The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

SARS-CoV-2 is highly pathogenic in humans and poses a great threat to public health worldwide. Clinical data shows a disturbed type I interferon (IFN) response during the virus infection. In this study, we discovered that the nucleocapsid (N) protein of SARS-CoV-2 plays an important role in the inhibition of interferon beta (IFN-β) production. N protein repressed IFN-β production induced by poly(I:C) or upon Sendai virus (SeV) infection. We noted that N protein also suppressed IFN-β production, induced by several signaling molecules downstream of the retinoic acid-inducible gene I (RIG-I) pathway, which is the crucial pattern recognition receptor (PRR) responsible for identifying RNA viruses. Moreover, our data demonstrated that N protein interacted with the RIG-I protein through the DExD/H domain, which has ATPase activity and plays an important role in the binding of immunostimulatory RNAs. These results suggested that SARS-CoV-2 N protein suppresses the IFN-β response through targeting the initial step, potentially the cellular PRR–RNA-recognition step in the innate immune pathway. Therefore, we propose that the SARS-CoV-2 N protein represses IFN-β production by interfering with RIG-I.

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients' sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.

BACKGROUND:Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions.RESULTS:We show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages.CONCLUSIONS:We have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.

During host-virus co-evolution, cells develop innate immune systems to inhibit virus invasion, while viruses employ strategies to suppress immune responses and maintain infection. Here, we reveal that Zika virus (ZIKV), a re-emerging arbovirus causing public concerns and devastating complications, restricts host immune responses through a distinct mechanism. ZIKV nonstructural protein 5 (NS5) interacts with the host retinoic acid-inducible gene I (RIG-I), an essential signaling molecule for defending pathogen infections. NS5 subsequently represses K63-linked polyubiquitination of RIG-I, attenuates the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), and inhibits the expression and production of interferon-beta (IFN-beta), thereby restricting the RIG-I signaling pathway. Interestingly, we demonstrate that the methyltransferase (MTase) domain of NS5 is required for the repression of RIG-I ubiquitination, IRF3 activation, and IFN-beta production. Detailed studies further reveal that the conservative active site D146 of NS5 is critical for the suppression of the RIG-I signaling. Therefore, we uncover an essential role of NS5 conservative site D146 in ZIKV-mediated repression of innate immune system, illustrate a distinct mechanism by which ZIKV evades host immune responses, and discover a potential target for anti-viral infection.

One of the fundamental reactions of the innate immune responses to pathogen infection is the release of pro-inflammatory cytokines, including IL-1β, processed by the NLRP3 inflammasome. The stimulator of interferon genes (STING) has the essential roles in innate immune response against pathogen infections. Here we reveal a distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1β secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and facilitates NLRP3 localization in the endoplasmic reticulum, thereby facilitating the inflammasome formation. Second, STING interacts with NLRP3 and attenuates K48- and K63-linked polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against HSV-1 infection.