SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored Plpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKK?-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKK?, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3- TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which Plpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.     

Activation of the STING‐IRF3 pathway involved in psoriasis with diabetes mellitus

Psoriasis and type 2 diabetes mellitus (T2DM) share similar inflammatory pathways in their pathogenesis. The stimulator of interferon genes (STING)‐interferon regulatory factor 3 (IRF3) pathway has recently been shown to play an important role in immune and metabolic diseases. In this study, we investigated the activation of the STING‐IRF3 pathway in human immortalized keratinocytes (HaCaT) cells treated with palmitic acid (PA) and imiquimod (IMQ). Additionally, we detected the STING‐IRF3 pathway in diabetic mice with imiquimod (IMQ)‐induced psoriasis and assessed the potential of STING inhibitor C‐176. Furthermore, skin samples from patients with psoriasis and diabetes were collected for immunohistochemical analysis. The results indicated that the STING‐IRF3 pathway was activated in HaCaT cells. Moreover, the STING pathway was also found to be induced in the skin tissue of diabetic mice with psoriasis; the inflammatory responses were ameliorated by treatment with C‐176. In the skin tissue samples of patients with psoriasis and diabetes, immunohistochemistry showed that the expression levels of STING and phosphorylated IRF3 were also significantly increased. Thus, we conclude that the STING‐IRF3 pathway is involved in the inflammatory response in the manifestation of psoriasis with T2DM. Inhibition of the activation of the STING pathway can ameliorate the development of psoriasis in diabetes and could be targeted for the development of therapeutic agents for these conditions.     

STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS)

Stimulator of interferon genes (STING) contributes to immune responses against tumors and may control viral infection including SARS-CoV-2 infection. However, activation of the STING pathway by airway silica or smoke exposure leads to cell death, self-dsDNA release, and STING/type I IFN dependent acute lung inflammation/ARDS. The inflammatory response induced by a synthetic non-nucleotide-based diABZI STING agonist, in comparison to the natural cyclic dinucleotide cGAMP, is unknown. A low dose of diABZI (1 µg by endotracheal route for 3 consecutive days) triggered an acute neutrophilic inflammation, disruption of the respiratory barrier, DNA release with NET formation, PANoptosis cell death, and inflammatory cytokines with type I IFN dependent acute lung inflammation. Downstream upregulation of DNA sensors including cGAS, DDX41, IFI204, as well as NLRP3 and AIM2 inflammasomes, suggested a secondary inflammatory response to dsDNA as a danger signal. DNase I treatment, inhibition of NET formation together with an investigation in gene-deficient mice highlighted extracellular DNA and TLR9, but not cGAS, as central to diABZI-induced neutrophilic response. Therefore, activation of acute cell death with DNA release may lead to ARDS which may be modeled by diABZI. These results show that airway targeting by STING activator as a therapeutic strategy for infection may enhance lung inflammation with severe ARDS. Open in a separate windowSTING agonist diABZI induces neutrophilic lung inflammation and PANoptosis A, Airway STING priming induce a neutrophilic lung inflammation with epithelial barrier damage, double-stranded DNA release in the bronchoalvelolar space, cell death, NETosis and type I interferon release. B, 1. The diamidobenzimidazole (diABZI), a STING agonist is internalized into the cytoplasm through unknown receptor and induce the activation and dimerization of STING followed by TBK1/IRF3 phosporylation leading to type I IFN response. STING activation also leads to NF-kB activation and the production of pro-inflammatory cytokines TNFα and IL-6. 2. The activation of TNFR1 and IFNAR1 signaling pathway results in ZBP1 and RIPK3/ASC/CASP8 activation leading to MLKL phosphorylation and necroptosis induction. 3. This can also leads to Caspase-3 cleavage and apoptosis induction. 4. Self-dsDNA or mtDNA sensing by NLRP3 or AIM2 induces inflammsome formation leading to Gasdermin D cleavage enabling Gasdermin D pore formation and the release mature IL-1β and pyroptosis. NLRP3 inflammasome formation can be enhanced by the ZBP1/RIPK3/CASP8 complex. 5. A second signal of STING activation with diABZI induces cell death and the release of self-DNA which is sensed by cGAS and form 2′3′-cGAMP leading to STING hyper activation, the amplification of TBK1/IRF3 and NF-kB pathway and the subsequent production of IFN-I and inflammatory TNFα and IL-6. This also leads to IFI204 and DDX41 upregulation thus, amplifying the inflammatory loop. The upregulation of apoptosis, pyroptosis and necroptosis is indicative of STING-dependent PANoptosis. Subject terms: Cell death and immune response, Respiratory tract diseases     

Cancer cell-specific cGAS/STING Signaling pathway in the era of advancing cancer cell biology

Pattern-recognition receptors (PRRs) are critical to recognizing endogenous and exogenous threats to mount a protective proinflammatory innate immune response. PRRs may be located on the outer cell membrane, cytosol, and nucleus. The cGAS/STING signaling pathway is a cytosolic PRR system. Notably, cGAS is also present in the nucleus. The cGAS-mediated recognition of cytosolic dsDNA and its cleavage into cGAMP activates STING. Furthermore, STING activation through its downstream signaling triggers different interferon-stimulating genes (ISGs), initiating the release of type 1 interferons (IFNs) and NF-κB-mediated release of proinflammatory cytokines and molecules. Activating cGAS/STING generates type 1 IFN, which may prevent cellular transformation and cancer development, growth, and metastasis. The current article delineates the impact of the cancer cell-specific cGAS/STING signaling pathway alteration in tumors and its impact on tumor growth and metastasis. This article further discusses different approaches to specifically target cGAS/STING signaling in cancer cells to inhibit tumor growth and metastasis in conjunction with existing anticancer therapies.     

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3C^pro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.     

Deubiquitinase USP35 restrains STING-mediated interferon signaling in ovarian cancer

Ovarian cancer is the most lethal malignant tumor of female reproductive system. It is well-known that induction of STING-mediated type I interferons can enhance the resultant antitumor activity. However, STING pathway is usually inactivated in cancer cells at multiple levels. Here, we identified deubiquitinase USP35 is upregulated in ovarian cancer tissues. High level of USP35 was correlated with diminished CD8⁺ T cell infiltration and poor prognosis in ovarian cancer patients. Mechanistically, we found that silencing USP35 reinforces the activation of STING-TBK1-IRF3 pathway and promotes the expression of type I interferons. Our data further showed that USP35 can directly deubiquitinate and inactivate STING. Interestingly, activation of STING promotes its binding to USP35 in a STING phosphorylation-dependent manner. Functionally, we found that knockdown of USP35 sensitizes ovarian cancer cells to the DNA-damage chemotherapeutic drug cisplatin. Overall, our study indicates that upregulation of USP35 may be a mechanism of the restricted STING activity in cancer cells, and highlights the significance of USP35 as a potential therapeutic target for ovarian cancer.Subject terms: Oncogenes, Cancer     

Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway

Crohn’s disease (CD) is an intestinal immune-dysfunctional disease. Extracellular vesicles (EVs) are membrane-enclosed particles full of functional molecules, e.g., nuclear acids. Recently, EVs have been shown to participate in the development of CD by realizing intercellular communication among intestinal cells. However, the role of EVs carrying double-strand DNA (dsDNA) shed from sites of intestinal inflammation in CD has not been investigated. Here we isolated EVs from the plasma or colon lavage of murine colitis and CD patients. The level of exosomal dsDNA, including mtDNA and nDNA, significantly increased in murine colitis and active human CD, and was positively correlated with the disease activity. Moreover, the activation of the STING pathway was verified in CD. EVs from the plasma of active human CD triggered STING activation in macrophages in vitro. EVs from LPS-damaged colon epithelial cells were also shown to raise inflammation in macrophages via activating the STING pathway, but the effect disappeared after the removal of exosomal dsDNA. These findings were further confirmed in STING-deficient mice and macrophages. STING deficiency significantly ameliorated colitis. Besides, potential therapeutic effects of GW4869, an inhibitor of EVs release were assessed. The application of GW4869 successfully ameliorated murine colitis by inhibiting STING activation. In conclusion, exosomal dsDNA was found to promote intestinal inflammation via activating the STING pathway in macrophages and act as a potential mechanistic biomarker and therapeutic target of CD.Subject terms: Acute inflammation, Monocytes and macrophages, Signal transduction     

The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling

Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF3 and IRF7 are activated in response to a variety of viruses or after engagement of Toll-like receptor (TLR) 3 and TLR4 by double-stranded RNA and lipopolysaccharide, respectively. The activation of IRF5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, interleukin 1 receptor-associated kinase 1, and tumor necrosis factor receptor-associated factor 6 are required for the activation of IRF5 and IRF7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF5 enabled type I interferon production in response to TLR7 signaling, whereas knockdown of IRF5 by small interfering RNA reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF5 and IRF7, therefore, emerge from these studies as critical mediators of TLR7 signaling.     

MyD88 Drives the IFN-β Response to Lactobacillus acidophilus in Dendritic Cells through a Mechanism Involving IRF1, IRF3, and IRF7

Type I IFNs are induced by pathogens to protect the host from infection and boost the immune response. We have recently demonstrated that this IFN response is not restricted to pathogens, as the Gram-positive bacterium Lactobacillus acidophilus, a natural inhabitant of the intestine, induces high levels of IFN-β in dendritic cells. In the current study, we investigate the intracellular pathways involved in IFN-β upon stimulation of dendritic cells with L. acidophilus and reveal that this IFN-β induction requires phagosomal uptake and processing but bypasses the endosomal receptors TLR7 and TLR9. The IFN-β production is fully dependent on the TIR adapter molecule MyD88, partly dependent on IFN regulatory factor (IRF)1, but independent of the TIR domain-containing adapter inducing IFN-β MyD88 adapter-like, IRF and IRF7. However, our results suggest that IRF3 and IRF7 have complementary roles in IFN-β signaling. The IFN-β production is strongly impaired by inhibitors of spleen tyrosine kinase (Syk) and PI3K. Our results indicate that L. acidophilus induces IFN-β independently of the receptors typically used by bacteria, as it requires MyD88, Syk, and PI3K signaling and phagosomal processing to activate IRF1 and IRF3/IRF7 and thereby the release of IFN-β.     

Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors

DNA-dependent activator of IFN regulatory factors (IRF; DAI, also known as ZBP1 or DLM-1) is a cytosolic DNA sensor that initiates IRF3 and NF-kappaB pathways leading to activation of type I IFNs (IFNalpha, IFNbeta) and other cytokines. In this study, induction of NF-kappaB is shown to depend on the adaptor receptor-interacting protein kinase (RIP)1, acting via a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI. DAI binds to and colocalizes with endogenous RIP1 at characteristic cytoplasmic granules. Suppression of RIP1 expression by RNAi abrogates NF-kappaB activation as well as IFNbeta induction by immunostimulatory DNA. DAI also interacts with RIP3 and this interaction potentiates DAI-mediated activation of NF-kappaB, implicating RIP3 in regulating this RHIM-dependent pathway. The role of DAI in activation of NF-kappaB in response to immunostimulatory DNA appears to be analogous to sensing of dsRNA by TLR3 in that both pathways involve RHIM-dependent signaling that is mediated via RIP1, reinforcing a central role for this adaptor in innate sensing of intracellular microbes.