Crassostrea gigas).

Autophagy, a highly conserved intracellular degradation system, is involved in numerous processes in vertebrate and invertebrate, such as cell survival, ageing, and immune responses. However, the detailed molecular mechanism of autophagy and its immune regulatory role in bivalves are still not well understood. In the present study, an autophagy-related protein ATG10 (designated as CgATG10) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgATG10 cDNA was of 621 bp, encoding a polypeptide of 206 amino acid residues with an Autophagy_act_C domain (from 96 to 123 amino acid), which shared high homology with that from C. virginica and Octopus bimaculoides. The mRNA transcripts of CgATG10 were widely expressed in all the tested tissues including mantle, gonad, gills, hemocytes and hepatopancreas, with the highest expression level in mantle. After the stimulation with poly (I:C), the mRNA expression level of CgATG10 in the mantle of oysters was significantly up-regulated (4.92-fold of that in Blank group, p < 0.05), and the LC3-conversion from LC3-I to LC3-II (LC3-II/LC3-I) also increased. After an additional injection of dsRNA to knock-down the expression of CgATG10 (0.33-fold and 0.10-fold compared respectively with Blank group and dsGFP group, p < 0.05), the downstream conversion of CgLC3 was inhibited significantly compared with that of the control dsGFP group, while the expression level of autophagy-initiator CgBeclin1 did not change significantly. In addition, the mRNA transcripts of interferon regulatory factor CgIRF-1 increased significantly in CgATG10-knockdown oysters at 12 h post poly (I:C) stimulation. All the results indicated that CgATG10 might participate in the immune response against poly (I:C) by regulating autophagosome formation and interferon system in oysters.

Molecular cloning, expression and functional analysis of Atg16L1 from orange-spotted grouper (Epinephelus coioides).

Autophagy related gene 16 (Atg16), which encodes a core protein for autophagosome formation, participates in autophagy activity, the ubiquitin proteasome system and inflammatory response in mammals. In this study, we cloned and characterized an Atg16 homolog from orange-spotted grouper (Epinephelus coioides) (EcAtg16L1). EcAtg16L1 encodes a 656-amino acid polypeptide, which shares 94.22% and 72.65% homology with large yellow croakers (Larimichthys crocea) and humans (Homo sapiens), respectively. EcAtg16L1 contains a conserved Atg16 domain and a WD-repeat-containing domain. Subcellular localization showed that EcAtg16L1 was distributed in the cytoplasm of grouper cells with a dot-like pattern. EcAtg16L1 overexpression promoted Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by the increase in viral gene transcription and viral coat protein. Furthermore, EcAtg16L1 overexpression negatively regulated interferon (IFN)-related molecules and proinflammatory cytokines, and decreased IFN, IFN-stimulated response element, and nuclear factor κB promoter activities. Taken together, aside from its function in autophagosome formation, EcAtg16L1 also plays role in promoting SGIV and RGNNV replication and the pro-viral effect might involve its down regulation to interferon and inflammatory responses.

ATG Genes Influence the Virulence of Cryptococcus neoformans through Contributions beyond Core Autophagy Functions.

The process of autophagy is conserved among all eukaryotes from yeast to humans and is mainly responsible for bulk degradation of cellular contents and nutrient recycling during starvation. Autophagy has been suggested to play a role in the pathogenesis of the opportunistic human fungal pathogen Cryptococcus neoformans, potentially through a contribution to the export of virulence factors. In this study, we showed that deletion of each of the ATG1, ATG7, ATG8, and ATG9 genes in C. neoformans leads to autophagy-related phenotypes, including impaired amino acid homeostasis under nitrogen starvation. In addition, the atgΔ mutants were hypersensitive to inhibition of the ubiquitin-proteasome system, a finding consistent with a role in amino acid homeostasis. Although each atgΔ mutant was not markedly impaired in virulence factor production in vitro, we found that all four ATG genes contribute to C. neoformans virulence in a murine inhalation model of cryptococcosis. Interestingly, these mutants displayed significant differences in their ability to promote disease development. A more detailed investigation of virulence for the atg1Δ and atg8Δ mutants revealed that both strains stimulated an exaggerated host immune response, which, in turn, contributed to disease severity. Overall, our results suggest that different ATG genes are involved in nonautophagic functions and contribute to C. neoformans virulence beyond their core functions in autophagy.

Proteomic and Bioinformatic Characterization of Extracellular Vesicles Released from Human Macrophages upon Influenza A Virus Infection.

Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.

Trimethylamine N-oxide prime NLRP3 inflammasome via inhibiting ATG16L1-induced autophagy in colonic epithelial cells.

Recently, the intricate relationship between Trimethylamine N-oxide (TMAO) and inflammatory bowel disease (IBD) is of growing interest. The NLRP3 inflammasome plays crucial roles in gut homeostasis and determining the severity of inflammation in IBD, however, the precise roles of the NLRP3 inflammasome in IBD are still debated. ATG16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with IBD. Whether TMAO prime NLRP3 inflammasome via ATG16L1-induced autophagy remains unclear. This study observed the expression of ATG16L1, LC3-II and p62 and activation of NLRP3 inflammasome stimulated by TMAO in fetal human colon cells (FHCs), aiming to elucidate the mechanism by which the TMAO may contribute to colonic epithelial inflammation. Our results demonstrated that TMAO significantly inhibited ATG16L1, LC3-II and p62 expression, and triggered the activated NLRP3 inflammasome and production of ROS in a dose- and time-dependent manner. Furthermore, TMAO-mediated effects were observably reversed by over-expression ATG16L1 and siRNA-mediated knockdown NLRP3.The present results support the hypothesis that TMAO may be involved in the pathogenesis of IBD by impacting ATG16L1-induced autophagy and activating NLRP3 inflammasome, suggesting a potential therapeutic targets for the treatment of IBD and TMAO-associated complications.

Role of autophagy in HIV infection and pathogenesis.

The aim of autophagy is to re-establish homeostasis in response to a variety of stress conditions. By forming double-membrane vesicles, autophagy engulfs damaged or superfluous cytoplasmic material and recycles degradation products for new synthesis or energy production. Of note, the same mechanism is used to capture pathogens and has important implications in both innate and adaptive immunity. To establish a chronic infection, pathogens have therefore evolved multiple mechanisms to evade autophagy-mediated degradation. HIV infection represents one of the best characterized systems in which autophagy is disarmed by a virus using multiple strategies to prevent the sequestration and degradation of its proteins and to establish a chronic infection. HIV alters autophagy at various stages of the process in both infected and bystander cells. In particular, the HIV proteins TAT, NEF and ENV are involved in this regulation by either blocking or stimulating autophagy through direct interaction with autophagy proteins and/or modulation of the mTOR pathway. Although the roles of autophagy during HIV infection are multiple and vary amongst the different cell types, several lines of evidence point to a potential beneficial effect of stimulating autophagy-mediated lysosomal degradation to potentiate the immune response to HIV. Characterization of the molecular mechanisms regulating selective autophagy is expected to be valuable for developing new drugs able to specifically enhance the anti-HIV response.

Autophagy as a target for therapeutic uses of multifunctional peptides.

The emergence of complex diseases is promoting a change from one-target to multitarget drugs and peptides are ideal molecules to fulfill this polypharmacologic role. Here we review current status in the design of polypharmacological peptides aimed to treat complex diseases, focusing on tuberculosis. In this sense, combining multiple activities in single molecules is a two-sided sword, as both positive and negative side effects might arise. These polypharmacologic compounds may be directed to regulate autophagy, a catabolic process that enables cells to eliminate intracellular microbes (xenophagy), such as Mycobacterium tuberculosis (MBT). Here we review some strategies to control MBT infection and propose that a peptide combining both antimicrobial and pro-autophagic activities would have a greater potential to limit MBT infection. This endeavor may complement the knowledge gained in understanding the mechanism of action of antibiotics and may lead to the design of better polypharmacological peptides to treat complex diseases such as tuberculosis.

NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells.

OBJECTIVE: The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD). METHODS: A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNFα, IL-8 and IL-1ßmRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn's disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot. RESULTS: AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children. CONCLUSIONS: The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients.

Autophagy proteins and its homeostasis in cellular environment.

Autophagy is a self-destructing mechanism of cell via lysosomal degradation, which helps to degrade/destroy hazardous substances, proteins, degenerating organelles and recycling nutrients. It plays an important role is cellular homeostasis and regulates internal environment of cell, moreover, when needed causes non-apoptotic programmed death of cell. Autophagy has been observed as one of the major factors in parasite clearance in leishmaniasis. Being an intra-cellular pathogen, the cell mediated response is the only alternative for adaptive immunity against Leishmania in host. Pro-inflammatory cytokines IL12 and TNFα generate Th2 response which helps in active phagocytosis of parasite whereas an anti-inflammatory cytokine like IL10 mediate parasite promotion by blocking autophagic pathways and inhibiting phagocytic actions. In the present chapter, through systems biology approach, we are trying to decipher the role of pro-inflammatory and anti-inflammatory cytokine in autophagy during leishmanial infection. TLR2/6 mediated signaling stimulated by LPG produces many pro-inflammatory cytokines like IL12, TNFα and IL6 etc. Among them TNFα, causes the activation of PI3P through a series of events, which results in activation of autophagic machinery, whereas, IL10 through ATG9 and mTOR activation, inhibits autophagy. The mathematical modeling of these pathways shows that, ATG9-PI3P act as a negative feedback loop in autophagic machinery of leishmaniasis.

Endothelial cell autophagy in homeostasis and cancer.

Autophagy, the major lysosomal pathway for the degradation and recycling of cytoplasmic materials, is increasingly recognized as a major player in endothelial cell (EC) biology and vascular pathology. Particularly in solid tumors, tumor microenvironmental stress such as hypoxia, nutrient deprivation, inflammatory mediators, and metabolic aberrations stimulates autophagy in tumor-associated blood vessels. Increased autophagy in ECs may serve as a mechanism to alleviate stress and restrict exacerbated inflammatory responses. However, increased autophagy in tumor-associated ECs can re-model metabolic pathways and affect the trafficking and surface availability of key mediators and regulators of the interplay between EC and immune cells. In line with this, heightened EC autophagy is involved in pathological angiogenesis, inflammatory, and immune responses. Here, we review major cellular and molecular mechanisms regulated by autophagy in ECs under physiological conditions and discuss recent evidence implicating EC autophagy in tumor angiogenesis and immunosurveillance.

A signature of 13 autophagy­related gene pairs predicts prognosis in hepatocellular carcinoma.

Growing evidences suggest that autophagy plays a momentous part in the tumorigenesis and development of hepatocellular carcinoma (HCC). However, there are not many researches to predict the prognosis of HCC using autophagy-related genes. Therefore, based on the clinical information and RNA-Seq data of The Cancer Genome Atlas data portal (TCGA), 13 autophagy­related gene pairs were screened to build the autophagy­related signature to predict the prognosis by least absolute shrinkage and selection operator (LASSO) regression analysis. Besides, the International Cancer Genome Consortium (ICGC) cohort was further applied to verify the autophagy­related prognostic signature. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were also used to predict the relevant function of the autophagy-related gene pairs signature. As shown in the results, the autophagy-related gene pairs were mainly involved in process utilizing autophagic mechanism, autophagy, macroautophagy and cellular response to oxidative stress. The immune cell levels in the high-risk and low-risk group were explored, which showed that the three immune cells were obviously increased in the high-risk group, while the five immune cells were obviously increased in the low-risk group. In conclusion, an autophagy­related prognostic signature was established to predict the prognosis of HCC patients with great accuracy and we found that autophagy­related prognostic signature was related to infiltrating immune cells.

Reduced Expression of Autophagy Markers and Expansion of Myeloid-Derived Suppressor Cells Correlate With Poor T Cell Response in Severe COVID-19 Patients.

Widespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed ~140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with ~30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-γ production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease.

Hepatitis C virus enhances Rubicon expression, leading to autophagy inhibition and intracellular innate immune activation.

Autophagy, a degradation system, works to maintain cellular homeostasis. However, as the impact of Hepatitis C virus (HCV) infection on hepatocyte autophagy and its effect on HCV replication remain unclear, we examined them. HCV infection suppressed late-stage autophagy and increased Rubicon. siRNA-mediated knockdown of Rubicon promoted autophagy in HCV-infected cells. In Huh-7 cells harbouring the HCV replicon, Rubicon knockdown downregulated the expression of type 1 interferon (IFN)-related genes and upregulated HCV replication. Rubicon overexpression or administration of bafilomycin A1 or chloroquine, an inhibitor of late-stage autophagy, suppressed autophagy and activated the type 1 IFN pathway. On the other hand, Atg7 knockout suppressed early-stage autophagy and did not activate the type 1 IFN pathway. In livers of humanized liver chimeric mice, HCV infection increased Rubicon and enhanced type 1 IFN signalling. Elimination of HCV in the mice reduced the increase in Rubicon due to HCV infection. The expression levels of Rubicon and IFN-stimulated genes in chronic hepatitis C patients were higher than those in non-B, non-C hepatitis patients. HCV infection increased Rubicon and suppressed hepatocyte autophagy, leading to activation of the intracellular immune response. Rubicon induction is involved in HCV replication via activation of the intracellular immune response.

Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes.

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5-12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200-but not of other essential autophagy components ATG5, ATG16L1 or ATG7-in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.