Abstract Introduction RIG-I-like receptors are cytosolic viral RNA sensors that are critical for type I interferon (IFN)-driven antiviral defense against RNA virus infections. RIG-I has been shown to survey viral RNA in subcellular compartments, including stress granules, mitochondria-associated membranes, microsomes, and the nucleus, after virus invasion. Our recent work revealed a novel RIG-I action mode in which RIG-I is recruited onto endosomes, the “gateway” of virus entry into a host cell, to detect viral RNA upon viral breaching the endosomes. Endosomes serve as signaling platforms for the ubiquitination and activation of RIG-I. To better understand this novel RIG-I action mode, we further explored the spatiotemporal regulation of RIG-I signaling by endosomes, as well as the potential interplay between MDA5 and endosomes. Methods Biochemical and microscopic analyses are employed to investigate the interaction between RLRs and endosomes. Genetic approaches are employed to investigate the role of the endosomal adaptor TAPE in antiviral defense in vivo. Results Our data suggest that endosomes act as signaling platforms for MDA5 ubiquitination and activation. Conclusion These novel action modes of RIG-I-like receptors reveal the host’s early engagement with viruses to trigger antiviral defense. Funding Source Taiwan National Science and Technology Council Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Abstract Introduction In systemic lupus erythematosus (SLE), accelerated cell death promotes autoantigen release and immune complex formation, leading to visceral deposition and alveolar hemorrhage (AH) and lupus nephritis (LN). Viral infection has been implicated in autoimmunity, and post-COVID-19 disease flares are increasingly reported in SLE. Elevated pro-inflammatory cytokines are observed in SARS-CoV-2—infected individuals and patients with active SLE; however, the mechanistic role of Toll-like receptor 2 (TLR2) sensing of the SARS-CoV-2 envelope (Env) protein in post-COVID-19 SLE flares remains unclear. Methods Peripheral blood, AH lung tissues, and LN kidney tissues were obtained from SLE experiencing post-COVID-19 flares. THP-1 and RAW264.7 cells were stimulated with Env protein, followed by TLR2 inhibition. In vivo, pristane-induced AH or LN mouse models received intratracheal Env pseudovirus infusion in TLR2-deficient mice or in wild-type mice with concomitant sh-TLR2 delivery. Results Molecular docking predicted TLR2—Env interaction, which was validated by co-immunoprecipitation and solid-phase binding assays. Env stimulation activated NF-κB/MAPK signaling, increased ROS production and pro-inflammatory cytokines in RAW264.7 cells, which were attenuated by TLR2 inhibition or knockdown. In patients with post-COVID-19 SLE flares, plasma IL-1β levels were elevated with increased NLRP3, caspase-1, and N-gasdermin D expression in peripheral blood mononuclear cells, lung, and kidney tissues. Env-induced pyroptosis in THP-1 cells increased LDH and IL-1β release and was suppressed by TLR2 inhibition. In vivo, Env pseudovirus infusion exacerbated pristane-induced alveolar hemorrhage and lupus nephritis via enhanced pyroptosis, whereas TLR2 suppression markedly alleviated disease severity. Conclusion Together, these findings indicate that TLR2 sensing of the SARS-CoV-2 envelope protein promotes NLRP3-dependent pyroptosis and contributes to post-COVID-19 disease flares in systemic lupus erythematosus. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

BACKGROUND:Dysregulation of the oxytocin (OXT) system is implicated in the pathophysiology of several neuropsychiatric and neurological disorders, particularly autism spectrum disorder (ASD). Our previous study using the Coiled-coil and C2 domain containing 1a (Cc2d1a) conditional knockout (cKO) mouse model of ASD showed that restoring OXT levels effectively ameliorates irritability-like behavior; however, the mechanisms by which loss of Cc2d1a in forebrain excitatory neurons leads to decreased OXT expression in the paraventricular nucleus of the hypothalamus (PVN) remain elusive. METHODS:We used the bottle-brush test (BBT) to assess irritability-like behavior in wild-type (WT) and Cc2d1a cKO mice. Retrograde and anterograde trans-synaptic viral tracing were used to map associated neural circuits, and chemogenetic methods were employed to modify neuronal activity. Fiber photometry monitored OXT dynamics in situ, and whole-cell voltage-clamp recordings in ex vivo brain slices assessed synaptic transmission onto PVN OXT neurons. RESULTS:We found that decreased OXT expression in both magnocellular and parvocellular PVN neurons is evident in adult male Cc2d1a cKO mice, without corresponding changes in mRNA levels. Chronic silencing of PVN OXT neurons during adolescence leads to fewer OXT-immunoreactive neurons and heightened irritability-like behavior in adult male WT mice. Using both retrograde and anterograde trans-synaptic viral tracing techniques, we identified that the prelimbic cortex (PrL) indirectly regulates PVN OXT neuronal activity. In male Cc2d1a cKO mice, there was a preferential decrease in excitatory synaptic transmission onto PVN OXT neurons. In vivo real-time measurements of OXT dynamics in the posteroventral medial amygdala revealed reduced OXT release during the BBT in male Cc2d1a cKO mice. Chronic chemogenetic silencing of the PrL-PVN pathway during adolescence reduces irritability-like behavior in adult male Cc2d1a cKO mice. CONCLUSIONS:Our study unveils how forebrain Cc2d1a loss reduces OXT expression in the PVN at both molecular and circuit levels, underscoring the critical role of the OXT system as a potential therapeutic target for managing irritability in individuals with ASD.

CC2D1A is a multidomain scaffold protein implicated in transcriptional regulation and autosomal recessive non-syndromic intellectual disability (NSID), yet its molecular mechanism is still poorly understood due to a lack of structural information. Here, we present the crystal structure of the human CC2D1A491-810 fragment, encompassing the fourth DM14 domain, a coiled-coil region, and a C-terminal C2 domain. These elements form a compact, integrated architecture, with the C2 domain mediating symmetric dimerization through conserved electrostatic interactions. In addition, a unique antiparallel β1-β10 sheet connects the coiled-coil and C2 domains, stabilizing the tertiary structure. Fluorescence polarization assays reveal micromolar DNA-binding affinity, likely mediated by the basic surface of the DM14 domain. Comparison with the Drosophila homolog Lgd highlights conserved topology with added structural features, offering insights into CC2D1A's vertebrate-specific functions and NSID-related mutations.

Abstract Background Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity. Although reduced production of reactive oxygen species (ROS) by neutrophils correlates with severe SLE, the specific mechanisms linking ROS deficiency to heightened renal inflammation remain unknown. We aimed to elucidate the role of NOX2-derived ROS in LN pathogenesis and identify potential therapeutic targets. Methods We conducted an in vivo study using a pristane-induced lupus model in Ncf1 −/− (NOX2-deficient) mice and wild-type controls. We assessed LN severity and characterized renal immune infiltration using flow cytometry and single-cell RNA sequencing (scRNA-seq). We performed transcriptomic analysis to evaluate the function of NOX2-deficient neutrophils. Finally, we tested the therapeutic efficacy of an IL-1 receptor antagonist in ameliorating disease severity in the NOX2-deficient mice. Results NOX2 deficiency exacerbated LN severity compared to wild-type controls, demonstrated by increased serum anti-dsDNA antibody titers and worsened LN scores. Through scRNA-seq, we identified a distinct, activated neutrophil subset in Ncf1 −/− mice featuring a robust interferon signature and high Nlrp3 expression. Transcriptomic analysis confirmed the upregulation of core NLRP3 pathway within these cells. Crucially, treating NOX2-deficient mice with an IL-1 receptor antagonist reduced LN activity scores. Conclusion Our results showed that NOX2 deficiency was associated with the expansion of a highly inflammatory renal neutrophil subset that may contribute to aggravated renal inflammation. These findings suggest that NOX2 functions as a negative regulator of the NLRP3 inflammasome and IL-1β blockade represents a promising precision therapeutic strategy for patients with LN who exhibit impaired ROS production.

Background Viral infection plays an important role in driving activity of autoimmune diseases. Preceding SARS-CoV-2 infection has been observed to trigger disease flares in systemic lupus erythematosus (SLE). Besides cytomegalovirus, SARS-CoV-2 can induce pulmonary capillaritis to develop diffuse alveolar hemorrhage (DAH), a respiratory emergency of SLE. Objective Owing to lack of comprehensive studies for SARS-CoV-2-triggered DAH in SLE, we conducted a monocentric analysis with mechanistic investigation. Methods Hospitalized SLE patients were retrospectively analyzed for the DAH manifestation in the COVID-19 era since December, 2019, focusing on those with preceding SARS-CoV-2 infection. Peripheral blood (PB) and lung tissues samples and immune/alveolus cell lines were used for mechanistic research. Results Twenty-five DAH episodes were identified in 21 SLE patients (3% occurrence), all in disease flares with high activity scores. During the domestic omicron variant outbreak, 7 patients (33%) had preceding SARS-CoV-2 infection, 3 to 7 weeks earlier to the onset of DAH, while they had elevated IL-6, nitro-oxidative stress- and cell death-associated molecules levels in PB and lung tissues with enhanced apoptosis formation. IL-6-stimulated alveolus/immune cells exhibited a dose-dependent increase in nitro-oxidative stress- and cell death-associated molecules levels, up-regulated p38MAPK/NF-κB-p65 phosphorylation, raised ROS expression and enhanced apoptosis formation. These findings implicated cell death induced by IL-6-activated nitro-oxidative stress to generate circulating immune complexes with pulmonary deposition as the mechanism for DAH preceded by SARS-CoV-2 infection in SLE. Conclusion The DAH manifestation preceded by SARS-CoV-2 infection is observed in SLE with disease flares, requiring careful monitor and management of COVID-19 in such patients.

The systemic lupus erythematosus (SLE) pathogenesis involves accelerated cell death, releasing autoantigens with immune complexes deposition to induce activity manifesting as alveolar hemorrhage (AH), lupus nephritis (LN), etc. TLR2 is shown to sense SARS-CoV-2-envelope (E), while SLE patients have post-COVID-19 disease flare. Mechanistic evidence of binding between TLR2 and E to induce lupus activity remains to be elucidated. In SLE, there were up-regulated TLR2, iNOS and Nox1 expression in blood mononuclear cells (BMCs), pulmonary and renal tissues with elevated plasma IL-6, IL-8 and IFN-γ levels. In situ cell death were found in the AH lungs and LN kidneys. Molecular docking predicted interaction between TLR2 or TLR4 and E, while co-immunoprecipitation confirmed stronger binding in TLR2 than TLR4. E protein-stimulated BMCs had elevated IL-6, IL-8, IFN-γ, iNOS and Nox1 levels. Increased IL-6, IL-8, IFN-γ, iNOS, Nox1, ROS, p-NF-κB-p65, p-ERK and apoptosis expression in E protein-stimulated RAW264.7 cells were reduced by TLR2 inhibitor or sh-TLR2 transfection. HL-60 cells infected with LV-CoV-2-E, a SARS-CoV-2-E pseudovirus, had enhanced NETosis formation. E protein or LV-CoV-2-E intra-pulmonary infusion enhanced pristane-induced mouse AH by increasing cell death, while AH was reduced by sh-TLR2 co-delivery. LV-CoV-2-E infusion aggravated pristane-induced mouse LN. These data suggest SLE disease flare induced by SARS-CoV-2-E via binding TLR2 to enhance cell death formation. Translational and Interventional Immunology (TI)

Embryonic development and tumor genesis share numerous similarities, with OCT4 standing out as a pivotal transcription factor in embryonic development. Expression of OCT4 is associated with poor prognosis of lung adenocarcinoma. VEGF-correlated chemokine-1 (VCC-1), also known as C-X-C motif chemokine ligand 17 (CXCL17), has been suggested to play a role in promoting tumor angiogenesis and metastasis. In the present study, we show a positive correlation between OCT4 expression levels and tumor metastatic potential, where an increase in OCT4 expression parallels an upregulation of VCC-1 in lung cancer. This relationship was substantiated through DNA microarray analysis and further confirmed by tissue staining of clinical lung cancer samples, demonstrating a positive correlation between OCT4 and VCC-1 expression. In A549 and H1299 human lung cancer cells, modulations in OCT4 expression directly influenced VCC-1 levels, as evidenced by the reporter assay of the VCC-1 promoter, indicating the regulatory role of OCT4 in transactivating VCC-1 expression. Furthermore, enhanced VCC-1 expression in H1299 cells promoted transforming growth factor-β (TGF-β) secretion, contributing to lung cancer cell aggressiveness. Additionally, VCC-1 secretion by H1299 cells could attract THP-1 macrophages, further implicating its role in tumor progression. NOD/SCID mice inoculated with VCC-1-knockdown A549 lung cancer cells exhibited significantly smaller tumors than those inoculated with control cells. On the basis of these findings, we highlight the importance of the OCT4-VCC-1 axis in lung cancer progression. Our findings also provide therapeutic targets for lung cancer.

BACKGROUND:The largest dengue virus 2 (DENV2) outbreak occurred in Taiwan in 2015, resulting in many fatalities. We therefore aim to identify crucial genetic variations which determine the virulence of the 2015 Taiwan outbreak strains. METHODS:We compared the 2015 Taiwan DENV2 sequences to the pre-2015 sequences. Reverse genetics (rg) viruses with substitutions were produced and the viral growth kinetics were investigated. We treated A549 cells with interferon (IFN) to determine the interferon-stimulated genes (ISGs) expression and STAT1 phosphorylation in the rg viral infection and plasmid transfection systems. IFN and pro-inflammatory cytokines levels were measured upon DENV infection using ELISA. RESULTS:The rgNS1-K272R mutant showed faster replication in IFN-I producing cells compared to wildtype (WT) virus. Results revealed that NS1-K272R substitution contributed to higher soluble NS1 secretion and evade the antiviral response by suppressing the expression of ISGs and STAT1 phosphorylation compared to NS1-WT. Infection with rgNS1-K272R induced higher secretion of pro-inflammatory cytokines through the activation of canonical nuclear factor-kappa B (NF-κB) signaling pathway. CONCLUSIONS:Our results revealed that the DENV NS1 amino acid substitution affects the NS1 ability in immune evasion, which may contribute to the largest dengue outbreak in Taiwan since the 1990s.

Irritability, a state of excessive reactivity to negative emotional stimuli, is common in individuals with autism spectrum disorder (ASD). Although it has a significant negative impact of patients' disease severity and quality of life, the neural mechanisms underlying irritability in ASD remain largely unclear. We have previously demonstrated that male mice lacking the Coiled-coil and C2 domain containing 1a (Cc2d1a) in forebrain excitatory neurons recapitulate numerous ASD-like behavioral phenotypes, including impaired social behaviors and pronounced repetitive behaviors. Here, using the bottle-brush test (BBT) to trigger and evaluate aggressive and defensive responses, we show that Cc2d1a deletion increases irritability-like behavior in male but not female mice, which is correlated with reduced number of oxytocin (OXT)-expressing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Intranasal OXT administration or chemogenetic activation of OXT neurons in the PVN rescues irritability-like behavior in Cc2d1a conditional knockout (cKO) mice. Administration of a selective melanocortin receptor 4 agonist, RO27-3225, which potentiates endogenous OXT release, also alleviates irritability-like behavior in Cc2d1a cKO mice, an effect blocked by a specific OXT receptor antagonist, L-368,899. We additionally identify a projection connecting the posterior ventral segment of the medial amygdala (MeApv) and ventromedial nucleus of the ventromedial hypothalamus (VMHvl) for governing irritability-like behavior during the BBT. Chemogenetic suppression of the MeApv-VMHvl pathway alleviates irritability-like behavior in Cc2d1a cKO mice. Together, our study uncovers dysregulation of OXT system in irritability-like behavior in Cc2d1a cKO mice during the BBT and provide translatable insights into the development of OXT-based therapeutics for clinical interventions.

RIG-I-like receptors (RLRs) are cytosolic RNA sensors critical for antiviral immunity. RLR activation is regulated by polyubiquitination and oligomerization following RNA binding. Yet, little is known about how RLRs exploit subcellular organelles to facilitate their posttranslational modifications and activation. Endosomal adaptor TAPE regulates the endosomal TLR and cytosolic RLR pathways. The potential interplay between RIG-I signaling and endosomes has been explored. Here, we report that endosomes act as platforms for facilitating RIG-I polyubiquitination and complex formation. RIG-I was translocated onto endosomes to form signaling complexes upon activation. Ablation of endosomes impaired RIG-I signaling to type I IFN activation. TAPE mediates the interaction and polyubiquitination of RIG-I and TRIM25. TAPE-deficient myeloid cells were defective in type I IFN activation upon RNA ligand and virus challenges. Myeloid TAPE deficiency increased the susceptibility to RNA virus infection in vivo. Our work reveals endosomes as signaling platforms for RIG-I activation and antiviral immunity.

SH3BGRL3 expression level in uroepithelial cell lines, xenografts and alternation of EMT markers by transient transfection of SH3BGRL3

Autism spectrum disorder (ASD) represents a heterogeneous group of neurodevelopmental disorders characterized by deficits in social communication, social interaction, and the presence of restricted repetitive behaviors. The cause of ASD involves complex interactions between genetic and environmental factors. Haploinsufficiency of the Coiled-coil and C2 domain containing 1A (Cc2d1a) gene is causally linked to ASD, and obesity has been associated with worse outcomes for ASD. High-fat diet (HFD) feeding leads to the development of obesity and metabolic dysfunction; however, the effect of HFD on pre-existing autistic-like phenotypes remains to be clarified. Here, we report that male Cc2d1a conditional knockout (cKO) mice fed with HFD, from weaning onwards and throughout the experimental period, show a marked aggravation in autistic-like phenotypes, manifested in increased restricted repetitive behaviors and impaired performance in the preference for social novelty, but not in sociability and cognitive impairments assessed using the object location memory, novel object recognition, and Morris water maze tests. HFD feeding also results in increased numbers of reactive microglia and astrocytes, and exacerbates reductions in dendritic complexity and spine density of hippocampal CA1 pyramidal neurons. Furthermore, we demonstrate that chronic treatment with minocycline, a semisynthetic tetracycline-derived antibiotic, rescues the observed behavioral and morphological deficits in Cc2d1a cKO mice fed with HFD. Collectively, these findings highlight an aggravating role of HFD in pre-existing autistic-like phenotypes and suggest that minocycline treatment can alleviate abnormal neuronal morphology and behavioral symptoms associated with ASD resulted from the interplay between genetic and environmental risk factors.

Background Influenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the β-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice. Methods We used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells. Results Galectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells. Conclusion We demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza.

The imbalance of mucosal immunity in the lower gastrointestinal tract can lead to chronic inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis. IBD is a chronic inflammatory disorder that causes small and/or large intestines ulceration. According to previous studies, recombinant interleukin (IL)-10 protein and genetically modified bacteria secreting IL-10 ameliorate dextran sulfate sodium (DSS)-induced colitis in mice. IL-19 is a transcriptional activator of IL-10 and can alter the balance of T helper 1 (Th)1/Th2 cells in favor of Th2. In this study, we aimed to investigate whether the expression of the murine IL-19 gene carried by Salmonella choleraesuis (S. choleraesuis) could ameliorate murine IBD. Our results showed that the attenuated S. choleraesuis could carry and express the IL-19 gene-containing plasmid for IBD gene therapy by reducing the mortality and clinical signs in DSS-induced acute colitis mice as compared to the untreated ones. We also found that IL-10 expression was induced in IL-19-treated colitis mice and prevented inflammatory infiltrates and proinflammatory cytokine expression in these mice. We suggest that S. choleraesuis encoding IL-19 provides a new strategy for treating IBD in the future.

Protein identification of urine from patients with BPH.

Supplementary Figure legend, Supplementary materials and methods

Univariate and stepwise multivariate regression analyses for cumulative incidences of progression in patients with bladder urothelial tumors treated by transurethral resection without intravesical instillation.

Co-localization of SH3BGRL3 with EGFR