Type I interferon (IFN) is an important component of antiviral innate immune signaling mediated by viral DNA and RNA recognition by the DNA sensor cGAS and RNA sensors RIG-I and MDA5. Activation of these DNA and RNA sensors leads to the recruitment of STING and MAVS, respectively, and converges on TANK-binding kinase 1 (TBK1) signaling for subsequent phosphorylation of IFN regulatory factor 3 (IRF3). However, the mechanisms that control TBK1 activation are still poorly defined. Here, we identify tripartite motif 9 short isoform (TRIM9s) as a positive regulator in type I IFN signaling. Upon viral infection, TRIM9s undergoes Lys-63-linked auto-polyubiquitination and serves as a platform to bridge GSK3β to TBK1, leading to the activation of IRF3 signaling. Interestingly, we found that TRIM9s selectively inhibits the production of pro-inflammatory cytokines, but enhances the expression of type I IFNs as well as IFN-stimulated genes, in response to viral infection. Our findings reveal novel dual functions of TRIM9s in antiviral immunity, which serve to balance pro-inflammatory response and production of type I IFNs.

Innate immune system is armed by several lines of pattern recognition receptors to sense various viral infection and to initiate antiviral immune response. This process is under a tight control and the negative feedback induced by infection and/or inflammation is critical to maintain immune homoeostasis and to prevent autoimmune disorders, however, the molecular mechanism is not fully understood. Here we report TRIM29, a ubiquitin E3 ligase, functions as an inducible negative regulator of innate immune response triggered by DNA virus and cytosolic DNA. DNA virus and cytosolic DNA stimulation induce TRIM29 expression robustly in macrophages and dendritic cells, although the basal level of TRIM29 is undetectable in those cells. TRIM29 deficiency elevates IFN-I and proinflammatory cytokine production upon viral DNA and cytosolic dsDNA stimulation. Consistently, in vivo experiments show that TRIM29-deficient mice are more resistant to HSV-1 infection than WT controls, indicated by better survival rate and reduced viral load in organs. Mechanism studies suggest that STING-TBK1-IRF3 signaling pathway in TRIM29 KO cells is significantly enhanced and the degradation of STING is impaired. Furthermore, we identify that TRIM29 targets STING for K48 ubiquitination and degradation. This study reveals TRIM29 as a crucial negative regulator in immune response to DNA virus and cytosolic DNA, preventing potential damage caused by overcommitted immune responses.

The Interleukin-1/Toll-like receptor signaling pathway is a crucial signaling pathway within the innate immune system and the use of mass spectrometric techniques became valuable to investigate signal transduction pathways. To date only a few reports exist that focus on the mass spectrometric identification of novel signaling intermediates within the TLR signal transduction pathway. Here we used this approach systematically to identify new interaction partners of the TLR signaling pathway and subsequently characterized them functionally. We identified 14-3-3 theta as a new member of the TLR signaling complex. With genetic complementation assays, we demonstrate that 14-3-3 negatively regulates TLR2-dependent NF-κB activity and amplifies the TLR4-dependent activation of the transcription factor. While 14-3-3 has no effect on TLR-induced apoptosis in innate immune cells, it controls the release of the inflammatory, IRF3-dependent cytokines like RANTES and IP-10 after stimulation with LPS. Most strikingly, 14-3-3 controls the production of proinflammatory cytokines like IL-6, IL-8, and TNFα in a different manner. Our results identify 14-3-3 theta as a new and important regulatory protein in the TLR signaling suppressing the MyD88-dependent pathway.

The mechanisms on metabolic regulation of immune responses are still elusive. We show here that viral infection induces immediate-early NF-κB activation independent of viral nucleic acid-triggered signaling, which triggers a rapid transcriptional induction of bile acid (BA) transporter and rate-limiting biosynthesis enzymes as well as accumulation of intracellular BAs in divergent cell types. The accumulated intracellular BAs activate SRC kinase via the TGR5-GRK-β-arrestin axis, which mediates tyrosine phosphorylation of multiple antiviral signaling components including RIG-I, VISA/MAVS, MITA/STING, TBK1 and IRF3. The tyrosine phosphorylation of these components by SRC conditions for efficient innate antiviral immune response. Consistently, TGR5 deficiency impairs innate antiviral immunity, whereas BAs exhibit potent antiviral activity in wild-type but not TGR5-deficient cells and mice. Our findings reveal an intrinsic and universal role of intracellular BA metabolism in innate antiviral immunity.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses^1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus^4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease^4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF^10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus^12-14.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis throughout the world. A majority (approx. 60%) of patients with IgAN experience disease exacerbations associated with an acute respiratory or gastrointestinal illness that appears to represent a viral infection. However, the exact mechanism of the disease exacerbation by viral infection is not understood, especially at the cellular and molecular levels. Here we report that glomerular podocytes express the major sensors for double-stranded RNA (dsRNA), a common byproduct of viral replication. In addition to these receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I)-like helicases (RLHs), podocytes express the collateral proteins required to support intracellular signaling. The pathways that mediate responses to dsRNA are fully functional in podocytes. The transcription factor interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-ĸB) are phosphorylated and translocate to the nucleus, and dsRNA increases synthesis of proteins driven by IRF3 (P54, P56 and P60) or NF-ĸB (interleukin 8 and A20). Furthermore, dsRNA suppresses podocyte cell migration, alters the expression of a panel of podocyte essential proteins (nephrin, podocin and CD2-associated protein or CD2AP) and changes transepithelial albumin flux. These effects are dsRNA sensor-specific: TLR3-/- podocytes do not respond to extracellular dsRNA, while intracellular dsRNA has no effect on podocytes bearing a dominant negative form of the major active RLH. These results demonstrate that innate responses to viruses can disturb podocyte cell function in vitro.

The production of type I interferon must be tightly regulated, and the aberrant production of this protein is harmful or even fatal to the host. The transcription factor IRF3 phosphorylation is a central regulator of type I interferon meditated antiviral response. Protein phosphatase-1 (PP1) has been reported to be important in many cell functions, including development, differentiation, and tumorigenesis. However, the roles of PP1 in Toll-like receptor (TLR)- or retinoic acid-inducible gene I like receptor (RLR)-triggered IRF-3 activation remain unclear. Here, we show that the activity of PP1 is downregulated in macrophages upon stimulation with TLR or RLR ligands, including lipopolysaccharide, and poly(I:C), or vesicular stomatitis virus (VSV), respectively. The overexpression of PP1 selectively inhibits TLR- and VSV-induced interferon regulatory factor 3 (IRF3) activation but has no substantial effect on TANK-binding kinase 1 (TBK1),ΚB kinase ε (IKKε) activation. Conversely, RNA interference of PP1 significantly promotes IRF3 activation. Consistently, The overexpression of PP1 inhibits TLR- and VSV-triggered IFN-β production while PP1 knockdown significantly increases the production of IFN-β in macrophages. We further demonstrate that PP1 directly interacts with IRF3 and dephosphorylates IRF3 at Ser385 and Ser396, resulting in the suppression of TLR- and RLR-triggered IFN-β production. Thus, PP1 functions as a negative feedback regulator of TLR- and RLR-triggered antiviral immune responses by acting as an IRF3 phosphatase.

The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ∆CTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ∆CTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.

Keywords: NF-κB; STING; TBK1; cGAS; interferon.

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.

OBJECTIVE

Activation of Toll-like receptor 4 (TLR4) triggers immune and inflammatory events by sensing endogenous danger signals as well as invading pathogens and contributes to the development of chronic inflammatory diseases. In this study, we investigated effect of 1-palmitoyl-2-(5-keto-6-octenedioyl)-sn-glycero-3-phosphocholine (KOdiA-PC), an oxidized phosphatidylcholine, on TLR4 activation and the underlying regulatory mechanism.

METHODS

RAW264.7 macrophages were used for the study. The levels of TNF-α, IFN-β, and COX-2 mRNA and protein were determined by quantitative PCR and ELISA, respectively. Activation of TLR4-signaling was examined by immunoblot and luciferase reporter assays. In vitro binding assay was performed to determine LPS binding to MD2. Macrophage migration was analyzed using a transwell-culture system.

RESULTS

KOdiA-PC prevented the activation of TLR4-signaling components including ERK, JNK, p38, NF-κB, and IRF3 leading to decrease of TNF-α, IFN-β, and COX-2 expression. In vitro binding assay revealed that KOdiA-PC interrupted LPS binding to MD2, a TLR4 co-receptor. Consistently, KOdiA-PC suppressed LPS-induced macrophage migration.

CONCLUSIONS

The results demonstrate that KOdiA-PC can modulate TLR4 activation by regulating ligand-receptor interaction. Therefore, endogenously generated, oxidized phospholipids may play a role in resolving inflammation by terminating TLR activation and macrophage recruitment to the inflamed site.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) has been characterized as the product of a novel antiviral gene and as an important modulator in innate immunity. However, the role of IFIT3 in PRRSV infection is scarcely understood. In this study, polyinosinic-polycytidylic acid (poly(I:C)) inhibited PRRSV replication in MARC-145 cells, following the appearance of increased IFIT3. Overexpression of porcine IFIT3 resulted in a decrease of PRRSV. Knockdown of IFIT3 in MARC-145 cells increased PRRSV replication and impaired the antiviral activity mediated by poly(I:C). Moreover, in the presence or absence of IFIT3, poly(I:C)-induced IFN-β promoter activity was significantly boosted or crippled, respectively. IFIT3, TBK1 and phosphorylation of IRF3 were activated in poly(I:C)-transfected MARC-145 cells. It demonstrated that IFIT3 plays an important role in IFN-β induction in MARC-145 cells, and, when activated, it can inhibit PRRSV replication.

In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-β mRNA transcription, while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response.IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon beta (IFN-β) by interacting with and degrading retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation. This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

The innate immune response is the first line of host defense to eliminate viral infection. Pattern recognition receptors in the cytosol, such as RIG-I-like receptors (RLR) and Nod-like receptors (NLR), and membrane bound Toll like receptors (TLR) detect viral infection and initiate transcription of a cohort of antiviral genes, including interferon (IFN) and interferon stimulated genes (ISGs), which ultimately block viral replication. Another mechanism to reduce viral spread is through RIPA, the RLR-induced IRF3-mediated pathway of apoptosis, which causes infected cells to undergo premature death. The transcription factor IRF3 can mediate cellular antiviral responses by both inducing antiviral genes and triggering apoptosis through the activation of RIPA. The mechanism of IRF3 activation in RIPA is distinct from that of transcriptional activation; it requires linear polyubiquitination of specific lysine residues of IRF3. Using RIPA-active, but transcriptionally inactive, IRF3 mutants, it was shown that RIPA can prevent viral replication and pathogenesis in mice.

The ORF3b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has a nuclear localization signal (NLS) at its C terminus and antagonizes interferon (IFN) function by modulating the activity of IFN regulatory factor 3 (IRF3). SARS-like coronaviruses (SL-CoVs) found in bats share an identical genome organization and high sequence identity for most of their gene products. In this study, ORF3b homologues were identified from three bat SL-CoV strains. These ORF3b homologues were C-terminally truncated and lacked the C-terminal NLS of SARS-CoV. IFN antagonist activities analysis demonstrated that one SL-CoV ORF3b still possessed IFN antagonist and IRF3-modulating activities. These results indicate that different ORF3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SL-CoV ORF3b function and viral pathogenesis.

BACKGROUND

Mesangial proinflammatory chemokine/cytokine expressions via innate immunity play a pivotal role in the pathogenesis of glomerulonephritis. CXCL1/GROα is a strong neutrophil chemoattractant cytokine and reportedly plays an important role in regional inflammatory reactions. However, detailed signaling of mesangial CXCL1 expression induced by viral or "pseudoviral" immunity remains to be determined.

METHODS

We treated normal human mesangial cells (MCs) in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of CXCL1 by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR and enzyme-linked immunosorbent assay. To elucidate the poly IC-induced signaling pathway for CXCL1 expression, we subjected the cells to RNA interference against Toll-like receptor (TLR) 3, retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), interferon (IFN)-β, nuclear factor (NF)-κB p65 and IFN regulatory factor (IRF) 3. We also conducted an immunofluorescence study to examine mesangial CXCL1 expression in biopsy specimens from patients with lupus nephritis (LN) and IgA nephropathy (IgAN).

RESULTS

We found that activation of TLR3 signaling could induce the expression of CXCL1 in MCs. NF-κB, IRF3 and IFN-β, but neither RIG-I nor MDA5, were found to be involved in mesangial CXCL1 expression in this setting. Induction of CXCL1 by poly IC was inhibited by pretreatment of cells with dexamethasone. Intense glomerular CXCL1 expression was observed in biopsy specimens from patients with LN, whereas only a trace staining occurred in specimens from patients with IgAN.

CONCLUSIONS

TLR3 signaling also contributes to the CXCL1 expression in MCs. These observations further support the implication of viral and "pseudoviral" immunity in the pathogenesis of inflammatory renal diseases, especially in LN.

Gastric cancer is one of the most common and deadly neoplasms with limited effective treatments. The emergence of the immunotherapy has brought great expectations for cancer patients. Sophoridine is extracted from the seeds of sophora alopecuroides and has various pharmacological actions including anti-tumor, anti-inflammatory, anti- arrhythmia and anti-virus. However, the effect of Sophoridine on gastric cancer microenvironment immunity and its underling mechanism remains poorly known. This study was aimed to investigate the effect of Sophoridine on the polarization status of gastric tumor-associated macrophages (TAMs) and its underlying mechanism. We isolated primary bone marrow-derived macrophages (BMDMs) and primary CD8⁺ T cells to perform coculture assay. Sophoridine educated TAMs polarize to M1-TAMs and suppressed M2-TAMs polarization through TLR4/IRF3 axis. Sophoridine-treated TAMs exhibited stronger pro-inflammatory function through upregulation the expression of INOS, IFN-β and IL-12α, and downregulation the expression of Arg-1, CD206 and IL-10. Sophoridine -primed TAMs increased the proliferation and cytotoxic function of CD8⁺ T by upregulating the expression of Granzyme-B, TNF-α and Perforin, and downregulated the expression of CD8⁺ T cells function exhaustion markers PD-1, Tim-3 and Lag-3. Furthermore, Sophoridine inhibited the migration ability of macrophage by decrease the CCR2 expression. Thus, Sophoridine acted on macrophages and CD8⁺ T cells to reshape gastric cancer immune microenvironment. Our studies provided preclinical basis for clinical application of Sophoridine.

The high-risk human papillomavirus E6 (hrHPV E6) protein has been widely studied due to its implication in cervical cancer. In response to viral threat, activated kinases phosphorylate the IRF3 autoinhibitory domain, inducing type1 interferon production. HPV circumvents the antiviral response through the possible E6 interaction with IRF3 and abrogates p53's apoptotic activity by recruiting E6-associated protein. However, the molecular mechanism of IRF3 inactivation by hrHPV E6 has not yet been delineated. Therefore, we explored this mechanism through in silico examination of protein-protein and protein-ligand docking, binding energy differences, and computational alanine mutagenesis. Our results suggested that the LxxLL motifs of IRF3 binds within the hydrophobic pocket of E6, precluding Ser-patch phosphorylation, necessary for IRF3 activation and interferon induction. This model was further supported by molecular dynamics simulation. Furthermore, protein-ligand docking and drug resistance modeling revealed that the polar patches in the pocket of E6, which are crucial for complex stability and ligand binding, are inconsistent among hrHPV species. Such variabilities pose a risk of treatment failure owing to point mutations that might render drugs ineffective, and allude to multi-drug therapy. Overall, this study reveals a novel perspective of innate immune suppression in HPV infections and suggests a plausible therapeutic intervention.

OBJECTIVE

The aim of this study was to evaluate the therapeutic potential of the phenethyl isothiocyanate (PEITC) in Toll-like receptor (TLR) signaling pathways.

METHODS

To evaluate the cytotoxic nature of PEITC in RAW 264.7 cells, cytotoxicity was determined using the MTS cell viability assay. RAW264.7 cells were transfected with a nuclear factor-κB (NF-κB), interferon β (IFNβ) PRDIII-I, or interferon inducible protein-10 (IP-10) luciferase plasmid and then luciferase enzyme activities were determined by luciferase assay. The expression of inducible nitric oxide synthase (iNOS) and phosphorylation of interferon regulatory factor 3 (IRF3) were determined by Western blotting. The levels of IP-10 were determined with culture medium by using an IP-10 enzyme-linked immunosorbent assay (ELISA) kit.

RESULTS

PEITC suppressed the activation of IRF3 and the expression of IP-10 induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly[I:C]).

CONCLUSIONS

TLRs play an important role in the induction of innate immune responses for host defense against invading microbial pathogens. PEITC found in cruciferous vegetables has an effect on treatment of many chronic diseases. Our results suggest that beneficial effects of PEITC on chronic inflammatory diseases are mediated through modulation of Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathway of TLRs.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, catalyzes the formation of the second messenger 2'3'-cGAMP that binds to STING and triggers the type I IFN signaling. Activation of cGAS can be modulated by several protein posttranslational modifications, including ubiquitination. However, the cGAS activation regulated by protein deubiquitination remains poorly understood. In this study, we identified that deubiquitinase USP27X could interact with cGAS and cleave K48-linked polyubiquitination chains from cGAS, leading to cGAS stabilization. Consistently, knockout of Usp27x in mice macrophages resulted in an accelerated turnover of cGAS, decreased cGAMP production, phosphorylation of TBK1 and IRF3, and IFN-β production. Furthermore, Usp27x knockout mice macrophages showed impaired innate antiviral responses against HSV type 1 infection. Our data suggest that USP27X is a novel regulator of the cGAS-STING cytosolic DNA sensing pathway.

In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.

Extracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) RNA is delivered into macrophage-derived EVs through an M.tb SecA2-dependent pathway and that EVs released from M.tb-infected macrophages stimulate a host RIG-I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG-I/MAVS-dependent manner, the maturation of M.tb-containing phagosomes through a noncanonical LC3 pathway, leading to increased bacterial killing. Moreover, treatment of M.tb-infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb-infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We hypothesize that EVs, which are preferentially removed by macrophages in vivo, can be combined with effective antibiotics as a novel approach to treat drug-resistant TB.

After viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear.

We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling.

Our results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection.