Interferon-independent Pathways Research Articles

An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2′−3′ cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

Virus-induced lncRNA-BTX allows viral replication by regulating intracellular translocation of DHX9 and ILF3 to induce innate escape

The roles of long noncoding RNA (lncRNA) and RNA-binding proteins (RBPs) in antiviral innate response warrant further investigation. Here, we identify an lncRNA, termed lncRNA-BTX (between Tbk1 and Xpot), which is upregulated upon viral infection via an IRF3-type I interferon-independent pathway, promoting viral innate immune escape. Deletion of lncRNA-BTX in cells or mice significantly reduces viral load invitro or invivo, respectively. Mechanistically, lncRNA-BTX strengthens the interactions between DHX9 or ILF3 (two RBPs that have opposite functions in regulating the replication of RNA virus) and their respective partner, JMJD6 or ILF2, which regulates intracellular translocations of DHX9 and ILF3 from the nucleus to the cytoplasm. Put simply, lncRNA-BTX facilitates DHX9's return to the cytoplasm and retains ILF3 within the nucleus, promoting viral replication. This work unveils a strategy developed by the virus to bypass host innate immunity, thus providing a potential target for antiviral therapeutics.

STING strengthens host anti-hantaviral immunity through an interferon-independent pathway

Hantaan virus (HTNV), the prototype virus of hantavirus, could escape innate immunity by restraining type I interferon (IFN) responses. It is largely unknown whether there existed other efficient anti-hantaviral tactics in host cells. Here, we demonstrate that the stimulator of interferon genes (STING) strengthens the host IFN-independent anti-hantaviral immunity. HTNV infection activates RIG-I through IRE1-XBP 1-mediated ER stress, which further facilitates the subcellular translocation and activation of STING. During this process, STING triggers cellular autophagy by interacting with Rab7A, thus restricting viral replication. To note, the anti-hantaviral effects of STING are independent of canonical IFN signaling. Additionally, neither application of the pharmacological antagonist nor the agonist targeting STING could improve the outcomes of nude mice post HTNV challenge in vivo. However, the administration of plasmids exogenously expressing the mutant C-terminal tail (ΔCTT) STING, which would not trigger the type I IFN responses, protected the nude mice from lethal HTNV infection. In summary, our research revealed a novel antiviral pathway through the RIG-I-STING-autophagy pathway, which offered novel therapeutic strategies against hantavirus infection.

Rapid interferon independent expression of IFITM3 following T cell activation protects cells from influenza virus infection.

Interferon-induced transmembrane protein 3 (IFITM3) is a potent antiviral protein that enhances cellular resistance to a variety of pathogens, including influenza virus. Classically defined as an interferon-stimulated gene, expression of IFITM3 on cells is rapidly up-regulated in response to type I and II interferon. Here we found that IFITM3 is rapidly up-regulated by T cells following their activation and this occurred independently of type I and II interferon and the interferon regulatory factors 3 and 7. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3 which, if targeted, could be an effective approach to harness the activity of IFITM3 for infection prevention.

Human Placental Syncytiotrophoblasts Restrict Toxoplasma gondii Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines

ABSTRACTToxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human midgestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at two distinct stages of the parasite lytic cycle—at the time of attachment and also during intracellular replication. Utilizing comparative transcriptome sequencing (RNA-seq) transcriptional profiling, we also show that human placental trophoblasts from both the second and third trimesters respond uniquely to T. gondii infection compared to trophoblast cell lines, typified by the upregulation of several immunity-related genes. One of the most differentially induced genes was the chemokine CCL22, which relies on the secretion of a parasite effector(s) either during or after invasion for its induction. Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.

The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression.

The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway.

RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism

Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism.

Interleukin-27 is a potent inhibitor of cis HIV-1 replication in monocyte-derived dendritic cells via a type I interferon-independent pathway.

IL-27, a member of the IL-12 family of cytokines, plays an important and diverse role in the function of the immune system. Whilst generally recognized as an anti-inflammatory cytokine, in addition IL-27 has been found to have broad anti-viral effects. Recently, IL-27 has been shown to be a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages. The main objective of this study was to see whether IL-27 has a similar inhibitory effect on HIV-1 replication in dendritic cells (DCs). Monocytes were differentiated into immature DCs (iDCs) and mature DCs (mDCs) with standard techniques using a combination of GM-CSF, IL-4 and LPS. Following differentiation, iDCs were infected with HIV-1 and co-cultured in the presence or absence of IL-27. IL-27 treated DCs were shown to be highly potent inhibitors of cis HIV-1, particularly of CCR5 tropic strains. Of note, other IL-12 family members (IL-12, IL-23 and IL-35) had no effect on HIV-1 replication. Microarray studies of IL-27 treated DCs showed no up-regulation of Type I (IFN) gene expression. Neutralization of the Type-I IFN receptor had no impact on the HIV inhibition. Lastly, IL-27 mediated inhibition was shown to act post-viral entry and prior to completion of reverse transcription. These results show for the first time that IL-27 is a potent inhibitor of cis HIV-1 infection in DCs by a Type I IFN independent mechanism. IL-27 has previously been reported to inhibit HIV-1 replication in CD4+ T cells and macrophages, thus taken together, this cytokine is a potent anti-HIV agent against all major cell types targeted by the HIV-1 virus and may have a therapeutic role in the future.

Trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes

Innate immune sensing of viral nucleic acids triggers type I interferon (IFN) production, which activates interferon-stimulated genes (ISGs) and directs a multifaceted antiviral response. ISGs can also be activated through IFN-independent pathways, although the precise mechanisms remain elusive. Here we found that the cytosolic exonuclease Trex1 regulates the activation of a subset of ISGs independently of IFN. Both Trex1−/− mouse and TREX1-mutant human cells express high levels of antiviral genes and are refractory to viral infections. The IFN-independent activation of antiviral genes in Trex1−/− cells requires STING, TBK1 and IRF3 and IRF7. We also found that Trex1-deficient cells display expanded lysosomal compartment, altered subcellular localization of the transcription factor EB (TFEB), and reduced mTORC1 activity. Together, our data identify Trex1 as a regulator of lysosomal biogenesis and IFN-independent activation of antiviral genes, and shows dysregulation of lysosomes can elicit innate immune responses.

Complement deficiencies and susceptibility to systemic lupus erythematosus revisited

Recent demonstration of the contribution of more than 30 different SNPs to lupus susceptibility has informed our understanding of pathogenesis of disease. Yet each of these genetic variants is commonly found in the general population and contributes a small effect to lupus susceptibility. In contrast, the almost universal association between C1q deficiency and SLE, as well as high relative risk of other classical components, provides a special opportunity to understand mechanisms of disease. The complement pathway was implicated in the immunopathogenesis of lupus and other autoimmune disorders decades ago. The apparent paradox that early complement component (C1q, C2 and C4) deficiencies predispose to lupus has been explained by the beneficial roles of these proteins in promoting the clearance of apoptotic cells and immune complexes (ICs). We recently showed that, in the absence of C1q, instead of ICs binding to monocytes, they preferentially engage plasmacytoid dendritic cells (pDC) so providing a powerful stimulus for the production of IFNα, the cytokine with potent immune adjuvant properties [1,2]. We confirmed and extended these findings using microarray analysis of total peripheral blood mononuclear cells and purified monocytes following incubation with SLE ICs in the presence or absence of C1q [3]. We observed that C1q suppressed SLE IC-induced interferon-stimulated genes such as TNFSF13B (BAFF) and TNFSF10 (TRAIL), which are associated with SLE pathogenesis. Interferon-independent pathways that were differentially affected by the presence or absence of C1q in SLE ICs included: multiple cytokines/chemokines (for example, CCL20, CCL23), receptors (for example, CD36, STAB1), and enzymes (for example, RNASE1,2,6, SOD2). Exposure of monocytes to SLE ICs was surprisingly non-inflammatory even when gene expression was examined by microarray. How then are the lower frequencies of lupus in C4, C2 and C3 deficient patients explained? First, isolated C1q has been shown to exert immunosuppressive properties that have not been identified with C4 or C2. Also, we and others have suggested that C3b may be the key complement protein required for removal of apoptotic cells but that C4b could potentially function in this regard. The lower prevalence of SLE in individuals with a deficiency of C4, C2 or C3 could then be due to only one pathway being defective and, possibly, a functional role for C4b in protection. Future studies will continue to address these questions and guide interventions to promote the safe handling of apoptotic debris and ICs.

Up-regulation of interferon-stimulated gene15 and its conjugates by tumor necrosis factor-α via type I interferon-dependent and -independent pathways

Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is strongly induced by type I interferons (IFN-α/β), bacterial endotoxin, and cellular stress. Up-regulation of ISG15 is observed in several cancer cell types and is associated with cancer progression. As many cytokines can influence all stages of tumorigenesis, the elevated expression of ISG15 system may be regulated in cancer cells by inflammatory cytokines. In this study, we showed that TNF-α, but not TGF-β and IL-6, up-regulates levels of both ISG15 and its conjugates in human lung carcinoma A549 and human squamous carcinoma HSC4 cell lines. Induction of ISG15 and its conjugates by TNF-α was dose-dependent and required mediation of p38 MAP kinase and Jak1 through up-regulation of endogenous type I interferon expression. SB202190 (p38 MAPK inhibitor) and Jak1 inhibitor suppressed TNF-α-induced expression of ISG15 and its conjugates. However, only SB202190 inhibited the expression of type I interferons by TNF-α. Although B18R, a soluble type I interferon receptor, totally abolished the effect of exogenous IFN-β, it was unable to inhibit completely the TNF-α-induced ISG15 production. In addition, the initiation of ISG15 induction by TNF-α was detected earlier than that of IFN-β induction. Taken together, TNF-α elicits the induction of ISG15 and ISG15 conjugates not only via the autocrine stimulation of type I interferon expression, but also through a type I interferon-independent pathway. These data provide a possible link between inflammatory response and cancer progression.

Induction of Indoleamine 2,3-Dioxygenase by Uropathogenic Bacteria Attenuates Innate Responses to Epithelial Infection

Uropathogenic Escherichia coli (UPEC) are the chief cause of urinary tract infections. Although neutrophilic inflammation is a hallmark of disease, previous data indicate that UPEC promotes local dampening of host innate immune responses. Here, we show that UPEC attenuates innate responses to epithelial infection by inducing expression of indoleamine 2,3-dioxygenase (IDO), a host enzyme with previously defined roles in adaptive immune regulation. UPEC induced IDO expression in human uroepithelial cells and polymorphonuclear leukocytes (PMN) in vitro and in bladder tissue during murine cystitis via a noncanonical, interferon-independent pathway. In the bladders of UPEC-infected IDO-deficient mice, we observed augmented expression of proinflammatory cytokines and local inflammation, correlated with reduced survival of extracellular bacteria. Pharmacologic inhibition of IDO also increased human PMN transepithelial migration. Stimulation of IDO expression therefore represents a pathogen strategy to create local immune privilege at epithelial surfaces, attenuating innate responses to promote colonization and the establishment of infection.

Lipopolysaccharide-induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-independent pathways

Our previous studies have shown that Toll-like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non-parenchymal liver cells and trigger the production of interferon (IFN) to inhibit hepatitis B virus replication in vivo and in vitro. However, little is known about TLR-mediated cellular responses in primary hepatocytes. By the model of woodchuck hepatitis virus (WHV) infected primary woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI-3k/Akt pathways in PWHs. Further, inhibitors of MAPK-ERK and PI-3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN- independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.

339 Toll-like receptor mediated innate immune response in hepatocytes suppressed woodchuck hepatitis virus replication via interferon-independent pathway

339 Toll-like receptor mediated innate immune response in hepatocytes suppressed woodchuck hepatitis virus replication via interferon-independent pathway

Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC- -dependent mechanism

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-alpha activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) pro-apoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-alpha in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

A role for IRF3-dependent RXRalpha repression in hepatotoxicity associated with viral infections.

Viral infections and antiviral responses have been linked to several metabolic diseases, including Reye's syndrome, which is aspirin-induced hepatotoxicity in the context of a viral infection. We identify an interferon regulatory factor 3 (IRF3)–dependent but type I interferon–independent pathway that strongly inhibits the expression of retinoid X receptor α (RXRα) and suppresses the induction of its downstream target genes, including those involved in hepatic detoxification. Activation of IRF3 by viral infection in vivo greatly enhances bile acid– and aspirin-induced hepatotoxicity. Our results provide a critical link between the innate immune response and host metabolism, identifying IRF3-mediated down-regulation of RXRα as a molecular mechanism for pathogen-associated metabolic diseases.

Interleukin-12 enhances antifungal activity of human mononuclear phagocytes against Aspergillus fumigatus: implications for a gamma interferon-independent pathway.

The potential of recombinant human interleukin-12 (IL-12) to enhance the capacity of human monocytes (MNC) to elicit an oxidative burst and damage hyphae of Aspergillus fumigatus was investigated. Incubation of peripheral blood mononuclear cells (PBMC) from healthy adults with 10 to 100 ng of IL-12/ml at 37 degrees C for 2 to 3 days enhanced the production of superoxide anion (O2-) in response to phorbol myristate acetate (PMA) (P = 0.04) and unopsonized A. fumigatus hyphae (P = 0.03) and further enhanced hyphal damage (P = 0.009). Anti-gamma interferon (anti-IFN-gamma) blocked secretion of IFN-gamma by IL-12-treated PBMC but did not inhibit IL-12-induced O2- production by these cells in response to PMA. In addition, IL-12-treated elutriated MNC secreted no IFN-gamma or tumor necrosis factor alpha but exhibited enhanced O2- production compared to controls (P = 0.013). These findings demonstrate that IL-12 augments oxidative antifungal activities of MNC via an IFN-gamma-independent route, suggesting a novel pathway of IL-12 action in antifungal defense.