The non-canonical caspase-4 inflammasome is a crucial anti-bacterial immune mechanism, yet, when dysregulated, may contribute to sepsis pathogenesis. Its regulation is heavily reliant on transcriptional control of caspase-4 expression. However, posttranslational regulation of the caspase-4 inflammasome remains poorly understood. Here, we report that UBX domain-containing protein 1 (UBXN1) facilitates the non-canonical inflammasome via unanchored lysine 48- or 63-linked polyUb (K48/63-Ub) chains. UBXN1 deficiency impairs the LPS-induced caspase-4 inflammasome and pyroptosis, renders mice resistant to LPS and polymicrobial sepsis. Depleting cellular unanchored polyUb with ubiquitin-specific proteinase 5 (USP5) reduces, while inhibiting USP5 enhances, caspase-4 activation in a UBXN1-dependent manner. In vitro , unanchored K48/63-Ub chains enhance LPS-induced caspase-4 enzymatic activity in a chain length- and UBXN1-dependent manner. Mechanistically, UBXN1 directly interfaces with and bridges unanchored K48/63-Ub and caspase-4, forming a tripartite complex to facilitate caspase-4 activation. Our findings uncover a previously unrecognized UBXN1- and unanchored K48/63-Ub-dependent regulatory layer in the caspase-4 inflammasome.

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.

Abstract Introduction Inflammasomes are crucial for innate immune defense to many microbial infections but are also subjected to stringent cellular regulation to avoid persistent, aberrant activation that could lead to lethal sepsis and autoinflammatory conditions. Ubiquitination is one of the most important posttranslational modifications involved in many immune signaling pathways, including the nucleotide-binding and oligomerization domain (NOD)-like receptor 3 (NLRP3) inflammasome. However, its role in the non-canonical caspase-4 inflammasomes is poorly understood. Methods To address this significant gap, we constructed 375 individual ubiquitin E3 ligase knockout lines by CRISPR-Cas9 and performed an unbiased screening. This library represents almost all the currently known definite E3 ligases (total ∼377). Results Our screen identified 15 positive regulators of the caspase-4 inflammasome; one of the top hits was Muscle Excess 3B (MEX3B), an RNA-binding protein with a ubiquitin ligase domain. We found that deletion of MEX3B inhibited caspase-4 and gasdermin D (GSDMD) activation, pyroptosis, and secretion of inflammasome-dependent inflammatory cytokines in human cell lines and murine primary macrophages upon priming with interferon gamma (IFN-γ) and transfection with lipopolysaccharide (LPS). Notably, MEX3B was also crucial for canonical inflammasome signaling such as NLRP3 and NLRC4. Mechanistically, MEX3B was required for caspase-1 activation but not NLRP3 oligomerization. The role of MEX3B in inflammasome signaling was reliant on its RNA-binding, but not E3 ligase activity. However, the expression of caspase-4 and GSDMD, IFN-γ and Toll-like receptor (TLR4) signaling, and apoptosis remained intact in MEX3B-/- cells. Conclusion Our results suggest that MEX3B is a pan-inflammasome regulator targeting inflammatory caspases. Ongoing work is to investigate both the NLRP3 and caspase-4 inflammasome signaling in Mex3b-/- mice and elucidate the molecular mechanism of action of MEX3B. Funding Source National Institute of Allergy and Infectious Diseases, USA Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Ubiquitination is a major posttranslational covalent modification that regulates numerous cellular processes including inflammasome signaling. Cells also contain unanchored ubiquitin chains (polyUb) that bind protein targets non-covalently, but their physiological functions in immunity have been appreciated only recently. Here, we report that ubiquitin regulatory x domain-containing protein 1 (UBXN1) activates the noncanonical inflammasome via unanchored Lysin (K) 48- or 63-linked polyUb. UBXN1 deficiency impairs the activation of caspase-4/11, secretion of inflammasome-dependent cytokines and pyroptosis in response to intracellular lipopolysaccharide (LPS). UBXN1-deficient mice are protected from LPS- and cecal-ligation-and-puncture-induced sepsis, evidenced by reduced mortality and systemic inflammation, compared to UBXN1-sufficient littermates. Mechanistically, UBXN1 together with unanchored K48/63-linked polyUb bind caspase-4/11, the intracellular sensors of LPS, and promote their assembly and activation. Depleting cellular unanchored polyUb with recombinant ubiquitin-specific proteinase 5 (USP5) reduces UBXN1 binding to caspase-4/11 and inflammasome signaling, while USP5 inhibitors enhance pyroptosis in an UBXN1-dependent manner. Thus, this study identifies a critical UBXN1-dependent posttranslational mechanism involved in noncanonical inflammasome activation and UBXN1 as a potential therapeutic target for sepsis and advances a fundamental understanding of unanchored polyUb biology. ### Competing Interest Statement The authors have declared no competing interest.

Ubiquitination is a major posttranslational covalent modification that regulates numerous cellular processes including inflammasome signaling. Cells also contain unanchored ubiquitin chains (polyUb) that bind protein targets non-covalently, but their physiological functions in immunity have been appreciated only recently. Here, we report that ubiquitin regulatory x domain-containing protein 1 (UBXN1) activates the noncanonical inflammasome via unanchored Lysin (K) 48- or 63-linked polyUb. UBXN1 deficiency impairs the activation of caspase-4/11, secretion of inflammasome-dependent cytokines and pyroptosis in response to intracellular lipopolysaccharide (LPS). UBXN1-deficient mice are protected from LPS- and cecal-ligation-and-puncture-induced sepsis, evidenced by reduced mortality and systemic inflammation, compared to UBXN1-sufficient littermates. Mechanistically, UBXN1 together with unanchored K48/63-linked polyUb bind caspase-4/11, the intracellular sensors of LPS, and promote their assembly and activation. Depleting cellular unanchored polyUb with recombinant ubiquitin-specific proteinase 5 (USP5) reduces UBXN1 binding to caspase-4/11 and inflammasome signaling, while USP5 inhibitors enhance pyroptosis in an UBXN1-dependent manner. Thus, this study identifies a critical UBXN1-dependent posttranslational mechanism involved in noncanonical inflammasome activation and UBXN1 as a potential therapeutic target for sepsis and advances our understanding unanchored polyUb biology. R01AI132526 R21AI170989 Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

BACKGROUND AND PURPOSE:Chronic kidney disease (CKD) is characterised by inflammation, which can lead to tubular atrophy and fibrosis. The molecular mechanisms are not well understood. In this study, we investigated the functional role of the cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) signalling in renal inflammation and fibrosis. EXPERIMENTAL APPROACH:Mice with global cGAS deficiency or global or myeloid cell-specific STING deficiency or wild-type mice treated with RU.521, a selective cGAS inhibitor, were used to examine the role of cGAS-STING signalling in renal inflammation and fibrosis in a preclinical model of obstructive nephropathy in vivo. Bone marrow-derived macrophages were used to determine whether tubular epithelial cell-derived DNA can activate cGAS-STING signalling in vitro. KEY RESULTS:Following obstructive injury, cGAS-STING signalling was activated in the kidneys during the development of renal fibrosis. Mice with deficiency of cGAS or STING exhibited significantly less macrophage proinflammatory activation, myofibroblast formation, total collagen deposition, and extracellular matrix (ECM) protein production in the kidneys following obstructive injury. Pharmacological inhibition of cGAS with RU.521 reduced macrophage proinflammatory activation, suppressed myofibroblast formation, and attenuated kidney fibrosis following obstructive injury. Mechanistically, cGAS-STING signalling in macrophages is activated by double-stranded DNA released from damaged tubular epithelial cells, which induces inflammatory responses. CONCLUSIONS AND IMPLICATIONS:Our study identifies the cGAS-STING signalling pathway as a critical regulator of macrophage proinflammatory activation during the development of renal fibrosis. Therefore, inhibition of cGAS-STING signalling may represent a novel therapeutic strategy for CKD.

Recent high-impact research has significantly advanced our understanding of inflammasomes as therapeutic targets for inflammatory diseases. Breakthrough studies have revealed new mechanisms of inflammasome regulation and innovative inhibition approaches. A key discovery identified NEK7 as an essential component for NLRP3 inflammasome activation, providing a new target for therapeutic intervention. Additionally, researchers developed CY-09, a small molecule inhibitor that directly binds to the ATP-binding site of NLRP3, offering a highly specific method for inflammasome inhibition. Further progress includes elucidating the role of metabolic reprogramming in inflammasome activation, with studies finding that itaconate can directly inhibit NLRP3 activation. This discovery bridges cellular metabolism and inflammasome regulation, suggesting new metabolic approaches to modulate inflammatory responses. Research has also highlighted the importance of the non-canonical inflammasome pathway in atherosclerosis progression, expanding therapeutic possibilities for cardiovascular diseases. In the field of targeted therapies, a nanoparticle-based delivery system for inhibiting AIM2 inflammasome in psoriasis demonstrated significant efficacy in preclinical models. This approach showcases the potential of nanotechnology in enhancing the specificity and effectiveness of inflammasome-targeted therapies. These latest advancements collectively underscore the rapid progress in understanding inflammasome biology and developing innovative therapeutic strategies, paving the way for more effective and precise treatments for a wide range of inflammatory diseases.

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs), whose role in antiviral immunity is elusive. Here, we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state, GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9, TUG). Following RNA virus infection, GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs, preventing them from activating IFN-β responses. UBXN9 deletion prompts constitutive GLUT4 trafficking, sequestration of RLRs, and attenuation of antiviral immunity, whereas GLUT4 deletion heightens RLR signaling. Notably, reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall, our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs.

The Retinoic acid-Inducible Gene I (RIG-I) like receptors (RLRs) are the major viral RNA sensors essential for the initiation of antiviral immune responses. RLRs are subjected to stringent transcriptional and posttranslational regulations, of which ubiquitination is one of the most important. However, the role of ubiquitination in RLR transcription is unknown. Here, we screen 375 definite ubiquitin ligase knockout cell lines and identify Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5) as a positive regulator of RLR transcription. UBR5 deficiency reduces antiviral immune responses to RNA viruses, while increases viral replication in primary cells and mice. Ubr5 knockout mice are more susceptible to lethal RNA virus infection than wild type littermates. Mechanistically, UBR5 mediates the Lysine 63-linked ubiquitination of Tripartite Motif Protein 28 (TRIM28), an epigenetic repressor of RLRs. This modification prevents intramolecular SUMOylation of TRIM28, thus disengages the TRIM28-imposed brake on RLR transcription. In sum, UBR5 enables rapid upregulation of RLR expression to boost antiviral immune responses by ubiquitinating and de-SUMOylating TRIM28.

ABSTRACT β3-adrenergic receptor (β3-AR) has been proposed as a new therapy for several myocardial diseases. However, the effect of β3-AR activation on sepsis-induced myocardial apoptosis is unclear. Here, we investigated the effect of β3-AR activation on the cardiomyocyte apoptosis and cardiac dysfunction in cecal ligation and puncture (CLP)-operated rats and lipopolysaccharide (LPS)-treated cardiomyocytes. We found that β3-AR existed both in adult rat ventricular myocytes (ARVMs) and H9c2 cells. The expression of β3-AR was upregulated in LPS-treated ARVMs and the heart of CLP rats. Pretreatment with β3-AR agonist, BRL37344, inhibited LPS-induced cardiomyocyte apoptosis and caspase-3, -8 and -9 activation in ARVMs. BRL37344 also reduced apoptosis and increased the protein levels of PI3K, p-AktSer473 and p-eNOSSer1177 in LPS-treated H9c2 cells. Inhibition of PI3K using LY294002 abolished the inhibitory effect of BRL37344 on LPS-induced caspase-3, -8, and -9 activation in H9c2 cells. Furthermore, administration of β3-AR antagonist, SR59230A (5 mg/kg), significantly decreased the maximum rate of left ventricular pressure rise (+dP/dt) in CLP-induced septic rats. SR59230A not only increased myocardial apoptosis, reduced p-AktSer473 and Bcl-2 contents, but also increased mitochondrial Bax, cytoplasm cytochrome c, cleaved caspase-9 and cleaved caspase-3 levels of the myocardium in septic rats. These results suggest that endogenous β3-AR activation alleviates sepsis-induced cardiomyocyte apoptosis via PI3K/Akt signaling pathway and maintains intrinsic myocardial systolic function in sepsis.

BACKGROUND:The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. METHODS:We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. FINDINGS:Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. INTERPRETATION:UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. FUNDING:United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Cardiomyopathy is particularly common in septic patients. Our previous studies have shown that activation of the alpha 1 adrenergic receptor (α1-AR) on cardiomyocytes inhibits sepsis-induced myocardial dysfunction. However, the role of cardiac endothelial α1-AR in septic cardiomyopathy has not been determined. Here, we identified α1-AR expression in mouse and human endothelial cells and showed that activation of α1-AR with phenylephrine (PE) improved cardiac function and survival by preventing cardiac endothelial injury in septic mice. Mechanistically, activating α1-AR with PE decreased the expression of ICAM-1, VCAM-1, iNOS, E-selectin, and p-p38MAPK, while promoting PKC and ERK1/2 phosphorylation in LPS-treated endothelial cells. These effects were abolished by a PKC inhibitor or α1-AR antagonist. PE also reduced p65 nuclear translocation, but this suppression is not blocked by PKC inhibition. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on p38MAPK phosphorylation. Our results demonstrate that cardiac endothelial α1-AR activation prevents sepsis-induced myocardial dysfunction in mice by inhibiting the endothelial injury via PKC-ERK/p38MAPK signaling pathway and a PKC-independent inhibition of p65 nuclear translocation. These findings offer a new perspective for septic patients with cardiac dysfunction by inhibiting cardiac endothelial cell injury through α1-AR activation.

The current dogma of RIG-I-like receptor (RLR) regulation states that dysregulated interferon (IFN) production is predominantly repressed by reversible posttranslational modifications (e.g., ubiquitination). However, RLR activation has profound outcomes on cellular metabolism, which governs the nature of these inflammatory immune responses. Here, we reveal a novel mechanism of immunometabolic regulation of RLR signaling by the glucose transporter 4 (GLUT4). In addition to its canonical role in maintaining organismal glucose homeostasis, GLUT4 attenuates innate immune signaling through sequestering RLRs into the plasma membrane. Translocation of GLUT4 from intracellular compartments to the plasma membrane is tightly regulated by UBXN9, a ubiquitin-domain containing protein (UBXN). Disruption of UBXN9 releases GLUT4, suppresses RLR signaling/IFN responses and enhances viral replication, while genetic ablation of GLUT4 improves antiviral immunity. Strikingly, these distinct phenotypes are independent of glycolysis, but dictated by the mobilization of GLUT4 to the plasma membrane. Further, GLUT4 colocalizes with RIG-I after insulin treatment (which stimulates GLUT4 trafficking) or viral infection to blunt RLR activation. Fine-tuning of this UBXN9-GLUT4 axis is critical for antiviral immunity, but dysregulation of these components may underlie inflammatory myopathies that are characterized by a hyperactive RLR-IFN pathway. Together, this study reveals a novel link between GLUTs and cytosolic immune sensors, and underscores the intersection of vesicular trafficking, glucose metabolism and antiviral immunity. Supported by grants from NIH (NIH R01AI132526, R21AI155820)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic named coronavirus disease 2019 (COVID-19) that has become the greatest worldwide public health threat of this century. Recent studies have unraveled numerous mysteries of SARS-CoV-2 pathogenesis and thus largely improved the studies of COVID-19 vaccines and therapeutic strategies. However, important questions remain regarding its therapy. In this review, the recent research advances on COVID-19 mechanism are quickly summarized. We mainly discuss current therapy strategies for COVID-19, with an emphasis on antiviral agents, neutralizing antibody therapies, Janus kinase inhibitors, and steroids. When necessary, specific mechanisms and the history of therapy are present, and representative strategies are described in detail. Finally, we discuss key outstanding questions regarding future directions of the development of COVID-19 treatment.

Background: Dexmedetomidine (DEX) administered before or at 30 min after sepsis induction was reported to alleviate septic cardiomyopathy in experimental models. However, sepsis is a life-threatening organ dysfunction due to infection-induced dysregulated host response, whether DEX treatment in the presence of organ dysfunction affects septic cardiomyopathy is unknown. This study investigated the effect of DEX posttreatment on septic cardiomyopathy.Methods: Male wild-type and alpha 2A-adrenergic receptor (AR) knockout mice were exposed to lipopolysaccharide (LPS) or cecal ligation puncture (CLP), and cultured cardiac endothelial cells were used. Mouse survival, myocardial function, inflammatory response and related signaling pathways were determined.Results: DEX treatment at 6, 9 h after LPS challenge significantly reduced survival rate of LPS-challenged mice, especially at 9 h. DEX administered at 9 h after LPS injection or CLP significantly reduced survival in LPS or CLP-induced sepsis in wild-type mice, but not in alpha 2A-AR knockout mice. LPS treatment for 20 h decreased the left ventricle + dp/dt, increased myocardial interleukin (IL)-1 beta and IL-6 concentrations as well as cardiac endothelial tumor necrosis factor (TNF)-alpha, vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1 expression, which were enhanced by DEX treated at 9 h after LPS injection in wild-type mice, but not in alpha 2A-AR knockout mice. Furthermore, DEX posttreatment increased p38 phosphorylation, c-Fos nuclear translocation and VCAM-1 expression in LPS-treated cardiac endothelial cells, which were eliminated by alpha 2A-AR knockout or PKC inhibitor.Conclusions: DEX posttreatment aggravates LPS-induced cardiac inflammation and myocardial dysfunction, at least in part, via activating cardiac endothelial alpha 2A-AR-mediated PKC signal pathway.

Cardiomyopathy is a common complication and significantly increases the risk of death in septic patients. Our previous study demonstrated that post-treatment with dexmedetomidine (DEX) aggravates septic cardiomyopathy. However, the mechanisms for the side effect of DEX post-treatment on septic cardiomyopathy are not well-defined. Here we employed a cecal ligation and puncture (CLP) model and α2A-adrenoceptor deficient (Adra2a-/-) mice to observe the effects of DEX post-treatment on myocardial metabolic disturbances in sepsis. CLP mice displayed significant cardiac dysfunction, altered mitochondrial dynamics, reduced cardiac lipid and glucose uptake, impaired fatty acid and glucose oxidation, enhanced glycolysis and decreased ATP production in the myocardium, almost all of which were dramatically enhanced by DEX post-treatment in septic mice. In Adra2a-/- mice, DEX post-treatment did not affect cardiac dysfunction and metabolic disruptions in CLP-induced sepsis. Additionally, Adra2a-/- mice exhibited impaired cardiac function, damaged myocardial mitochondrial structures, and disturbed fatty acid metabolism and glucose oxidation. In sum, DEX post-treatment exacerbates metabolic disturbances in septic cardiomyopathy in a α2A-adrenoceptor dependent manner.

Innate immune signaling plays a significant role in the rapid cellular responses against foreign entities. An inflammasome is a large cytosolic polymer of a pattern recognition receptor with/without an adaptor protein, formed in response to these entities. Canonically, an inflammasome can recruit and lead to auto-activation of caspase-1, subsequent maturation and secretion of inflammatory cytokines, and pyroptosis. One particular inflammasome, the noncanonical inflammasome, is formed by caspase-4 or -5 (mouse caspase-11) upon binding of lipopolysaccharide and is essential for controlling gram-negative bacterial infection. However, prolonged hyper-activation of the non-canonical inflammasome has been implicated in the pathogenesis of inflammatory diseases and endotoxemia sepsis. This review will summarize the recent advances on the noncanonical inflammasome, its mechanism of activation, key cellular regulators and role in health and disease.

The human genome encodes 13 ubiquitin regulatory X (UBX) domain-containing proteins (UBXN) that might participate in diverse cellular processes. However, their in vivo physiological functions remain largely elusive.