IFN-I Signaling Research Articles

Inflammasome Activation Dampens Type I IFN Signaling to Strengthen Anti-Toxoplasma Immunity.

Innate immunity acts as the first line of defense against pathogen invasion. During Toxoplasma gondii infection, multiple innate immune sensors are activated by invading microbes or pathogen-associated molecular patterns (PAMPs). However, how inflammasome is activated and its regulatory mechanisms during T. gondii infection remain elusive. Here, we showed that the infection of PRU, a lethal type II T. gondii strain, activates inflammasome at the early stage of infection. PRU tachyzoites, RNA and soluble tachyzoite antigen (STAg) mainly triggered the NLRP3 inflammasome, while PRU genomic DNA (gDNA) specially activated the AIM2 inflammasome. Furthermore, mice deficient in AIM2, NLRP3, or caspase-1/11 were more susceptible to T. gondii PRU infection, and the ablation of inflammasome signaling impaired antitoxoplasmosis immune responses by enhancing type I interferon (IFN-I) production. Blockage of IFN-I receptor fulfilled inflammasome-deficient mice competent immune responses as WT mice. Moreover, we have identified that the suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator induced by inflammasome-activated IL-1β signaling and inhibits IFN-I production by targeting interferon regulatory factor 3 (IRF3). In general, our study defines a novel protective role of inflammasome activation during toxoplasmosis and identifies a critical regulatory mechanism of the cross talk between inflammasome and IFN-I signaling for understanding infectious diseases. IMPORTANCE As a key component of innate immunity, inflammasome is critical for host antitoxoplasmosis immunity, but the underlying mechanisms are still elusive. In this study, we found that inflammasome signaling was activated by PAMPs of T. gondii, which generated a protective immunity against T. gondii invasion by suppressing type I interferon (IFN-I) production. Mechanically, inflammasome-coupled IL-1β signaling triggered the expression of negative regulator SOCS1, which bound to IRF3 to inhibit IFN-I production. The role of IFN-I in anti-T. gondii immunity is little studied and controversial, and here we also found IFN-I is harmful to host antitoxoplasmosis immunity by using knockout mice and recombinant proteins. In general, our study identifies a protective role of inflammasomes to the host during T. gondii infection and a novel mechanism by which inflammasome suppresses IFN-I signaling in antitoxoplasmosis immunity, which will likely provide new insights into therapeutic targets for toxoplasmosis and highlight the cross talk between innate immune signaling in infectious diseases prevention.

Expression Profiles of Differentially Expressed Circular RNAs and circRNA-miRNA-mRNA Regulatory Networks in SH-SY5Y Cells Infected with Coxsackievirus B5.

Coxsackievirus B5 (CVB5) is the causative agent of hand, foot, and mouth disease (HFMD) that can cause neurological complications and fatalities. Circular RNA (circRNA) has been shown to play an important role in regulating pathogenic processes. However, the functions of circRNA in response to CVB5 infection remain unclear. In our research, RNA-seq was employed to analyze the expression profiles of circRNAs in SH-SY5Y cells with or without CVB5 infection. Out of 5,665 circRNAs identified to be expressed in SH-SY5Y cells, 163 circRNAs were found to be differentially expressed significantly. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially expressed circRNAs were mainly involved in ubiquitin-mediated proteolysis and signaling pathways during CVB5 infection. Additionally, RT-qPCR was used to validate the RNA-seq data, and a circRNA–miRNA–mRNA interaction network was constructed based on two circRNAs, such as hsa_circ_0008378 and novel_circ_0014617, which were associated with the regulation of innate immune response in host cells. Additionally, we confirmed the two circRANs up-regulated the key factors in the IFN-I signaling pathway, hampering viral replication. Our data provide a new perspective that facilitates further understanding of the virus-host mechanism.

Apabetalone, a BET inhibitor, attenuates inflammation induced by viral RNA mimetic and reduces SARS-CoV-2 spike protein binding regardless of variants

Abstract Background/Introduction Hyperinflammatory responses to SARS-CoV-2 can cause myocarditis and cardiac dysfunction including congestive heart failure [1]. SARS-CoV-2 RNA induces type I interferon (IFN-I), activating IFN regulatory factors (IRFs) and downstream IFN stimulated genes (ISGs) to initiate inflammatory processes. SARS-CoV-2 variants may develop immune escape, undercutting benefits of vaccinations. These challenges highlight the need of variant-independent therapies to improve COVID-19 outcomes. Apabetalone is an epigenetic BD2-selective BET inhibitor in phase 3 trials for cardiovascular disease [2]. Apabetalone has the potential to treat COVID-19. It counters inflammatory signals caused by cytokine storm (CS), preventing cardiac dysfunction associated with severe COVID-19 symptoms in cardiac organoids [3]. It also downregulates angiotensin-converting enzyme 2 (ACE2) expression, the main host cell receptor for SARS-CoV-2 spike protein thus impeding propagation of wild-type SARS-CoV-2 [3,4]. Purpose 1) Evaluate apabetalone's effect on inflammatory processes induced by viral-RNA mimetic in human lung cells; 2) Assess apabetalone's ability to prevent binding of the highly contagious delta variant spike protein to human lung cells. Methods Inflammatory gene expression was examined by real-time PCR in apabetalone treated human bronchial epithelial cells (Calu-3) stimulated with poly I:C, a well-accepted viral RNA mimetic that elicits inflammatory signals similar to SARS-CoV-2 RNA [5]. Binding of SARS-CoV-2 delta or wild-type spike protein to apabetalone treated Calu-3 cells was determined by flow cytometry. Results In Calu-3 cells, apabetalone dose-dependently downregulated poly I:C induced transcription of key COVID-19 associated cytokines (IL6, CXCL10, CCL2) to a similar extent as baricitinib (up to 86%, p<0.0001), an anti-inflammatory agent in emergency use for COVID-19 treatment. Moreover, apabetalone but not baricitinib diminished IL1B mRNA levels (up to 66%, p<0.0001). Apabetalone and baricitinib opposed poly I:C induced expression of IFNB1 (an IFN-I), IRF1 and IRF9 (upstream regulators) as well as IFIT1 and IFIT2 (downstream ISGs that regulate CXCL10 expression; up to 90%, p<0.0001). Clinically relevant doses of apabetalone did not alter expression of anti-viral IFITM2, an ISG that blocks SARS-CoV-2, particularly omicron, endosomal entry [6]. Therefore, apabetalone counters the expression of inflammatory factors with roles in CS and IFN-I signaling in response to poly I:C. Additionally, apabetalone reduced delta and wild-type spike protein binding to unstimulated Calu-3 cells (up to 72%, p<0.0001). Conclusions Apabetalone's dual anti-viral and anti-inflammatory mechanism positions it as a variant-independent COVID-19 therapeutic. Together with an established safety profile from >2000 treatment-years with apabetalone, the data provide rationale for an ongoing clinical trial (NCT04894266) which includes analysis of cardiac damage. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Resverlogix Corp

STAT3 Inhibits Autocrine IFN Signaling in Type I Conventional Dendritic Cells.

Type I conventional dendritic cells (cDC1s) are an essential Ag-presenting population required for generating adaptive immunity against intracellular pathogens and tumors. While the transcriptional control of cDC1 development is well understood, the mechanisms by which extracellular stimuli regulate cDC1 function remain unclear. We previously demonstrated that the cytokine-responsive transcriptional regulator STAT3 inhibits polyinosinic:polycytidylic acid [poly(I:C)]-induced cDC1 maturation and cDC1-mediated antitumor immunity in murine breast cancer, indicating an intrinsic, suppressive role for STAT3 in cDC1s. To probe transcriptional mechanisms regulating cDC1 function, we generated novel RNA sequencing datasets representing poly(I:C)-, IL-10-, and STAT3-mediated gene expression responses in murine cDC1s. Bioinformatics analyses indicated that poly(I:C) stimulates multiple inflammatory pathways independent of STAT3, while IL-10-activated STAT3 uniquely inhibits the poly(I:C)-induced type I IFN (IFN-I) transcriptional response. We validated this mechanism using purified cDC1s deficient for STAT3 or IFN signaling. Our data reveal IL-10-activated STAT3 suppresses production of IFN-β and IFN-γ, accrual of tyrosine phosphorylated STAT1, and IFN-stimulated gene expression in cDC1s after poly(I:C) exposure. Moreover, we found that maturation of cDC1s in response to poly(I:C) is dependent on the IFN-I receptor, but not the type II IFN receptor, or IFN-λ. Taken together, we elucidate an essential role for STAT3 in restraining autocrine IFN-I signaling in cDC1s elicited by poly(I:C) stimulation, and we provide novel RNA sequencing datasets that will aid in further delineating inflammatory and anti-inflammatory mechanisms in cDC1s.

Sensing of SARS-CoV-2 by pDCs and their subsequent production of IFN-I contribute to macrophage-induced cytokine storm during COVID-19.

Lung-infiltrating macrophages create a marked inflammatory milieu in a subset of patients with COVID-19 by producing a cytokine storm, which correlates with increased lethality. However, these macrophages are largely not infected by SARS-CoV-2, so the mechanism underlying their activation in the lung is unclear. Type I interferons (IFN-I) contribute to protecting the host against SARS-CoV-2 but may also have some deleterious effect, and the source of IFN-I in the lungs of infected patients is not well defined. Plasmacytoid dendritic cells (pDCs), a key cell type involved in antiviral responses, can produce IFN-I in response to SARS-CoV-2. We observed the infiltration of pDCs in the lungs of SARS-CoV-2-infected patients, which correlated with strong IFN-I signaling in lung macrophages. In patients with severe COVID-19, lung macrophages expressed a robust inflammatory signature, which correlated with persistent IFN-I signaling at the single-cell level. Hence, we observed the uncoupling in the kinetics of the infiltration of pDCs in the lungs and the associated IFN-I signature, with the cytokine storm in macrophages. We observed that pDCs were the dominant IFN-α-producing cells in response to the virus in the blood, whereas macrophages produced IFN-α only when in physical contact with infected epithelial cells. We also showed that IFN-α produced by pDCs, after the sensing of SARS-CoV-2 by TLR7, mediated changes in macrophages at both transcriptional and epigenetic levels, which favored their hyperactivation by environmental stimuli. Together, these data indicate that the priming of macrophages can result from the response by pDCs to SARS-CoV-2, leading to macrophage activation in patients with severe COVID-19.

Menthone inhibits type-I interferon signaling by promoting Tyk2 ubiquitination to relieve local inflammation of rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease. RA development is mediated by the abnormal activation of multiple signaling pathways. Recent studies have revealed that type-I interferon (IFN-I) signaling plays an essential role in the occurrence and development of RA. However, how to target IFN-I signaling to develop anti-rheumatoid arthritis drugs remains largely unexplored. Here, our study showed that IFN-I signaling was over-activated in articular synovial cells from collagen II-induced arthritis (CIA) mice. Interestingly, we found that a small molecule compound, menthone, strongly inhibited the activation of the IFN-I signaling pathway. Further studies revealed that menthone promoted K48-linked polyubiquitination of Tyk2, thus lowering the protein level and stability of Tyk2. Importantly, menthone administration in the local articulus of CIA mice significantly attenuated the local inflammation in CIA mice. This study could promote our understanding of rheumatoid arthritis, and also suggests a potential strategy to develop anti-RA drugs.

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

ABSTRACTXIAP-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.

E3 ubiquitin ligase MID1 ubiquitinates and degrades type‐I interferon receptor 2

Type I interferon (IFN-I) is a common biological molecule used for the treatment of viral diseases. However, the clinical antiviral efficacy of IFN-I needs to be greatly improved. In this study, IFN-I receptor 2 (IFNAR2) was revealed to undergo degradation at the protein level in cells treated with IFN-I for long periods of time. Further studies found a physical interaction between the E3 ubiquitin ligase midline-1 (MID1) and IFNAR2. As a consequence, MID1 induced both K48- and K63-linked polyubiquitination of IFNAR2, which promoted IFNAR2 protein degradation in a lysosome-dependent manner. Conversely, knockdown of MID1 largely restricted IFN-I-induced degradation of IFNAR2. Importantly, MID1 regulated the strength of IFN-I signalling and IFN-I-induced antiviral activity. These findings reveal a regulatory mechanism of IFNAR2 ubiquitination and protein stability in IFN-I signalling, which could provide a potential target for improving the antiviral efficacy of IFN-I.

A comprehensive evaluation of the immune system response and type-I Interferon signaling pathway in hospitalized COVID-19 patients

BackgroundThe COVID-19 pandemic has become the world’s main life-threatening challenge in the third decade of the twenty-first century. Numerous studies have been conducted on SARS-CoV2 virus structure and pathogenesis to find reliable treatments and vaccines. The present study aimed to evaluate the immune-phenotype and IFN-I signaling pathways of COVID-19 patients with mild and severe conditions.Material and methodsA total of 100 COVID-19 patients (50 with mild and 50 with severe conditions) were enrolled in this study. The frequency of CD4 + T, CD8 + T, Th17, Treg, and B lymphocytes beside NK cells was evaluated using flow cytometry. IFN-I downstream signaling molecules, including JAK-1, TYK-2, STAT-1, and STAT-2, and Interferon regulatory factors (IRF) 3 and 7 expressions at RNA and protein status were investigated using real-time PCR and western blotting techniques, respectively. Immune levels of cytokines (e.g., IL-1β, IL-6, IL-17, TNF-α, IL-2R, IL-10, IFN-α, and IFN-β) and the existence of anti-IFN-α autoantibodies were evaluated via enzyme-linked immunosorbent assay (ELISA).ResultsImmune-phenotyping results showed a significant decrease in the absolute count of NK cells, CD4 + T, CD8 + T, and B lymphocytes in COVID-19 patients. The frequency of Th17 and Treg cells showed a remarkable increase and decrease, respectively. All signaling molecules of the IFN-I downstream pathway and IRFs (i.e., JAK-1, TYK-2, STAT-1, STAT-2, IRF-3, and IRF-7) showed very reduced expression levels in COVID-19 patients with the severe condition compared to healthy individuals at both RNA and protein levels. Of 50 patients with severe conditions, 14 had anti-IFN-α autoantibodies in sera. Meanwhile, this result was 2 and 0 for patients with mild symptoms and healthy controls, respectively.ConclusionOur results indicate a positive association of the existence of anti-IFN-α autoantibodies and immune cells dysregulation with the severity of illness in COVID-19 patients. However, comprehensive studies are necessary to find out more about this context.5mo_nwLJpK7s-uZity9h4-Video abstract

Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis.

Depression is a serious public-health issue. Recent reports have suggested higher susceptibility to viral infections in depressive patients. However, how depression affects antiviral innate immune signaling remains unknown. Here, we revealed a reduction in expression of Abelson helper integration site 1 (AHI1) in the peripheral blood mononuclear cells (PBMCs) and macrophages from the patients with major depressive disorder (MDD), which leads to attenuated antiviral immune response. We found that depression-related arginine vasopressin (AVP) induces reduction of AHI1 in macrophages. Further studies demonstrated that AHI1 is a critical stabilizer of basal type-I-interferon (IFN-I) signaling. Mechanistically, AHI1 recruits OTUD1 to deubiquitinate and stabilize Tyk2, while AHI1 reduction downregulates Tyk2 and IFN-I signaling activity in macrophages from both MDD patients and depression model mice. Interestingly, we identified a clinical analgesic meptazinol that effectively stimulates AHI1 expression, thus enhancing IFN-I antiviral defense in depression model mice. Our study promotes the understanding of the signaling mechanisms of depression-mediated antiviral immune dysfunction, and reveals meptazinol as an enhancer of antiviral innate immunity in depressive patients.

Angiotensin-Converting Enzyme 2 Potentiates SARS-CoV-2 Infection by Antagonizing Type I Interferon Induction and Its Down-Stream Signaling Pathway.

ABSTRACTThe innate interferon (IFN) response constitutes the first line of host defense against viral infections. It has been shown that IFN-I/III treatment could effectively contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in vitro. However, how SARS-CoV-2 survives through the innate antiviral mechanism remains to be explored. Our study uncovered that human angiotensin-converting enzyme 2 (ACE2), identified as a primary receptor for SARS-CoV-2 entry, can disturb the IFN-I signaling pathway during SARS-CoV-2 infection in human lung cells. We identified that ACE2 was significantly upregulated by SARS-CoV-2 and Sendai virus (SeV) infection, and exogenous expression of ACE2 suppressed IFN-I production in a dose-dependent manner. Mechanistically, ACE2 disrupted poly (I:C)-mediated inhibition of SARS-CoV2 replication by antagonizing IFN-I production by blocking IRF3 phosphorylation and nuclear translocation. Moreover, ACE2 quenched the IFN-mediated antiviral immune response by degrading endogenous STAT2 protein, inhibiting STAT2 phosphorylation and nuclear translocation. Interestingly, IFN-inducible short ACE2 (dACE2 or MIRb-ACE2) can also be induced by virus infection and inhibits the IFN signaling. Thus, our findings provide mechanistic insight into the distinctive role of ACE2 in promoting SARS-CoV-2 infection and enlighten us that the development of interventional strategies might be further optimized to interrupt ACE2-mediated suppression of IFN-I and its signaling pathway.IMPORTANCE Efficient antiviral immune responses against SARS-CoV-2 infection play a key role in controlling the coronavirus diseases 2019 (COVID-19) caused by this virus. Although SARS-CoV-2 has developed strategies to counteract the IFN-I signaling through the virus-derived proteins, our knowledge of how SARS-CoV-2 survives through the innate antiviral mechanism remains poor. We herein discovered the distinctive role of ACE2 as a restraining factor of the IFN-I signaling in facilitating SARS-CoV-2 infection in human lung cells. Both full-length ACE2 and truncated dACE2 can antagonize IFN-mediated antiviral response. These findings are key to understanding the counteraction between SARS-CoV-2 pathogenicity and the host antiviral defenses.

Type I interferons and TGF-β cooperate to induce liver fibrosis during HIV-1 infection under antiretroviral therapy.

Liver diseases have become a major comorbidity health concern for people living with HIV-1 (PLWH) treated with combination antiretroviral therapy (cART). To investigate if HIV-1 infection and cART interact to lead to liver diseases, humanized mice reconstituted with progenitor cells from human fetal livers were infected with HIV-1 and treated with cART. We report here that chronic HIV-1 infection with cART induced hepatitis and liver fibrosis in humanized mice, associated with accumulation of M2-like macrophages (M2LMs), elevated TGF-β, and IFN signaling in the liver. Interestingly, IFN-I and TGF-β cooperatively activated human hepatic stellate cells (HepSCs) in vitro. Mechanistically, IFN-I enhanced TGF-β–induced SMAD2/3 activation in HepSCs. Finally, blockade of IFN-I signaling reversed HIV/cART-induced liver diseases in humanized mice. Consistent with the findings in humanized mice with HIV-1 and cART, we detected elevated markers of liver injury, M2LMs, and of IFN signaling in blood specimens from PLWH compared with those of healthy individuals. These findings identify the IFN-I/M2LM/HepSC axis in HIV/cART-induced liver diseases and suggest that inhibiting IFN-I signaling or M2LM may provide a novel therapeutic strategy for treating HIV/cART-associated liver diseases in PLWH treated with antiretroviral therapy.

P1441: SINGLE-CELL TRANSCRIPTOMIC CHARACTERIZATIONS OF BONE MARROW MONONUCLEAR CELLS AND CAR-T PRODUCTS ASSOCIATED WITH EFFICACY AND TOXICITY IN B-ALL PATIENTS

Background: Chimeric antigen receptor-T (CAR-T) cell therapy has transformed B-cell acute lymphoblastic leukemia (B-ALL) treatment, yielding outstanding clinical results. However, less than half of patients achieve long-term remission, with treatment-associated adverse events a clinical challenge. Aims: This study aims to investigate the transcriptomic characterizations of CAR-T products and bone marrow mononuclear cells, and identify predictive biomarkers for efficacy and toxicity. Methods: Seventeen B-ALL patients were enrolled in this study. Single-cell RNA sequencing was performed on BMMCs (baseline and post-infusion) and IPs in 4 patients, and 120,780 residual cells were collected for bioinformatic analysis to identify biomarkers for the early clinical forecast. Another 13 patients-cohort was used for bulk RNA-sequencing validation. Results: An overview of our approach for sc-RNA-seq is shown in Figure A. Twenty-one distinctly transcriptional cell clusters were identified after extensive, unbiased clustering of all BMMCs and IPs (Figure B). Cell clusters were classified into five broad cell types -- IPs, HSPCs, NK/T cells, myeloid cells, and B-ALL cells (Figure C). For severe CRS prediction, 4 patients were divided into two groups based on tumor burden. Baseline cytokine-mediated signaling increased in high-tumor burden patients, mainly in B-ALL blasts, HSPCs (Pt 10 vs Pt 8); and low tumor burden patients, primarily in NK/T and Myeloid cells (Pt 11 vs Pt 9) (Figure D). IFITM1, a gene in IFN-related signaling pathways, was significantly upregulated in the above-mentioned cell types and validated in the bulk-RNA cohort (Figure E). Unlike complete response patients, NR (Pt 8) patients had diminished SRGN expression in every BMMC-1 cell type (Figure F), with probable arrests of IPs at the G1 phase (Figure G). In the NR patient tumor subsets, treatment-resistant tumor clusters (C1) in the IFN-I signaling pathway were enriched, whereas treatment-sensitive tumor cells (C3/5/10) underwent apoptosis (Figure H/I/J). Other ways, including the extracellular structure organization that predicts extramedullary relapse, were amplified (Figure K). The bulk RNA-sequencing cohort produced identical results. Image:Summary/Conclusion: IP and BMMC transcriptional feature heterogeneity possibly contributes to diverse clinical outcomes post-anti-CD19 CAR-T cell therapy and could be predictive.

Anti-Inflammatory Effects of Red Rice Bran Extract Ameliorate Type I Interferon Production via STING Pathway.

Type I interferons (IFNs-I) are inflammatory cytokines that play an essential role in the pathogenesis of inflammation and autoimmune diseases. Signaling through nucleic acid sensors causes the production of IFNs-I. A stimulator of interferon genes (STING) is a DNA sensor that signals transduction, leading to the production of IFNs-I after their activation. This study aims to determine the anti-inflammatory effects of red rice bran extract (RRBE) on macrophages through the activation of STING signaling. RAW264.7 macrophage cells were stimulated with STING agonist (DMXAA) with and without RRBE. Cells and supernatant were collected. The level of mRNA expression was determined by qPCR, and inflammatory cytokine production was investigated by ELISA. The results indicate that RRBE significantly lowers the transcription of STING and interferon-stimulated genes (ISGs). Moreover, RRBE suppresses the phosphorylation of STING, leading to a decrease in the expression of Irf3, a transcription factor that initiates IFN-I signaling. Our results provide evidence that red rice bran extract may be a protective compound for inflammatory diseases by targeting STING signaling.

Regulation and function of Id2 in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that promote anti-viral immune responses and contribute to autoimmunity. Development of pDCs requires the transcriptional regulator E2–2 and is opposed by inhibitor of DNA binding 2 (Id2). Prior work indicates Id2 is induced in pDCs upon maturation and may affect pDC IFN-I production via suppression of E2–2, suggesting an important yet uncharacterized role in this lineage. We found TLR7 agonists stimulate Id2 mRNA and protein expression in pDCs. We further show that transcriptional activation of Id2 is dependent on the E2 ubiquitin-conjugating enzyme Ubc13, but independent of IFN-I signaling in response to TLR7 agonist stimulation. Nonetheless, conditional Id2 depletion in pDCs indicates Id2 is dispensable for TLR7 agonist-induced maturation and inhibition of E2–2 expression. Thus, we identify new mechanisms of Id2 regulation by Ubc13, which may be relevant for understanding Id2 gene regulation in other contexts, while ruling out major roles for Id2 in pDC responses to TLR7 agonists.

Autophagy Protects Ocular Surface Against Overactivated Inflammation by Degrading Retinoic Acid-Induced Gene-I in Human Conjunctival Epithelial Cells.

Purpose: To evaluate the pathological role of autophagy in dry eye diseases by detecting the autophagic degradation of RIG-I, a master RNA-sensing receptor in cells. Methods: RNA-sequencing analysis and qPCR analysis of the expression level of genes related to IFN-I signaling pathway was used to evaluate the inflammatory level of cells overexpressed with RIG-I or empty vector, which was further confirmed by WB analysis. Chemical treatment (3-methyladenine, chloroquine, NH4Cl, rapamycin, torin 1 or trehalose) or gene knockdown was used to modulate autophagy. When the autophagy level was regulated, the autophagic degradation of RIG-I and its pathological role in dry eye diseases were determined by detecting the protein level of RIG-I and the level of cell inflammation. Results: Cells that overexpressed RIG-I showed increased expression of genes involved in the IFN-I signaling pathway compared with cells transfected with an empty vector. Inhibition of autophagy leaded to the accumulation of RIG-I in HCECs, combined with the aggravation of the RIG-I-mediated IFN-I signaling pathway. Contrarily, promoting the autophagic degradation of RIG-I by trehalose treatment could alleviate IFN-I signaling pathway. Conclusions: Autophagy could protect the ocular surface against IFN-I signaling pathway by degrading RIG-I in HCECs. This process may restrict the overactivation of inflammation in the pathological development of dry eye disease.

The host inflammatory response contributes to disease severity in Crimean-Congo hemorrhagic fever virus infected mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.

Dysregulation of the IFN-I signaling pathway by Mycobacterium tuberculosis leads to exacerbation of HIV-1 infection of macrophages.

While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb), the agent of TB in humans, worsens HIV-1 pathogenesis still need to be fully elucidated. Recently, we showed that HIV-1 infection and spread are exacerbated in macrophages exposed to TB-associated microenvironments. Transcriptomic analysis of macrophages conditioned with medium of Mtb-infected human macrophages (cmMTB) revealed an up-regulation of the typeI interferon (IFN-I) pathway, characterized by the overexpression of IFN-inducible genes. Historically, IFN-I are well known for their antiviral functions, but our previous work showed that this is not the case in the context of coinfection with HIV-1. Here, we show that the IFN-I response signature in cmMTB-treated macrophages matches the one observed in the blood of active TB patients, and depends on the timing of incubation with cmMTB. This suggests that the timing of macrophage's exposure to IFN-I can impact their capacity to control HIV-1 infection. Strikingly, we found that cmMTB-treated macrophages are hyporesponsive to extrastimulation with exogenous IFN-I, used to mimic HIV-1 infection. Yet, depleting STAT1 by gene silencing to block the IFN-I signaling pathway reduced TB-induced exacerbation of HIV-1 infection. Altogether, by aiming to understand why TB-derived IFN-I preexposure of macrophages did not induce antiviral immunity against HIV-1, we demonstrated that these cells are hyporesponsive to exogenous IFN-I, a phenomenon that prevents macrophage activation against HIV-1.

Induction of Autophagy reduces IFN-I mediated Inflammation and restores anti-HIV-1 T Cell response in vivo

Abstract A hallmark of HIV-1 infection is chronic inflammation. Chronic immune activation drives the pathogenesis of HIV-1 infection, leading to loss of CD4+ T cells and immune exhaustion. Previously, we demonstrated that persistent type I interferon (IFN-I) signaling drives T cell exhaustion during chronic HIV infection and combined anti-retroviral therapy (ART) and IFN-I receptor blockade could lead to reduced inflammation, accelerated viral suppression and HIV reservoir. However, as IFN-Is are key regulators for antiviral immunity, more specific interventions to fine-tune IFN-I signaling and chronic inflammation are needed. Autophagy is a homeostatic mechanism for disposal of damaged cellular organelles and elimination of intracellular pathogens, which is pivotal for cellular homeostasis, T cell development and function. Autophagy is also impaired during HIV-1 infection. Here we demonstrate that autophagy is directly linked to IFN-I signaling. Impairment of autophagy leads to accumulation of damaged mitochondria and spontaneous IFN-I signaling. Autophagy inducers reduce IFN-I signaling in activated macrophages and restore functions of exhausted anti-HIV-1 T cells in vitro. Importantly, autophagy inducer treatment in HIV-1 infected humanized bone marrow/liver/thymus (BLT) mice significantly reduced persistent IFN-I signaling and immune activation, restored exhausted antiviral T cell responses, and accelerated viral suppression by ART. Autophagy inducer treatment also led to reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach for treating persistent immune activation and improve immune control of HIV replication. This work was funded by NIAID 1R21AI140866 (to Zhen), NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie&Zhen), NCI 1R01CA239261-01 (to Kitchen), NIH Grants NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504(PIs: Kitchen & Chen), CIRM DISC2-10748, UPLIFT: UCLA Postdocs’ Longitudinal Investment in Faculty (Award # K12 GM106996) (to Carrillo), R01 AG052340 (to Jamieson). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation. We would also like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study.

Type I Interferon remodels intrahepatocytic signaling to promote T cell dysfunction during liver stage Plasmodium infection

Abstract Malaria is caused by Plasmodium parasites and accounted for 627,000 deaths in 2021. Upon deposition into the dermis, Plasmodium parasites traffic to, infect, and develop within hepatocytes before egressing to cause symptomatic erythrocytic infection. We recently reported that liver stage Plasmodium replication induces type I Interferon (IFN-I) signaling that compromises anti-malaria T cells. We now show that this IFN-I-mediated, inferior T cell response is mostly limited to hepatic CD8 T cells and is characterized by diminished cytokine production, impaired homeostatic CD8 T cell survival, and the expression of receptors and transcription factors associated with T cell exhaustion. We set out to identify the cells and molecular events involved in the induction of T cell exhaustion during the transient and non-persistent liver stage Plasmodium infection and observed that infected mice lacking IFNAR expression solely on hepatocytes do not generate significant frequencies of exhausted T cells. Furthermore, transcriptomic analyses of hepatocytes isolated during infection indicate that IFN-I signaling coincides with a remodeling of the hepatocyte transcriptome such that antigen processing and presentation, chemokine transcripts and immunoregulatory receptors and soluble factors are upregulated. We hypothesize that IFN-I signaling reshapes the hepatocyte transcriptome to promote an immunosuppressive liver microenvironment that impairs anti-malaria T cells. Together, our studies represent the first description of a negative role for liver stage IFN-I signaling on anti-malaria adaptive immunity and may necessitate a paradigm shift in the rational design of efficacious anti-malaria whole parasite vaccines. Supported by 1U01AI42001