SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

Molecular profile reveals immune-associated markers of medulloblastoma for different subtypes.

Medulloblastoma, a common pediatric malignant tumor, has been recognized to have four molecular subgroups [wingless (WNT), sonic hedgehog (SHH), group 3, group 4], which are defined by the characteristic gene transcriptomic and DNA methylomic profiles, and has distinct clinical features within each subgroup. The tumor immune microenvironment is integral in tumor initiation and progression and might be associated with therapeutic responses. However, to date, the immune infiltrative landscape of medulloblastoma has not yet been elucidated. Thus, we proposed MethylCIBERSORT to estimate the degree of immune cell infiltration and weighted correlation network analysis (WGCNA) to find modules of highly correlated genes. Synthesizing the hub genes in the protein-protein interaction (PPI) network and modules of the co-expression network, we identify three candidate biomarkers [GRB2-associated-binding protein 1 (GAB1), Abelson 1 (ABL1), and CXC motif chemokine receptor type 4 (CXCR4)] via the molecular profiles of medulloblastoma. Given this, we investigated the correlation between these three immune hub genes and immune checkpoint blockade response and the potential of drug prediction further. In addition, this study demonstrated a higher presence of endothelial cells and infiltrating immune cells in Group 3 tumor bulk. The above results will be conducive to better comprehending the immune-related pathogenesis and treatment of medulloblastoma.

XAF1 Protects Host against Emerging RNA Viruses by Stabilizing IRF1-Dependent Antiviral Immunity.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.

PLCL1 regulates fibroblast-like synoviocytes inflammation via NLRP3 inflammasomes in rheumatoid arthritis.

BACKGROUND: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). METHODS: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1ß and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1ß in FLS by WB. RESULT: When PLCL1 was silenced, the level of IL-6, IL-1ß and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1ß and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. CONCLUSION: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.

Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell-specific manner.

Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell-specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell-specific Hem-1-deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) - alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.

XIAP promotes melanoma growth by inducing tumour neutrophil infiltration.

Elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been frequently reported in malignant melanoma suggesting that XIAP renders apoptosis resistance and thereby supports melanoma progression. Independent of its anti-apoptotic function, XIAP mediates cellular inflammatory signalling and promotes immunity against bacterial infection. The pro-inflammatory function of XIAP has not yet been considered in cancer. By providing detailed in vitro analyses, utilising two independent mouse melanoma models and including human melanoma samples, we show here that XIAP is an important mediator of melanoma neutrophil infiltration. Neutrophils represent a major driver of melanoma progression and are increasingly considered as a valuable therapeutic target in solid cancer. Our data reveal that XIAP ubiquitylates RIPK2, involve TAB1/RIPK2 complex and induce the transcriptional up-regulation and secretion of chemokines such as IL8, that are responsible for intra-tumour neutrophil accumulation. Alteration of the XIAP-RIPK2-TAB1 inflammatory axis or the depletion of neutrophils in mice reduced melanoma growth. Our data shed new light on how XIAP contributes to tumour growth and provides important insights for novel XIAP targeting strategies in cancer.

Anti-DFS70 Antibodies Are Associated With Proliferative Lupus Nephritis and Renal Pathological Activity.

Objective: The significance of anti-dense fine speckles 70 (DFS70) antibodies in systemic lupus erythematosus (SLE) is still unclear, especially in lupus nephritis (LN) patients. We investigated the prevalence, clinical and pathological relevance of anti-DFS70 antibodies in LN patients. Methods: Anti-DFS70 antibodies were measured using enzyme-linked immunosorbent assays in 377 biopsy-proven LN patients, 268 non-LN SLE patients, 232 chronic kidney disease (CKD) patients, and 78 healthy individuals (HI). Demographic, clinical, and pathological parameters were compared between LN patients with and without anti-DFS70 antibodies. Stepwise multivariable logistic regression was performed to identify covariates associated with anti-DFS70 antibodies. Results: The prevalence of anti-DFS70 antibodies in LN (19.6%) was comparable to non-LN SLE patients (19.8%, P=0.9630), but was significantly higher than CKD patients (13.4%, P=0.0468) and HI (9.0%, P=0.0252). Using multivariable logistic regression analysis, the titer of anti-double-stranded DNA (dsDNA) antibodies (adjusted odds ratio=1.002, 95% confidence interval 1.001-1.003, P=0.004) was associated with positive anti-DFS70 antibodies in LN patients. In addition, anti-DFS70 antibodies were more prevalent in proliferative LN (22.0%, 68/309) compared to membrane LN patients (10.2%, 6/59, P=0.0376). Furthermore, LN patients with positive anti-DFS70 antibodies had significantly higher activity index (AI) compared to patients who were negative (8.0 vs 6.0, P=0.0131). However, the chronicity index was similar between the groups (3.0 vs 3.0, P=0.8412). Conclusion: Anti-DFS70 antibodies were not associated with LN development in SLE patients but were associated with anti-dsDNA antibodies, proliferative LN, and renal AI. This suggests their potential to serve as a non-histological biomarker for LN subclass and activity status.

Massive digital gene expression analysis reveals different predictive profiles for immune checkpoint inhibitor therapy between adenocarcinoma and squamous cell carcinoma of advanced lung cancer.

BACKGROUND: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. METHODS: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test. RESULTS: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. CONCLUSIONS: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) are closely related to unfavorable prognosis of patients with clear cell renal cell carcinoma (ccRCC). However, the important molecules in the interaction between ccRCC and TAMs are unclear. METHODS: TCGA-KIRC gene expression data of tumor tissues and normal tissues adjacent to tumor were compared to identify differentially expressed genes in ccRCC. TAMs related genes were discovered by analyzing the correlation between these differentially expressed genes and common macrophage biomarkers. Gene set enrichment analysis was performed to predict functions of TAMs related gene. The findings were further validated using RNA sequencing data obtained from the CheckMate 025 study and immunohistochemical analysis of samples from 350 patients with ccRCC. Kaplan-Meier survival curve, Cox regression analysis and Harrell's concordance index analysis were used to determine the prognostic significance. RESULTS: In this study, we applied bioinformatic analysis to explore TAMs related differentially expressed genes in ccRCC and identified 5 genes strongly correlated with all selected macrophage biomarkers: STAC3, LGALS9, TREM2, FCER1G, and PILRA. Among them, FCER1G was abundantly expressed in tumor tissues and showed prognostic importance in patients with ccRCC who received treatment with Nivolumab; however, it did not exhibit prognostic value in those treated with Everolimus. We also discovered that high expression levels of FCER1G are related to T cell suppression. Moreover, combination of FCER1G and macrophage biomarker CD68 can improve the prognostic stratification of patients with ccRCC from TCGA-KIRC. Based on the immunohistochemical analysis of samples from patients with ccRCC, we further validated that FCER1G and CD68 are both highly expressed in tumor tissue and correlate with each other. Higher expression of CD68 or FCER1G in ccRCC tissue indicates shorter overall survival and progression-free survival; patients with high expression of both CD68 and FCER1G have the worst outcome. Combining CD68 and FCER1G facilitates the screening of patients with a worse prognosis from the same TNM stage group. CONCLUSIONS: High expression of FCER1G in ccRCC is closely related to TAMs infiltration and suppression of T cell activation and proliferation. Combining the expression levels of FCER1G and macrophage biomarker CD68 may be a promising postoperative prognostic index for patients with ccRCC.

Functional dissection of inherited non-coding variation influencing multiple myeloma risk.

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-ß pathway in Nile tilapia (Oreochromis niloticus).

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).

BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration.

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.

Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30-40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses.

Host engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effectors of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. In-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knockout mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ELMO1 generates differential pro-inflammatory cytokines between pathogenic and nonpathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1-SifA interaction affects bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1-SifA interaction increases TNF-α and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map and induces inflammation after challenge with microbes or microbial ligands. ELMO1 generates a differential response through interaction with the WxxxE motif, which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response.

The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

REIC/Dkk-3 Gene Therapy Induces Immunogenic Cell Death in a Mouse Model of Malignant Mesothelioma.

BACKGROUND/AIM: The adenovirus vector- carrying reduced expression in immortalized cell (REIC) gene (Ad-REIC) increases endoplasmic reticulum stress chaperone GRP78/BiP expression and induces the JNK-mediated apoptotic pathway. We aimed to determine whether Ad-REIC-induced apoptotic cell death can trigger immunogenic cell death (ICD). MATERIALS AND METHODS: We examined the emission of damage-associated molecular patterns in vitro and the vaccination effect in vivo. We determined the immunological changes in the tumour microenvironment by putative ICD inducers and the combined effects of immune checkpoint blockade therapies. RESULTS: Ad-REIC induced the release of high-mobility group box 1 and adenosine triphosphate and the translocation of calreticulin in murine mesothelioma AB12 cells. The vaccination effect was elicited by Ad-REIC treatment in vivo. The effect of Ad-REIC was potentiated by anti-cytotoxic T-lymphocyte-associated protein 4 antibody treatment in a murine mesothelioma AB1-HA cell model. CONCLUSION: Ad-REIC induces ICD in malignant mesothelioma.

Complement Potentiates Immune Sensing of HIV-1 and Early Type I Interferon Responses.

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

gga-miR-142-3p negatively regulates Mycoplasma gallisepticum (HS strain)-induced inflammatory cytokine production via the NF-κB and MAPK signaling by targeting TAB2.

OBJECTIVE: Mycoplasma gallisepticum (MG), a notorious avian pathogen, leads to considerable economic losses in the poultry industry. MG infection is characterized by severe, uncontrollable inflammation and host DNA damage. Micro ribonucleic acids (miRNAs) have emerged as important regulators in microbial pathogenesis. However, the role of miRNAs in MG infection is poorly characterized. In this study, we validated the functional roles of gga-miR-142-3p. METHODS: The relative expression of gga-miR-142-3p in the lungs of the MG-infected chicken embryos and the MG-infected chicken embryonic fibroblast cell line (DF-1) was determined by reverse transcription quantitative real-time PCR analysis. Bioinformatics database was used to analysis the target gene of gga-miR-142-3p. The luciferase reporter assay as well as gene expression analysis were conducted to validate the target gene. To further explore the biological functions of gga-miR-142-3p upon MG infection, the cell proliferation was quantified using Cell Counting Kit-8 (CCK-8). Meanwhile, cell cycle analysis and apoptosis were measured using a flow cytometer. RESULTS: gga-miR-142-3p was significantly upregulated in both MG-infected chicken-embryo lungs and the DF-1 cells. gga-miR-142-3p over expression significantly downregulated the expression of pro-inflammatory cytokines, including interleukin-1ß, interleukin-6 and tumor necrosis factor alpha after MG infection. Meanwhile, gga-miR-142-3p enhanced the host defense against MG infection by facilitating cell proliferation, promoting cell progression and inhibiting cell apoptosis. Interestingly, TAB2 knockdown groups show similar results, whereas, TAB2 over-expression groups and gga-miR-142-3p inhibitor groups had thoroughly opposite results. The expression of p-p65 in nuclear factor kappa B (NF-κB) and p-p38 in the mitogen-activated protein kinase (MAPK) pathway was decreased when gga-miR-142-3p was over-expressed. CONCLUSION: Upon MG infection, upregulation of gga-miR-142-3p alleviates inflammation by negatively regulating the signaling pathways of NF-κB and MAPKs by targeting TAB2 and facilitates cell proliferation by inhibiting cell apoptosis and promoting cell cycle progression to defend against MG infection.

Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses.

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.

Cereblon regulates NK cell cytotoxicity and migration via Rac1 activation.

Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co-receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F-actin in chemokine-treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN-depleted NK cells, chemokine-mediated or target cell-mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells.