Anthony T. Vella

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.

Monocytes are critical in controlling tissue infections and inflammation. Monocyte dysfunction contributes to the inflammatory pathogenesis of cystic fibrosis (CF) caused by CF transmembrane conductance regulator (CFTR) mutations, making CF a clinically relevant disease model for studying the contribution of monocytes to inflammation. Although CF monocytes exhibited adhesion defects, the precise mechanism is unclear. Herein, superresolution microscopy showed that an integrin clustering but not an integrin activation defect determines the adhesion defect in CFTR-deficient monocytes, challenging the existing paradigm emphasizing an integrin activation defect in CF patient monocytes. We further found that the clustering defect is accompanied by defects in CORO1A membrane recruitment, actin cortex formation, and CORO1A engagement with integrins. Complementing canonical studies of leukocyte adhesion focusing on integrin activation, we highlight the importance of integrin clustering in cell adhesion and report that integrin clustering and activation are distinctly regulated, warranting further investigation for selective targeting in therapeutic strategy design involving leukocyte-dependent inflammation.

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 trapped 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 translocation, 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.

T cell receptor (TCR) sensitivity to peptide–major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2β in mouse and human. TRA2 β - PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2 β-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2 β-PE reinclusion allowed T cell survival. Finally, we found that TRA2 β-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2 β - PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases.

The search for dementia treatments, including treatments for neuropsychiatric lupus (NPSLE), has not yet uncovered useful therapeutic targets that mitigate underlying inflammation. Currently, NPSLE’s limited treatment options are often accompanied by severe toxicity. Blocking toll-like receptor (TLR) and IL-1 receptor signal transduction by inhibiting interleukin-1 receptor-associated kinase 4 (IRAK4) offers a new pathway for intervention. Using a pre-clinical NPSLE model, we compare lupus-like B6.MRL-Faslpr (MRL) mice with B6.MRL-Faslpr-IRAK4 kinase-dead (MRL-IRAK4-KD) mice, which are were less prone to ‘general’ lupus-like symptoms. We demonstrate that lupus-prone mice with a mutation in the kinase domain of IRAK4 no longer display typical lupus hallmarks such as splenomegaly, inflammation, production of hormones, and anti-double-stranded (ds)DNA antibody. water maze behavioral testing, which measures contextual associative learning, revealed that mice without functional IRAK4 displayed a recovery in memory acquisition deficits. RNA-seq approach revealed that cytokine and hormone signaling converge on the JAK/STAT pathways in the mouse hippocampus. Ultimately, the targets identified in this work may result in broad clinical value that can fill the significant scientific and therapeutic gaps precluding development of cures for dementia.

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.

Globoid cell leukodystrophy (GLD) or Krabbe’s disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

MicroRNA-150 (miR-150) has been shown to play a general role in the immune system, but very little is known about its role on CD4⁺ T cell responses. During T cell responses against superantigen Staphylococcal Enterotoxin A, miR-150 expression was down-regulated in antigen-specific CD4⁺ T cells but up-regulated in CD8⁺ T cells. CD4⁺ and CD8⁺ T cell clonal expansion was greater in miR-150-KO mice than in WT mice, but miR-150 selectively repressed IL-2 production in CD4⁺ T cells. Transcriptome analysis of CD4⁺ T cells demonstrated that apoptosis and mTOR pathways were highly enriched in the absence of miR-150. Mechanistic studies confirmed that miR-150 promoted apoptosis specifically in antigen-specific CD4⁺ T cells, but not in bystander CD4⁺ nor in CD8⁺ T cells. Furthermore, inhibition of mTOR-linked mitochondrial superoxidedismutase-2 increased apoptosis in miR-150^-/- antigen-specific CD4⁺ T. Thus, miR-150 impacts CD4⁺ T cell helper activity by attenuating IL-2 production along with clonal expansion, and suppresses superoxidedismutase to promote apoptosis.