TRIM21 drives stress-induced neuroinflammation and behavioral deficits via gasdermin D- dependent IL-1β release in a rat model of repeated social defeat

TRIM21 drives stress-induced neuroinflammation and behavioral deficits via gasdermin D- dependent IL-1β release in a rat model of repeated social defeat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TRIM21 drives stress-induced neuroinflammation and behavioral deficits via gasdermin D- dependent IL-1β release in a rat model of repeated social defeat Soni Tiwari, Zaidan Mohammed, Santi Ranjan Atta, Aryan Tiwari, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8260120/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The upstream mechanism that transduces psychosocial stress into the release of the pivotal pro-inflammatory cytokine IL-1β has remained a fundamental gap in understanding neuropsychiatric disorders. Here, we identify tripartite motif-containing protein 21 (TRIM21) as the critical trigger. In a rat model of repeated social defeat, TRIM21 was upregulated in the hippocampus, brain microvessels, and peripheral blood mononuclear cells (PBMCs), correlating with anxiety-like behavior and memory deficits, with females exhibiting a more pronounced pathophysiology. Mechanistically, TRIM21 directly binds to and promotes the cleavage of gasdermin D, facilitating the formation of membrane pores for IL-1β release. This pathway was active in PBMCs, where TRIM21 drove sub-lytic pore formation and IL-1β release, linking peripheral inflammation to behavioral deficits. Critically, in vivo TRIM21 knockdown abrogated this pathway systemically, reducing IL-1β release and NF-κB signaling, which in turn rescued blood-brain barrier integrity, restored synaptic density, and normalized behavior. Using an in vitro blood-brain barrier model, we pinpointed this effect to a TRIM21-IL-1β-p65 axis. We therefore define TRIM21 as the critical node in a stress-responsive circuit, bridging peripheral immune activation to GSDMD-dependent neuroinflammation and blood-brain barrier disruption, thereby providing a mechanistic framework that redefines the pathophysiology of stress-related neuropsychiatric disorders and reveals new avenues for their treatment. Stress anxiety inflammasomes TRIM21 Gasdermin D Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The escalating global burden of stress-related neuropsychiatric disorders represents a paramount challenge to modern medicine, with anxiety and depression constituting a leading cause of disability worldwide [ 1 ]. This crisis is compounded by the well-documented inadequacy of monoamine-centric pharmacotherapies, which fail to achieve remission in a substantial proportion of patients, highlighting a fundamental gap in our understanding of disease etiology [ 2 ]. This therapeutic impasse has catalysed a paradigm shift toward alternative pathophysiological models, with the neuroimmune interface emerging as a critical frontier. It is now established that psychosocial stress propagates its deleterious effects by instigating a maladaptive neuroimmune response, wherein neuroinflammation and blood-brain barrier (BBB) dysfunction arise as central, interconnected pathological hubs [ 3 – 5 ]. This feedforward cascade drives a coordinated assault on CNS homeostasis, characterized by peripheral immune cell infiltration, microglial activation, and a disintegration of vascular tight junctions, which collectively disrupt synaptic plasticity, compromise neurotrophic signaling, and ultimately manifest as behavioral pathology [ 4 , 6 , 7 ]. A critical insight from recent research is that stress-induced inflammation and BBB deterioration are not sequential events but are mechanistically coupled in a self-perpetuating vicious cycle [ 8 ]. The breach of the neurovascular unit is not merely a passive leakage but an active driver of pathology, facilitating the unregulated entry of pro-inflammatory cytokines and immune cells into the brain parenchyma. For instance, seminal studies demonstrate that social defeat stress promotes the recruitment of monocytes from the spleen and bone marrow [ 9 , 10 ]. This process is contingent upon the stress-induced upregulation of adhesion molecules (e.g., ICAM-1, VCAM-1) and chemokines on brain endothelial cells [ 11 , 12 ]. This facilitated leukocyte trafficking not only amplifies the central inflammatory milieu but also directly contributes to the manifestation of anxiety-like behavior. The resulting convergence of peripheral and central immune signals creates a self-amplifying inflammatory loop, establishing and sustaining a persistent pathological state that is refractory to conventional treatments. Orchestrating this complex neuroimmune crosstalk is the potent pleiotropic cytokine, interleukin-1β (IL-1β), which sits at the nexus of stress and pathology. Elevated levels of IL-1β in both the central nervous system and periphery are a reproducible hallmark of Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD) [ 13 , 14 ]. The clinical relevance of this pathway is underscored by findings that peripheral blood mononuclear cells (PBMCs) from affected patients spontaneously produce IL-1β, while in animal models, psychological stress activates IL-1β-producing monocytes and microglia, which subsequently localize to the neurovasculature to promote anxiogenesis [ 15 , 16 ]. Furthermore, IL-1β acts as a direct disruptor of the neurovascular unit, downregulating critical tight junction proteins like claudin-5 and ZO-1 while simultaneously inducing adhesion molecules that facilitate further leukocyte-endothelial adherence, thereby accelerating the cycle of BBB breakdown [ 17 ]. The release of mature, bioactive IL-1β is a tightly regulated molecular event, requiring a two-step process of priming and activation. The second signal involves inflammasome-mediated activation of caspase-1, which cleaves the inactive pro-IL-1β precursor into its active form—a process our group previously demonstrated is potentiated by stress-induced BTK-NLRP3 signaling [ 18 ]. A pivotal breakthrough revealed that caspase-1 also cleaves the protein gasdermin D (GSDMD), and the resulting N-terminal fragments oligomerize to form plasma membrane pores that serve as the principal conduit for the export of mature IL-1β [ 19 , 20 ]. While the stress-IL-1β axis is well-characterized, the fundamental upstream sensor that transduces a psychological stimulus into this specific proteolytic cascade has remained one of the most critical, unresolved questions in the field [ 15 ]. The identification of this trigger is essential for understanding the etiology of these disorders and for developing targeted interventions. Among the potential upstream regulators, the tripartite motif (TRIM) family of E3 ubiquitin ligases represent compelling candidates, given their established roles as nodal regulators of innate immune signaling and inflammatory outcomes [ 21 ]. TRIM21, in particular, has well-defined functions in autoimmunity and intracellular antibody sensing [ 22 , 23 ]. Critically, it has recently been identified as a direct regulator of GSDMD-mediated pyroptosis in macrophages, where it promotes GSDMD oligomerization [ 24 ]. However, this finding exists in isolation, and its potential significance for the brain under psychological stress is entirely unexplored. Specifically, it remains unknown whether TRIM21 regulates GSDMD in this context, and most importantly, whether a TRIM21-GSDMD axis is the missing molecular link that directly couples psychosocial stress to IL-1β-driven neuroinflammation, BBB failure, and the ensuing behavioral deficits. Although GSDMD itself has been recently implicated in stress pathophysiology [ 25 – 27 ], the upstream mechanisms governing its activation in the brain remain a fundamental black box. Here, we bridge this critical knowledge gap by demonstrating that TRIM21 functions as the essential upstream trigger of stress-induced pathology. Employing a repeated social defeat (RSD) paradigm in rats, combined with in vivo knockdown, cell-type-specific analyses, and sophisticated in vitro BBB modelling, we systematically dissect this pathway. We identify TRIM21 as the key regulator that physically and functionally bridges the psychological stress response to GSDMD pore formation, IL-1β-driven neuroinflammation, and neurovascular dysfunction. We thus define TRIM21 as the critical missing trigger that transduces psychosocial stress into GSDMD–IL-1β–mediated pathology, thereby providing a new mechanistic framework for understanding and treating stress-related neuropsychiatric disorders. Methods Animals Male and female Sprague Dawley rats (two months old) were procured from the National Institute of Biologicals (Noida, India) and the All India Institute of Medical Sciences (New Delhi, India). Upon arrival, animals were acclimatized for 20 days prior to any experimentation. At the onset of the study, rats were two months of age. They were group-housed (maximum of three per cage) in standard ventilated cages with corn cob bedding, maintained in a climate-controlled vivarium under a 12-hour light/dark cycle (lights on 07:00–19:00), with ambient temperature at 21 ± 2°C, relative humidity at 55 ± 10%, and a controlled light intensity of 20–22 lux. Standard rodent diet and filtered water were provided ad libitum. All animal procedures strictly adhered to the 3Rs principles (Replacement, Reduction, Refinement), and efforts were made to utilize in vitro approaches to complement and inform the in vivo work. In vitro and in vivo knockdown of Trim21 In Vitro knockdown : A pool of four siRNA sequences targeting Trim21 (Rat Gene ID: 308901; Eurofins Genomics, Germany) and a corresponding non-targeting scrambled siRNA pool (control) were first validated for knockdown efficiency in rat peripheral blood mononuclear cells (PBMCs). Rat PBMCs or rat brain microvascular endothelial cells were transfected with 300 pmol of the siRNA pool using the Amaxa Nucleofector system (Lonza, Basel, Switzerland), following the manufacturer's protocol and established methods [ 28 , 29 ]. Knockdown efficiency was assessed by measuring TRIM21 protein levels 96 hours post-transfection. The sequences for all siRNA constructs are listed in the Supplementary Materials. In Vivo knockdown For in vivo studies, to enable non-invasive delivery of siRNA across the BBB to target central expression of TRIM21, an intranasal delivery route was employed to administer the siRNA pool to the brain of two-month-old rats, as previously described [ 30 ]. Briefly, the pool of four Trim21 siRNAs or the scrambled control was complexed with the in vivo -jetPEI cationic polymer transfection reagent (Sartorius, Germany). The siRNA- in vivo -jetPEI complex, prepared in a 10% glucose solution, was administered dropwise (15 µg siRNA per nostril; total 30 µg/rat/day) into each nostril using a Hamilton syringe. To ensure sustained and efficient knockdown, this intranasal administration was performed once daily for seven consecutive days. Repeated social defeat stress The repeated social defeat (RSD) paradigm was employed to induce psychosocial stress in a same-strain intruder-aggressor paradigm, as previously validated [ 31 – 33 ]. To minimize physical injury, we used SD rats for both resident aggressors and intruder test subjects, a strategy shown to reduce the severity of attacks compared to more aggressive strains [ 34 , 35 ]. Resident aggressors (5-month-old male SD rats) were pre-selected for consistent aggressive behavior, i.e. attacks and victories in screening sessions. For stress induction, 2-month-old test rats (intruders) were introduced into the aggressor's home cage for 10 minutes, allowing for physical confrontation characterized by pursuits, sideways threats, and bites. To maintain psychosocial stress while preventing injury, intruders were then housed within a protective wire mesh enclosure inside the aggressor's cage for an additional 20 minutes. This 30-minute procedure was repeated daily for 7 consecutive days, with intruders facing a novel aggressor each day to prevent habituation. Only intruders displaying consistent submissive postures were included in the study. Control animals were handled but not subjected to defeat stress. Pharmacological interventions and behavioral testing During the 7-day repeated social defeat paradigm, rats concurrently received daily intranasal administration of Trim21 siRNA or intraperitoneal injections of disulfiram (50 mg/kg). Behavioral assessments commenced 72 hours after the final stress session to evaluate anxiety-like behavior, social interaction, and working memory. All tests were conducted during the dark (active) phase of the light cycle (19:00–23:30) to align with the rodents' natural behavioral state. Apparatuses were thoroughly cleaned with 70% ethanol between trials to remove olfactory cues. Open field test (OFT) Anxiety-like behavior was assessed in an open-air black polycarbonate arena (80 × 80 × 60 cm) as previously described [ 36 ]. Individual rats were placed in a corner and allowed to explore freely for 5 minutes. The time spent in the central zone (40 × 40 cm) was quantified, with less time indicating higher anxiety-like behavior. Elevated plus maze test (EPM) Anxiety-like behavior was further assessed using an elevated plus maze, consisting of two open arms and two enclosed arms (each 60 × 15 cm) elevated 100 cm above the floor. Rats were placed in the central platform and allowed to explore the maze for 5 minutes under 100 lux illumination. Anxiety levels were quantified as the percentage of time spent in the open arms, with less time indicating higher anxiety [ 37 ]. Light-dark test (LDT) The innate conflict between rodent exploratory drive and aversion to brightly lit areas was assessed using a two-chamber apparatus [ 38 ]. The box featured identical compartments (25 × 25 × 45 cm), one dark (opaque) and one illuminated (transparent), connected by a small doorway (10 × 10 cm). Rats were allowed to explore freely for 10 minutes, and anxiety-like behavior was quantified as the ratio of time spent in the dark versus light chamber. Three-chamber social interaction test Social behavior was assessed using a three-chamber polycarbonate apparatus. Following a 10-minute habituation, test rats could interact for 10 minutes with a familiar conspecific (Intruder 1) in one chamber and a novel conspecific (Intruder 2) in the opposite chamber. A social preference index was calculated as the ratio of time spent investigating the novel versus the familiar rat, with investigation defined as nose-point proximity within ~ 1 cm. Novel object recognition test (NOR) Working memory was assessed using a novel object recognition paradigm [ 39 ]. After habituation, rats were exposed to two identical objects for 10 minutes (familiarization). Following a 20-minute retention interval, one object was replaced with a novel one. Rats were given 5 minutes to explore, and working memory was quantified as the percentage of time spent investigating the novel object. Y-maze test Spatial working memory was assessed using a Y-maze as described earlier [ 40 ]. During a 15-minute acquisition trial, one arm was blocked. After a 30-minute inter-trial interval, rats were allowed to explore all three arms for 15 minutes. Memory was quantified by the number of entries into the novel arm versus total arm entries, with reduced novel arm exploration indicating stress-induced memory impairment. In vivo blood-brain barrier permeability assay BBB integrity was assessed using two tracer molecules with distinct molecular weights based on an earlier protocol [ 41 ]. Control and RSD-stressed female rats were administered sodium fluorescein (376 Da) and Evans blue (which binds serum albumin to form a ~ 68 kDa complex) intraperitoneally. After 30 minutes, rats were transcardially perfused with PBS to remove intravascular dye. For sodium fluorescein quantification, a low-mass tracer of BBB integrity, brain homogenate supernatants were analyzed fluorometrically against a standard curve. For the Evans Blue-albumin complex, a high-molecular-weight tracer indicating severe leakage, dye was extracted from homogenates with trichloroacetic acid and quantified spectrophotometrically at 610 nm. Tissue collection On day 14 post-stress, rats were anesthetized (ketamine/xylazine, 80/10 mg/kg). Cerebrospinal fluid (150 µl) was collected from the cisterna magna. Blood was obtained via cardiac puncture and centrifuged to isolate plasma. Following transcardial perfusion with ice-cold PBS, brains were rapidly extracted. The hippocampus and amygdala were micro-dissected on ice and snap-frozen for subsequent analysis. Golgi-Cox staining and dendritic spine analysis Hippocampal dendritic spine density and morphology in the CA3 region were analyzed using Golgi-Cox staining as described earlier [ 42 ]. In brief, brains were impregnated for 40 days, sectioned at 100 µm, and processed for color development. Apical dendrites were imaged at 100x magnification. Spine density was quantified per 10 µm of dendrite using ImageJ by an investigator blinded to experimental groups. PBMC isolation and culture PBMCs from experimental rats were isolated from heparinized blood by density gradient centrifugation using OptiPrep. Cells (≥ 99% pure) were cultured in DMEM/10% FBS and primed with LPS (100 ng/ml, Sigma-Aldrich) and nigericin (10 µM, Sigma-Aldrich). Where indicated, cells were pre-treated with Trim21 siRNA or GSDMD inhibitor disulfiram (100 nM). Supernatants and cell lysates were collected for analysis. In Vitro blood-brain barrier modelling Rat brain microvascular endothelial cells (RBECs) were isolated from SD rats brain microvessels by collagenase type 2 digestion and Percoll density gradient centrifugation, as adapted from established protocol [ 43 ]. To model stress-induced neuroinflammation, RBECs were cultured to confluency on Transwell inserts. The luminal side was then exposed to PBMCs (2x10⁵) and 2% sera, collected from either control or repeated social defeat (RSD) rats, mimicking the pro-inflammatory peripheral milieu. After 72 hours of co-culture, RBECs and media were collected for analysis. For functional BBB assessment, a co-culture model was employed where RBECs on Transwell inserts were cultured above a layer of primary rat astrocytes [ 44 ]. After 72-hour exposure to control or RSD PBMCs/sera, barrier integrity was quantified by measuring the apical-to-basolateral flux of 70 kDa FITC-dextran [ 45 ]. Please see ‘Supplementary Materials’ for the detailed protocol. Cell viability and membrane permeabilization assays PBMC viability was assessed using a WST-8 assay, where metabolic reduction of the tetrazolium salt to formazan was quantified by absorbance at 460 nm. Plasma membrane integrity was evaluated using the impermeant nucleic acid stain SYTOX Green (2.5 µM, Sigma-Aldrich). Dye influx, indicating pore formation, was measured by an increase in fluorescence (485/528 nm). Immunoblot analysis Protein lysates from brain tissues, PBMCs, and RBECs were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Total protein concentration was determined by BCA assay. Equal amounts of protein were resolved by SDS-PAGE on 4–12% Bis-Tris gels and transferred to PVDF membranes. After blocking with 5% BSA, membranes were probed overnight at 4°C with primary antibodies against targets of interest, followed by incubation with appropriate HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence. A complete list of antibodies and their catalogue identifiers is provided in the Supplementary Materials. Quantitative real-time PCR Total RNA was isolated from tissues and cells using a RNeasy kit (Qiagen) with on-column DNase digestion. RNA integrity was verified by spectrophotometry (A260/A280 and A260/A230 > 1.7). cDNA was synthesized from 1 µg RNA using SuperScript III reverse transcriptase and oligo(dT) primers. Quantitative PCR was performed using SYBR Green qPCR master mix on an Applied Biosystems instrument. Gene expression was normalized to GAPDH and calculated via the ΔΔCT method. Primer sequences are listed in the Supplementary Materials. Structural prediction and interface analysis To generate testable hypotheses for potential molecular interactions, we used AlphaFold 3 to predict the structures of rat GSDMD-TRIM21 and p65-TRIM21 complexes [ 46 ]. From the five generated models, one representative model was selected for each complex. It is important to note that these models had low confidence scores (GSDMD-TRIM21, model M2: ipTM = 0.13, pTM = 0.43; p65-TRIM21, model M0: ipTM = 0.17, pTM = 0.34), where scores below 0.5 are generally considered low-confidence. These in silico predictions, though not confirmatory, provided a rationale for experimentally testing these interactions via co-immunoprecipitation. The predicted structures were visualized and analyzed in UCSF ChimeraX [ 47 ]. Putative intermolecular interfaces were characterized by identifying residue pairs with atomic distances < 3.5 Å, which were visualized and labelled. Domains involved in the predicted interactions were highlighted for documentation. Co-immunoprecipitation Protein-protein interactions were analyzed by co-immunoprecipitation. Hippocampal lysates from RSD rats were prepared and pre-cleared by centrifugation. Pre-cleared lysates were incubated overnight at 4°C with a TRIM21-specific antibody or with a species-matched non-specific IgG (control). Immune complexes were captured using Protein G-Sepharose beads, eluted, and subsequently analyzed by immunoblotting for TRIM21, GSDMD, and NF-κB p65. Enzyme-linked immunosorbent assays (ELISA) Plasma corticosterone (Arbor Assays, K014-H1), IL-1β in brain homogenates/plasma/PBMC lysates (R&D Systems, RLB00), and MCP-1 in RBEC conditioned media (R&D Systems, DY3144-05) were quantified using commercial ELISA kits per manufacturer protocols. Caspase-1 activity assay Caspase-1 enzymatic activity in tissue and PBMC lysates was determined by a colorimetric assay (Merck Millipore) as per manufacturer’s instructions. Lysates were incubated with the substrate Ac-YVAD-pNA for 2 hours at 37°C. Cleavage of the p-nitroaniline (p-NA) moiety was quantified by measuring absorbance at 405 nm. Statistical analysis Data were analyzed using JAMOVI (v2.7.6) with factorial ANOVA. Significant interaction effects were prioritized, with effect sizes reported as partial eta-squared (ηp²). Where no significant interaction was found, main effects are reported. All post-hoc comparisons for significant effects were conducted using Bonferroni-corrected t-tests, with t-statistics and p-values reported. Results RSD induces a sexually dimorphic anxiety phenotype and upregulation of TRIM21 and inflammasome signaling We established a repetitive social defeat model in SD rats, validating its anxiogenic effect using the elevated plus maze and open field test (Fig. 1 A). RSD significantly reduced time spent in the EPM open arms and OFT center (Fig. 1 B, C). A two-way ANOVA of EPM data showed a significant main effect of stress (F(1, 40) = 126.85, p < 0.001) and a stress × sex interaction (F(1, 40) = 11.13, p = 0.002). Post-hoc analysis revealed that RSD provoked a more severe anxiety-like response in females, which spent significantly less time in the EPM open arms than stressed males (mean difference = − 7.02, SE = 1.88, t(40) = − 3.73, p = 0.004). This finding was corroborated in the OFT, where stressed females spent less time in the center than males (mean difference = − 5.02, SE = 1.18, t(40) = − 4.26, p < 0.001). Corticosterone levels were also significantly more elevated in RSD females versus males (Supplementary Fig. 1A). Molecular analysis revealed a parallel sexual dimorphism. A two-way ANOVA of TRIM21 protein levels showed significant main effects of stress (F(1, 28) = 294.0, p < 0.001, ηp² = 0.913) and sex (F(1, 28) = 11.3, p = 0.002, ηp² = 0.288), and a significant stress × sex interaction (F(1, 28) = 15.0, p < 0.001, ηp² = 0.394). Post-hoc tests confirmed RSD increased TRIM21 in both sexes (all p < 0.001), with levels higher in stressed females than males (mean difference = 0.87, SE = 0.17, t(28) = 5.12, p < 0.001; Fig. 1 D, E). Post-hoc tests confirmed that RSD females exhibited higher levels of GSDMD-N (p < 0.001), NLRP3 (p < 0.001), and caspase-1 (p < 0.001) compared to RSD males (Fig. 1 F, G; Supplementary Fig. 1B). Furthermore, qRT-PCR confirmed greater Trim21 mRNA upregulation in RSD females versus males in both amygdala (mean difference = 0.733, SE = 0.135, t(40) = 5.44, p < 0.001) and hippocampus (mean difference = 0.740, SE = 0.128, t(40) = 5.77, p < 0.001; Fig. 1 H). Notably, elevated Trim21 mRNA was significantly correlated with increased anxiety-like behavior (Fig. 1 I, J), demonstrating negative associations with time spent in the EPM open arms (males: r = -0.656, p = 0.028; females: r = -0.672, p = 0.017) and OFT center (males: r = -0.629, p = 0.038; females: r = -0.644, p = 0.024). TRIM21 knockdown attenuates RSD-induced GSDMD cleavage and synaptic deficits To investigate the causal role of TRIM21 in RSD pathogenesis, we knocked down Trim21 in rats using siRNA for one week (Fig. 2 A), which was confirmed by immunoblot analysis (Fig. 2 B, C). Critically, RSD-exposed rats with TRIM21 knockdown exhibited significantly lower levels of cleaved GSDMD compared to scrambled siRNA controls (mean difference = 1.60, SE = 0.24, t(28) = 6.74, p < 0.001; Fig. 2 D, E). This attenuation extended to downstream inflammatory markers. In the hippocampus of RSD rats, TRIM21 knockdown significantly reduced IL-1β protein levels (mean difference = 230.1, SE = 25.2, t(68) = 9.14, p < 0.001; Fig. 2 F), IL-1β levels in the CSF (mean difference = 4.98, SE = 0.36, t(68) = 13.86, p < 0.001; Fig. 2 G), and caspase-1 activity (mean difference = 1.18, SE = 0.14, t(68) = 8.23, p < 0.001; Fig. 2 H). Crucially, the functional consequences of RSD were also rescued by TRIM21 knockdown. Golgi staining demonstrated that the loss of dendritic spine density following RSD was significantly attenuated in TRIM21-knockdown rats (mean difference = 3.07, SE = 0.49, t(68) = 6.22, p < 0.001; Fig. 2 I, J). Furthermore, the RSD-induced phospho-p65 (NF-κB) levels in the hippocampus was also reduced following TRIM21 silencing (mean difference = 2.07, SE = 0.37, t(24) = 5.61, p < 0.001; Fig. 2 K, L). TRIM21 knockdown rescues RSD-Induced behavioral deficits We next assessed its effect on RSD-induced behavioral deficits. TRIM21 knockdown significantly attenuated anxiety-like behaviours in the RSD model. Compared to RSD rats treated with scrambled siRNA, TRIM21-knockdown rats spent more time in the open arms of the EPM (mean difference = 9.02, SE = 1.34, t(76) = 6.71, p < 0.001; Fig. 3 A), exhibited a lower D/L ratio in the LDT (mean difference = 1.71, SE = 0.20, t(76) = 8.69, p < 0.001; Fig. 3 B), and spent more time in the center of the OFT (mean difference = 8.86, SE = 1.46, t(76) = 6.08, p < 0.001; Fig. 3 C, D). TRIM21 knockdown produced a comprehensive rescue of RSD-induced behavioral deficits, restoring function in both cognitive and social domains. Silencing TRIM21 robustly improved working memory, as evidenced by significantly enhanced performance on two independent tests: the NOL (F(1, 76) = 104.20, p < 0.001, ηp² = 0.578; Post-hoc: RSD + knockdown vs. RSD + scrambled, p < 0.001) and the Y-maze (F(1, 76) = 30.90, p < 0.001, ηp² = 0.289; Post-hoc: RSD + knockdown vs. RSD + scrambled, p < 0.001) (Fig. 3 E, F). This cognitive recovery was accompanied by a significant improvement in social interaction, with knockdown animals spending significantly more time interacting with a novel conspecific (F(1, 76) = 34.73, p < 0.001, ηp² = 0.314; Post-hoc: p < 0.001; Fig. 3 G). Together, these behavioral data demonstrate that TRIM21 silencing produces a comprehensive rescue, normalizing anxiety-like behavior, working memory, and social interaction in RSD-exposed animals. TRIM21 knockdown attenuates RSD-induced BBB dysfunction We next investigated whether TRIM21 contributes to RSD-induced BBB disruption. Analysis of isolated hippocampal microvessels revealed that RSD significantly increased both TRIM21 (F(1, 30) = 104.67, p < 0.001, ηp² = 0.777) and cleaved GSDMD (F(1, 30) = 134.14, p < 0.001, ηp² = 0.817) protein levels, localizing this pathway to the cerebral vasculature (Fig. 4 A-C). To define the functional role of vascular TRIM21 in RSD, we performed targeted knockdown in female rats (Fig. 4 D, Supplementary Fig. 2A, B). TRIM21 silencing significantly protected BBB integrity, as shown by markedly reduced sodium fluorescein extravasation (mean difference = 4396.75, SE = 665, t(74) = 6.61, p < 0.001; Fig. 4 E, F) and Evans blue leakage (p < 0.001; Fig. 4 G) compared to RSD controls. The functional relevance of this TRIM21-GSDMD axis was confirmed using the GSDMD inhibitor disulfiram, which phenocopied the protective effect of TRIM21 knockdown by similarly reducing both sodium fluorescein and Evans blue leakage (both p < 0.001; Fig. 4 F, G). Mechanistically, both interventions significantly restored claudin-5 protein levels compared to RSD controls (post-hoc: both p < 0.001), with no statistical difference between their efficacy (Fig. 4 H). Collectively, these results define a novel pathway in which RSD-upregulated TRIM21 promotes GSDMD-mediated cleavage of tight junctions, directly compromising the BBB. This pathology is tractable, as demonstrated by its rescue through inhibition at either the level of TRIM21 or its effector, GSDMD. TRIM21 forms complexes with GSDMD and NF-κB p65 To elucidate the mechanism by which TRIM21 coordinates inflammasome and NF-κB signaling, we investigated its potential physical interactions with GSDMD and p65. We first employed AlphaFold 3 multimer for structural prediction, which generated low-confidence models (ipTM < 0.2) suggesting plausible complexes. These in silico data proposed a broad interaction interface between the TRIM21 PRY-SPRY domain and GSDMD, and a more localized binding mode with p65 (Fig. 5 A-D). While not confirmatory, these predictions provided a rationale to experimentally test for these interactions. We therefore performed co-immunoprecipitation from hippocampal lysates of RSD-stressed rats. Immunoprecipitation with a TRIM21-specific antibody, but not with a control IgG, successfully co-precipitated both cleaved GSDMD and the p65 subunit of NF-κB (Fig. 5 E), demonstrating a specific physical interaction. This result provides direct experimental evidence that TRIM21 physically interacts with both GSDMD and p65 in vivo , confirming the central role of TRIM21 as a molecular hub in the stress-induced inflammatory cascade. TRIM21 knockdown in PBMC attenuates RSD-induced GSDMD pore formation and efflux of IL-1β We first investigated the systemic role of TRIM21 by measuring plasma IL-1β in RSD-exposed rats. TRIM21 knockdown significantly attenuated the RSD-induced increase in plasma IL-1β in both females (mean difference = 14.53, t(79) = 7.71, p < 0.001) and males (mean difference = 8.28, t(79) = 4.40, p < 0.001) (Fig. 6 A), indicating a key role for TRIM21 in stress-induced systemic inflammation. Since peripheral immune cells are a primary source of plasma IL-1β, we analyzed PBMCs. Immunoblot and qPCR analyses revealed that RSD stress significantly induced TRIM21 and GSDMD expression in PBMCs, with females exhibiting markedly higher levels of both TRIM21 protein (mean difference = 1.134, t(28) = 5.40, p < 0.001; Fig. 6 B, C) and GSDMD protein (mean difference = 1.243, t(28) = 5.51, p < 0.001; Fig. 6 D), as well as Trim21 mRNA (mean difference = 0.814, t(40) = 5.05, p < 0.001; Fig. 6 E) than males. Critically, elevated Trim21 mRNA expression correlated strongly with increased anxiety-like behavior in the EPM test (males: r(9) = − 0.64, p = 0.034; females: r(10) = − 0.65, p = 0.022; Fig. 6 F), linking PBMC TRIM21 to a core behavioral outcome. To establish the mechanistic role of TRIM21 in peripheral inflammation, we performed in vitro knockdown in PBMCs from RSD rats (Fig. 6 G, H). TRIM21 silencing significantly suppressed the NLRP3 inflammasome pathway, reducing both caspase-1 activation (mean difference = − 2.92, t(66) = − 7.06, p < 0.001; Fig. 6 I) and IL-1β release (mean difference = -208.3, t(67) = 7.44, p < 0.001; Fig. 6 J). The NLRP3 inhibitor disulfiram phenocopied this effect (both p < 0.001), positioning TRIM21 upstream of inflammasome activation in immune cells. We then asked if TRIM21 facilitates IL-1β release via GSDMD pore formation. A SYTOX green uptake assay confirmed that PBMCs from RSD rats exhibited significantly increased plasma membrane permeability (t(25) = − 9.78, p < 0.001; Fig. 6 K). This pore formation was abolished by both TRIM21 knockdown and disulfiram treatment (both p < 0.001). The absence of cell death (Supplementary Fig. 3A) confirms that these were sub-lytic pores, sufficient for the efflux of IL-1β without resulting in pyroptosis. Our findings establish TRIM21 as a critical regulator of the RSD stress response, where it drives GSDMD-mediated, sub-lytic pore formation in PBMCs to facilitate IL-1β release, thereby linking peripheral immune activation to systemic inflammation and anxiety-like behavior. TRIM21 is a critical mediator of RSD-induced BBB dysfunction To investigate the role of TRIM21 in RSD-induced BBB impairment, we employed an in vitro BBB model where rat brain endothelial cell monolayers were exposed to PBMCs and sera from RSD rats (Fig. 7 A). This exposure robustly induced TRIM21 expression in RBECs [F(1, 24) = 342.9, p < 0.001, ηp² = .935], with females exhibiting significantly higher TRIM21 levels than males (mean difference = 1.41, t(24) = 5.60, p < 0.001; Fig. 7 B, C). qRT-PCR confirmed this induction, showing a significant upregulation of Trim21 mRNA (Supplementary Fig. 4A). This was accompanied by a significant female-predominant increase in NF-κB activation, as measured by p-p65 (mean difference = 1.25, t(24) = 3.61, p = 0.008; Fig. 7 D). Cell viability remained unaffected across all groups (Supplementary Fig. 4B), indicating that the effects were not due to cytotoxicity. Strikingly, TRIM21 knockdown in RBECs dramatically attenuated the pathogenic cascade triggered by RSD exposure. It significantly reduced the cleavage of GSDMD (mean difference = -2.43, t(24) = 7.48, p < 0.001; Fig. 7 E, F), indicating suppression of downstream signaling. Furthermore, TRIM21 depletion protected BBB integrity and suppressed the neurovascular inflammatory response. This was evidenced by the restoration of key endothelial proteins, including a decrease in the adhesion molecule ICAM-1 (mean difference = -2.15, t(24) = 5.38, p < 0.001; Fig. 7 G) and an increase in the tight junction protein ZO-1 (mean difference = 0.39, t(24) = -3.19, p = 0.024; Fig. 7 H). Concomitantly, TRIM21 knockdown attenuated endothelial activation, significantly reducing levels of the chemokine MCP-1 (mean difference = -179.3, t(24) = 3.56, p = 0.010; Fig. 7 I) and downregulating the expression of MMP9 and E-selectin (both p < 0.001; Supplementary Fig. 4C, D). Critically, in a functional BBB permeability assay, exposure to RSD components caused a profound increase in FITC-dextran leakage (mean difference = 58.30, t(56) = -11.65, p < 0.001; Fig. 7 J, K). TRIM21 knockdown effectively rescued this barrier impairment, significantly reducing dextran leakage (p < 0.001), thereby demonstrating that TRIM21 is a central driver of RSD-induced vascular hyperpermeability. Discussion This study identifies TRIM21 as a key molecular regulator that transduces psychosocial stress into a coordinated multi-system pathology, bridging peripheral inflammation, BBB dysfunction, and behavioral impairment. We delineate a previously unrecognized signaling axis—TRIM21-GSDMD—that operates as a critical amplifier of neuroinflammation, functioning both in parallel to and upstream of inflammasome pathways (Fig. 8 ). The discovery that an E3 ubiquitin ligase directly facilitates GSDMD-mediated IL-1β release provides a mechanistic explanation for how a psychological stimulus is converted into a sustained inflammatory response, positioning TRIM21 as a nodal point for therapeutic intervention. TRIM21 as a key inflammatory hub in the stressed brain Our data demonstrate that RSD induces a robust upregulation of TRIM21 across a distributed network, including emotion-regulating brain regions (hippocampus, amygdala), the cerebral vasculature, and peripheral blood mononuclear cells (PBMCs). This widespread induction suggests TRIM21 is a core component of the stress-responsive transcriptome. While the upstream signals driving Trim21 transcription following stress remain to be fully elucidated, its promoter contains binding sites for stress-responsive transcription factors like NF-κB and AP-1 [ 48 ], potentially creating a feed-forward loop that is initiated by the primary stress response and amplified by subsequent inflammation. Our most significant mechanistic insight is that TRIM21's role in facilitating GSDMD activation, previously defined in peripheral immunity, is a central driver of pathology in the stressed brain. While it was previously shown that TRIM21 binds the GSDMD C-terminal domain via its PRY-SPRY region to promote N-terminal oligomerization in macrophages during bacterial infection [ 24 ], its function in the brain under psychological stress was unknown. Our co-immunoprecipitation data demonstrates that this interaction occurs in vivo in the brain following RSD, a finding supported by our AlphaFold structural predictions. The multi-domain interaction we observed suggests a model where TRIM21 binding may not only promote oligomerization but also potentially displace the autoinhibitory C-terminal domain, lowering the activation threshold for GSDMD [ 49 , 50 ]. Furthermore, our finding that TRIM21 interacts with the Rel Homology Domain (RHD) of the p65 subunit of NF-κB positions it as a potent inflammatory amplifier. This interaction suggests TRIM21 is poised to influence NF-κB DNA-binding capacity and transcriptional activity [ 51 ]. This interaction could stabilize p65 or promote its ubiquitination in a manner that enhances its transcriptional activity, as suggested in other systems [ 52 ]. This creates a powerful, self-reinforcing inflammatory circuit: TRIM21-driven IL-1β release activates NF-κB, which in turn can further upregulate TRIM21, pro-IL-1β, and NLRP3, perpetuating a chronic inflammatory state characteristic of stress-related psychiatric disorders [ 5 , 25 ]. Sublytic pore formation: a mechanism for chronic neuroinflammation A pivotal finding is the demonstration that TRIM21 activation leads to sublytic GSDMD pore formation. The significant SYTOX Green uptake in the absence of cell death indicates a state of controlled membrane permeabilization. This sublytic signaling is emerging as a crucial mode of inflammatory signaling, allowing for the sustained release of IL-1β and other alarmins without committing the cell to pyroptotic death [ 53 , 54 ]. It provides a mechanistic basis for the chronic neuroinflammation observed in major depressive disorder and PTSD, where a continuous, low-grade release of inflammatory mediators from glial and endothelial cells could disrupt neural circuitry without causing overt cell loss [ 55 ]. Sexual dimorphism: a molecular basis for differential vulnerability The pronounced sexual dimorphism in the TRIM21 response is a finding with profound implications. The consistently higher upregulation of TRIM21, cleaved GSDMD, and IL-1β in female rats provides a plausible molecular substrate for the well-documented increased prevalence of anxiety and depressive disorders in women [ 56 , 57 ]. This dimorphism may be driven by interactions between the TRIM21 pathway and sex hormones. This aligns with foundational research demonstrating that susceptibility to chronic stress is associated with profoundly sex-specific transcriptomic signatures in key limbic brain regions [ 58 ]. Our data extend this principle by identifying a specific, inducible signaling node (the TRIM21-GSDMD axis) within the neuroinflammatory cascade that exhibits a female-predominant activation pattern. For instance, estradiol has been shown to potentiate NF-κB signaling in certain contexts [ 59 ], which could synergize with the TRIM21-p65 interaction we identified to create a more robust inflammatory response in females. From synapses to behavior: preserving circuit integrity The behavioral rescue achieved through in vivo TRIM21 knockdown underscores its central role in the pathophysiology of stress-induced behavioral deficits. The restoration of hippocampal dendritic spine density provides a structural correlate for the recovery of cognitive function [ 60 ]. Critically, these structural changes reflect the potential restoration of functional synaptic plasticity, the cellular basis of learning and memory, which is fundamentally governed by the dynamic regulation of AMPA receptor trafficking at the postsynaptic density [ 61 ]. The well-established ability of IL-1β to disrupt long-term potentiation and promote long-term depression and spine loss [ 62 ] strongly supports a model where TRIM21 knockdown preserves synaptic integrity and function primarily by dampening this key inflammatory mediator. This is consistent with foundational evidence demonstrating that stress-induced NF-κB activation within limbic brain circuits is a critical driver of synaptic remodeling and maladaptive behavior [ 63 ]. Therefore, by inhibiting the TRIM21-GSDMD-IL-1β axis, we likely restore the molecular environment necessary for normal synaptic plasticity, learning, and emotional regulation. TRIM21 as a mediator of BBB dysfunction Perhaps our most significant advance is in elucidating the active role of TRIM21 in breaching the BBB. We move beyond correlation to demonstrate causality, showing that TRIM21 within brain endothelial cells is both necessary and sufficient for stress-induced barrier breakdown. The discovery of elevated TRIM21 and cleaved GSDMD in isolated brain microvessels localizes this pathway directly to the BBB. Our in vitro BBB model mechanistically dissects this process: inflammatory signals from the periphery require endothelial TRIM21 to initiate a cascade of tight junction loss (ZO-1, claudin-5), adhesion molecule induction (ICAM-1, VCAM-1), chemokine production (MCP-1), and MMP9 activation. Consequently, TRIM21 emerges as a novel upstream regulator of a pathogenic cascade whose endpoint—BBB failure—is a recognized therapeutic target across neurological disorders [ 64 ]. The protection afforded by TRIM21 knockdown, which was comparable to direct GSDMD inhibition with disulfiram, underscores that GSDMD pore formation is a critical, and likely the predominant, downstream executor of TRIM21 signaling in endothelial dysfunction. The subsequent upregulation of ICAM-1 and MCP-1 creates a chemotactic gradient and adhesion platform, facilitating the firm adhesion of peripheral immune cells to the vessel wall, a critical step in their transmigration into the brain parenchyma, as observed in stress models [ 9 , 4 ]. This positions TRIM21 as the key mediator of a pathway that directly links psychological stress to the recruitment of peripheral immune cells into the brain. Implications and future directions In summary, we propose a unified model wherein psychosocial stress upregulates TRIM21, which then acts as a proteostatic hub, directly promoting GSDMD activation and NF-κB signaling to drive a self-amplifying inflammatory loop that disrupts the BBB, compromises synaptic integrity, and manifests as behavioral pathology. From a clinical standpoint, TRIM21 represents a uniquely promising target. Its upstream position means its inhibition could simultaneously dampen multiple inflammatory pathways (IL-1β release, NF-κB signaling, NLRP3 expression) and preserve BBB integrity, offering a more comprehensive strategy. Future work should focus on several key areas: First, identifying the precise upstream initiators of TRIM21 induction by stress, potentially involving glucocorticoid receptor signaling or catecholamine surges. Second, developing and testing specific pharmacological TRIM21 inhibitors in chronic stress models to validate its therapeutic potential. Third, the pronounced sex differences demand that these future strategies be evaluated and optimized with biological sex as a critical variable. Fourth, investigating cell-type-specific functions of TRIM21 (e.g., in microglia, neurons, endothelia) using conditional knockout models. Finally, longitudinal studies are needed to determine if TRIM21 inhibition can promote stress resilience or reverse established pathology. Conclusion Our findings fundamentally advance the understanding of neuroimmune mechanisms in stress-induced neuropsychological disorders by identifying TRIM21 as the critical missing link that translates psychological stress into a structured biological response. By orchestrating GSDMD-mediated release of IL-1β and downstream neurovascular dysfunction, the TRIM21 pathway integrates multiple pathological hallmarks of stress-related disorders. This work establishes a new mechanistic framework for stress-induced inflammation and provides a compelling impetus for developing therapeutics that intercept this stress-induced pathological cascade at its origin. Statements & Declarations Acknowledgments The authors extend their gratitude to the institutional core facilities that supported this work, specifically the Ashoka-ZEISS Core Imaging Facility at Ashoka University and the University Science Instrumentation Centre (USIC) at the University of Delhi. This research was made possible through funding from Ashoka University (S.G. and Z.M.), the Brain Aneurysm Foundation (I.S.), a DBT-Ramalingaswamy Fellowship (I.S.), and the Savrdh Foundation (S.T.). Funding This research was funded by Ashoka University (S.G. and Z.M.), the Brain Aneurysm Foundation (I.S.), a DBT-Ramalingaswamy Fellowship (I.S.), and the Savrdh Foundation (S.T.). Author contributions S.T., Z.M., E.Y., S.K., and S.R.A. conducted the animal experiments and behavioral testing. S.T. carried out the in vitro studies and histological staining. S.R.A. performed the in-silico interaction studies. A.T. conducted data quantification and formal statistical analysis. S.T., Z.M., S.R.A., A.T., A.Y., S.K., A.C., I.S., and S.G. were involved in writing – original draft preparation, review, and editing. I.S. and S.R.A. prepared the schematic figures. S.G. conceived the study, designed the methodology, and provided overall supervision. All authors have read and approved the final version of the manuscript. Data availability No large datasets were generated or analysed during the current study. All data supporting the findings of this study are available within the paper and its ‘Supplementary Material’. Additional data related to this study are available from the corresponding author upon reasonable request. 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16:35:04","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188869,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/964723c2117c173ce665938a.html"},{"id":98037406,"identity":"c9418c36-fb46-4da8-b73e-152b823c9a43","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepeated social defeat stress in SD rats induces sexually dimorphic anxiety-like behavior and upregulation of the TRIM21-inflammasome pathway. \u003c/strong\u003eExperimental timeline of the repeated social defeat (RSD) paradigm \u003cstrong\u003e(A)\u003c/strong\u003e. Anxiety-like behavior assessed by time spent in the open arms of the elevated plus maze (EPM) \u003cstrong\u003e(B)\u003c/strong\u003e and the center of the open field test (OFT) \u003cstrong\u003e(C) \u003c/strong\u003ein control and RSD-exposed male and female rats (n=10-12/group). Representative immunoblots \u003cstrong\u003e(D) \u003c/strong\u003eand quantification \u003cstrong\u003e(E)\u003c/strong\u003eof hippocampal TRIM21 protein levels, cleaved GSDMD (GSDMD-N) \u003cstrong\u003e(F) \u003c/strong\u003eand NLRP3 \u003cstrong\u003e(G)\u003c/strong\u003e (n=8/group). \u003cem\u003eTrim21\u003c/em\u003e mRNA expression in the amygdala and hippocampus measured by qRT-PCR\u003cstrong\u003e (H) \u003c/strong\u003e(n=10-12/group). Correlation between hippocampal \u003cem\u003eTrim21\u003c/em\u003e mRNA levels and time spent in the EPM open arms \u003cstrong\u003e(I) \u003c/strong\u003eand OFT center \u003cstrong\u003e(J)\u003c/strong\u003e for individual animals. Data are presented as mean with 95% CI; Factorial ANOVAs have been used for analysis; p-values are indicated for specific pairwise comparisons post-hoc. Correlation analyzed by Pearson's test and Pearson’s r and p values are reported.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/5f3afa45c642782af4c51dcf.png"},{"id":98037407,"identity":"bb3d67f6-abeb-4da4-810a-595eef021728","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":298124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e TRIM21 knockdown attenuates RSD-induced GSDMD cleavage, neuroinflammation, and synaptic deficits in SD rats. \u003c/strong\u003eExperimental timeline for \u003cem\u003ein vivo\u003c/em\u003eTRIM21 knockdown during RSD \u003cstrong\u003e(A)\u003c/strong\u003e. Validation of TRIM21 knockdown efficiency by representative immunoblot \u003cstrong\u003e(B) \u003c/strong\u003eand quantification \u003cstrong\u003e(C) \u003c/strong\u003ein the hippocampus (n=9/group). Representative immunoblot \u003cstrong\u003e(D) \u003c/strong\u003eand quantification \u003cstrong\u003e(E) \u003c/strong\u003eof hippocampal cleaved GSDMD (GSDMD-N) (n=8/group). Hippocampal IL-1β protein levels \u003cstrong\u003e(F)\u003c/strong\u003e, cerebrospinal fluid (CSF) IL-1β levels \u003cstrong\u003e(G)\u003c/strong\u003e, and hippocampal caspase-1 activity \u003cstrong\u003e(H)\u003c/strong\u003e(n=9-11/group). Quantification of dendritic spine density and representative images of Golgi-Cox stained hippocampal CA1 dendrites \u003cstrong\u003e(I)\u003c/strong\u003eand \u003cstrong\u003e(J)\u003c/strong\u003e (n=9-11/group). Scale bar = 5 µm. Representative immunoblot \u003cstrong\u003e(K) \u003c/strong\u003eand quantification \u003cstrong\u003e(L) \u003c/strong\u003eof hippocampal phospho-NF-κB p65 (p-p65) and total p65 (n=7/group Data are presented as mean with 95% CI; Data analyzed with Factorial ANOVAs; p-values are indicated for specific pairwise comparisons post-hoc.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/4688dcfd6624afb6d3112a20.png"},{"id":98037409,"identity":"6b52acbc-d88b-42d6-a3ef-a18a89b49926","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM21 knockdown rescues RSD-induced behavioral deficits in anxiety, working memory, and social interaction in SD rats. \u003c/strong\u003eAnxiety-like behavior assessed by time spent in the EPM open arms \u003cstrong\u003e(A)\u003c/strong\u003e, the dark/light (D/L) ratio in the light-dark test (LDT) \u003cstrong\u003e(B)\u003c/strong\u003e, time in the OFT center \u003cstrong\u003e(C)\u003c/strong\u003e, and representative OFT track plot \u003cstrong\u003e(D)\u003c/strong\u003e in RSD rats treated with scrambled (\u003cem\u003esc-siRNA\u003c/em\u003e) or \u003cem\u003eTrim21\u003c/em\u003e siRNA (\u003cem\u003esiTrim21\u003c/em\u003e, n=10-11/group). Working memory assessed by the percentage of time exploring the novel object in the Novel Object Location (NOL) test \u003cstrong\u003e(E) \u003c/strong\u003eand the number of novel arm entries in the Y-maze test \u003cstrong\u003e(F)\u003c/strong\u003e(n=17-22/group). Social interaction time with a novel conspecific in the three-chamber test (n=10-11/group). Data are presented as mean with 95% CI; Factorial ANOVAs have been used for analysis; p-values are indicated for specific pairwise comparisons post-hoc.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/2fded0b9fdc71227f0f15648.png"},{"id":98037413,"identity":"95640565-bbc1-4b43-b72a-63ccdd7cbd00","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":270121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM21 drives RSD-induced blood-brain barrier dysfunction via a GSDMD-dependent pathway in SD rats. \u003c/strong\u003eRepresentative immunoblots \u003cstrong\u003e(A) \u003c/strong\u003eand quantification of TRIM21 \u003cstrong\u003e(B) \u003c/strong\u003eand cleaved GSDMD \u003cstrong\u003e(C)\u003c/strong\u003e protein levels in isolated hippocampal microvessels from control and RSD rats (n=8/group). Experimental timeline for TRIM21 knockdown and disulfiram treatment in RSD female rats \u003cstrong\u003e(D)\u003c/strong\u003e. BBB permeability assessed by sodium fluorescein extravasation \u003cstrong\u003e(E, F) \u003c/strong\u003eand Evans Blue leakage \u003cstrong\u003e(G)\u003c/strong\u003e(n=9-12/group). \u003cstrong\u003e(H)\u003c/strong\u003e Quantification of claudin-5 mRNA levels by qRT-PCR in hippocampal microvessels (n=9-10/group). Data are presented as mean with 95% CI (n=10-12); Data analyzed with Factorial ANOVAs; p-values are indicated for specific pairwise comparisons posthoc.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/22e85f109ded4c17115fc4b1.png"},{"id":98037419,"identity":"f963767f-a26a-4f84-bbd5-6f0380ce609f","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":353021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM21 physically interacts with GSDMD and NF-κB p65. \u003c/strong\u003eZoomed in view of interactions at \u0026lt;3.5 Å of TRIM21 PRY SPRY domain (281-480, orange) and GSDMD N-terminal (1-276, cyan), linker region (278-287, cornflower blue), and C-terminal (288-488, pink) \u003cstrong\u003e(A)\u003c/strong\u003e. Bonds are shown in green dashed line and GSDMD cleavage site (Asp277) colored red. Interacting residues and numbers are shown with bonds and side chains. GSDMD-TRIM21 multimer structures are colored with pLDDT scores \u003cstrong\u003e(B)\u003c/strong\u003e. Zoomed in view of interactions at \u0026lt;3.5 Å of TRIM21 PRY SPRY domain (281-480, orange) and p65 Rel homology domain (19-308, blue), transactivation domain 1 (428-521, pink) \u003cstrong\u003e(C)\u003c/strong\u003e. Other regions are shown in light grey. p65-TRIM21 multimer structures are colored with pLDDT scores \u003cstrong\u003e(D)\u003c/strong\u003e. Co-immunoprecipitation (Co-IP) from hippocampal lysates of RSD-stressed rats \u003cstrong\u003e(E)\u003c/strong\u003e. Immunoprecipitation was performed with an anti-TRIM21 antibody or a control IgG, followed by immunoblotting for the indicated proteins. Input represents 5% of total lysate. Data are representative of five independent experiments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/cfd844124ff0d16d8e485435.png"},{"id":98426999,"identity":"4c4ae4c0-9766-4aa1-b527-b0b598e5182d","added_by":"auto","created_at":"2025-12-17 16:39:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM21 in PBMCs drives GSDMD-dependent sub-lytic pore formation and IL-1β release.\u003c/strong\u003e Plasma IL-1β levels in control and RSD rats with or without TRIM21 knockdown (n=10-12/group) \u003cstrong\u003e(A)\u003c/strong\u003e. Analysis of PBMCs from control and RSD rats: Representative immunoblots \u003cstrong\u003e(B) \u003c/strong\u003eand quantification of TRIM21 \u003cstrong\u003e(C)\u003c/strong\u003eand GSDMD\u003cstrong\u003e (D) \u003c/strong\u003eprotein levels (n=8/group). \u003cem\u003eTrim21\u003c/em\u003e mRNA expression by qRT-PCR (n=10-12/group) \u003cstrong\u003e(E)\u003c/strong\u003e. Correlation between PBMC \u003cem\u003eTrim21\u003c/em\u003e mRNA levels and time spent in the EPM open arms for individual animals\u003cstrong\u003e (F)\u003c/strong\u003e. Validation of \u003cem\u003ein vitro\u003c/em\u003e TRIM21 knockdown in PBMCs by representative immunoblot \u003cstrong\u003e(G)\u003c/strong\u003e and quantification \u003cstrong\u003e(H)\u003c/strong\u003e. Caspase-1 activity \u003cstrong\u003e(I)\u003c/strong\u003e in PBMCs and IL-1β release in culture media \u003cstrong\u003e(J)\u003c/strong\u003e of PBMCs isolated from RSD rats and treated \u003cem\u003ein vitro\u003c/em\u003e with scrambled siRNA, \u003cem\u003eTrim21\u003c/em\u003esiRNA, or disulfiram (n=8-12/group). Plasma membrane permeability measured by SYTOX Green uptake in the same treatment groups (n=7-8/group) \u003cstrong\u003e(K)\u003c/strong\u003e. Data are presented as mean with 95% CI (n=10-12); Data analyzed with Factorial ANOVAs; p-values are indicated for specific pairwise comparisons posthoc.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/46b4660f9b82c7b47237c627.png"},{"id":98426944,"identity":"3b28b1bc-7507-43e9-be89-d71ece4b304b","added_by":"auto","created_at":"2025-12-17 16:39:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":234380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelial TRIM21 is a critical mediator of RSD-induced blood-brain barrier dysfunction \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eSchematic of the \u003cem\u003ein vitro\u003c/em\u003e BBB model: rat brain endothelial cells (RBECs) were exposed to PBMCs and serum from RSD rats \u003cstrong\u003e(A)\u003c/strong\u003e. Representative immunoblot\u003cstrong\u003e (B) \u003c/strong\u003eand quantification of TRIM21 protein \u003cstrong\u003e(C)\u003c/strong\u003ephospho-NF-κB p65 (p-p65) \u003cstrong\u003e(D)\u003c/strong\u003e in RBECs (n=7/group). Representative immunoblot \u003cstrong\u003e(E)\u003c/strong\u003e and quantification \u003cstrong\u003e(F) \u003c/strong\u003eof cleaved GSDMD in RBECs (n=7/group). Expression of endothelial activation markers: ICAM-1 protein \u003cstrong\u003e(G)\u003c/strong\u003e, ZO-1 protein \u003cstrong\u003e(H)\u003c/strong\u003e by immunoblot analysis, and secreted MCP-1 chemokine in culture media by ELISA \u003cstrong\u003e(I)\u003c/strong\u003e (n=7/group). Schematic of functional BBB integrity assessment by FITC-dextran flux across the BBB model: rat brain endothelial cells were exposed to PBMCs and serum from RSD rats \u003cstrong\u003e(J)\u003c/strong\u003e. Leakage of FITC-dextran across RBEC barrier following treatment with \u003cem\u003ein vitro\u003c/em\u003e with scrambled siRNA, \u003cem\u003eTrim21\u003c/em\u003e siRNA, or disulfiram (n=8/group) \u003cstrong\u003e(K)\u003c/strong\u003e. Data are presented as mean with 95% CI; Data analyzed with Factorial ANOVAs; p-values are indicated for specific pairwise comparisons post-hoc.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/2471f5f434f3420eebdb0082.png"},{"id":98427196,"identity":"77de89c5-2f95-460d-b234-5e388146f7ac","added_by":"auto","created_at":"2025-12-17 16:39:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":184911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model: TRIM21 as a key node transducing psychosocial stress into neurovascular and behavioral pathology. \u003c/strong\u003eSchematic summarizing the proposed mechanism. Psychosocial stress (RSD) upregulates TRIM21 in the brain (microvasculature, hippocampus) and peripheral immune cells (PBMCs). TRIM21 acts as a molecular hub, physically interacting with and promoting the cleavage/activation of GSDMD, leading to sub-lytic pore formation and IL-1β release. TRIM21 also interacts with NF-κB p65, potentially amplifying the inflammatory mediators such as E-selectin, MCP-1, ICAM-1 and MMP9. This cascade culminates in blood-brain barrier disruption, neuroinflammation, synaptic deficits, and the manifestation of anxiety-like behavior, working memory impairments, and social withdrawal. Targeting TRIM21 or its effector GSDMD (e.g., with disulfiram) rescues this pathology.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/50e648d063b3cccee82fa6b1.png"},{"id":98444523,"identity":"3f2da075-cd03-4cd5-b3c7-b2efe10dea4e","added_by":"auto","created_at":"2025-12-17 17:16:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3392446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/48438fb7-9596-4ea0-a248-c17ee18ccb57.pdf"},{"id":98037410,"identity":"cf495667-f5a3-4807-8066-ec35b188848a","added_by":"auto","created_at":"2025-12-12 06:35:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":169622,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialSG.docx","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/0ac73d3fd392024ebcc496d3.docx"},{"id":98427806,"identity":"1d776b8d-0d3f-47a1-ae05-949c7ddab753","added_by":"auto","created_at":"2025-12-17 16:41:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4356120,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileOriginalBlots.docx","url":"https://assets-eu.researchsquare.com/files/rs-8260120/v1/d9b86aac4937984e386e60fe.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TRIM21 drives stress-induced neuroinflammation and behavioral deficits via gasdermin D- dependent IL-1β release in a rat model of repeated social defeat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe escalating global burden of stress-related neuropsychiatric disorders represents a paramount challenge to modern medicine, with anxiety and depression constituting a leading cause of disability worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This crisis is compounded by the well-documented inadequacy of monoamine-centric pharmacotherapies, which fail to achieve remission in a substantial proportion of patients, highlighting a fundamental gap in our understanding of disease etiology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This therapeutic impasse has catalysed a paradigm shift toward alternative pathophysiological models, with the neuroimmune interface emerging as a critical frontier. It is now established that psychosocial stress propagates its deleterious effects by instigating a maladaptive neuroimmune response, wherein neuroinflammation and blood-brain barrier (BBB) dysfunction arise as central, interconnected pathological hubs [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This feedforward cascade drives a coordinated assault on CNS homeostasis, characterized by peripheral immune cell infiltration, microglial activation, and a disintegration of vascular tight junctions, which collectively disrupt synaptic plasticity, compromise neurotrophic signaling, and ultimately manifest as behavioral pathology [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA critical insight from recent research is that stress-induced inflammation and BBB deterioration are not sequential events but are mechanistically coupled in a self-perpetuating vicious cycle [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The breach of the neurovascular unit is not merely a passive leakage but an active driver of pathology, facilitating the unregulated entry of pro-inflammatory cytokines and immune cells into the brain parenchyma. For instance, seminal studies demonstrate that social defeat stress promotes the recruitment of monocytes from the spleen and bone marrow [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This process is contingent upon the stress-induced upregulation of adhesion molecules (e.g., ICAM-1, VCAM-1) and chemokines on brain endothelial cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This facilitated leukocyte trafficking not only amplifies the central inflammatory milieu but also directly contributes to the manifestation of anxiety-like behavior. The resulting convergence of peripheral and central immune signals creates a self-amplifying inflammatory loop, establishing and sustaining a persistent pathological state that is refractory to conventional treatments.\u003c/p\u003e \u003cp\u003eOrchestrating this complex neuroimmune crosstalk is the potent pleiotropic cytokine, interleukin-1β (IL-1β), which sits at the nexus of stress and pathology. Elevated levels of IL-1β in both the central nervous system and periphery are a reproducible hallmark of Major Depressive Disorder (MDD) and Post-Traumatic Stress Disorder (PTSD) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The clinical relevance of this pathway is underscored by findings that peripheral blood mononuclear cells (PBMCs) from affected patients spontaneously produce IL-1β, while in animal models, psychological stress activates IL-1β-producing monocytes and microglia, which subsequently localize to the neurovasculature to promote anxiogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, IL-1β acts as a direct disruptor of the neurovascular unit, downregulating critical tight junction proteins like claudin-5 and ZO-1 while simultaneously inducing adhesion molecules that facilitate further leukocyte-endothelial adherence, thereby accelerating the cycle of BBB breakdown [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe release of mature, bioactive IL-1β is a tightly regulated molecular event, requiring a two-step process of priming and activation. The second signal involves inflammasome-mediated activation of caspase-1, which cleaves the inactive pro-IL-1β precursor into its active form\u0026mdash;a process our group previously demonstrated is potentiated by stress-induced BTK-NLRP3 signaling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A pivotal breakthrough revealed that caspase-1 also cleaves the protein gasdermin D (GSDMD), and the resulting N-terminal fragments oligomerize to form plasma membrane pores that serve as the principal conduit for the export of mature IL-1β [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While the stress-IL-1β axis is well-characterized, the fundamental upstream sensor that transduces a psychological stimulus into this specific proteolytic cascade has remained one of the most critical, unresolved questions in the field [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The identification of this trigger is essential for understanding the etiology of these disorders and for developing targeted interventions.\u003c/p\u003e \u003cp\u003eAmong the potential upstream regulators, the tripartite motif (TRIM) family of E3 ubiquitin ligases represent compelling candidates, given their established roles as nodal regulators of innate immune signaling and inflammatory outcomes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. TRIM21, in particular, has well-defined functions in autoimmunity and intracellular antibody sensing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Critically, it has recently been identified as a direct regulator of GSDMD-mediated pyroptosis in macrophages, where it promotes GSDMD oligomerization [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, this finding exists in isolation, and its potential significance for the brain under psychological stress is entirely unexplored. Specifically, it remains unknown whether TRIM21 regulates GSDMD in this context, and most importantly, whether a TRIM21-GSDMD axis is the missing molecular link that directly couples psychosocial stress to IL-1β-driven neuroinflammation, BBB failure, and the ensuing behavioral deficits. Although GSDMD itself has been recently implicated in stress pathophysiology [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the upstream mechanisms governing its activation in the brain remain a fundamental black box.\u003c/p\u003e \u003cp\u003eHere, we bridge this critical knowledge gap by demonstrating that TRIM21 functions as the essential upstream trigger of stress-induced pathology. Employing a repeated social defeat (RSD) paradigm in rats, combined with \u003cem\u003ein vivo\u003c/em\u003e knockdown, cell-type-specific analyses, and sophisticated \u003cem\u003ein vitro\u003c/em\u003e BBB modelling, we systematically dissect this pathway. We identify TRIM21 as the key regulator that physically and functionally bridges the psychological stress response to GSDMD pore formation, IL-1β-driven neuroinflammation, and neurovascular dysfunction. We thus define TRIM21 as the critical missing trigger that transduces psychosocial stress into GSDMD\u0026ndash;IL-1β\u0026ndash;mediated pathology, thereby providing a new mechanistic framework for understanding and treating stress-related neuropsychiatric disorders.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale and female Sprague Dawley rats (two months old) were procured from the National Institute of Biologicals (Noida, India) and the All India Institute of Medical Sciences (New Delhi, India). Upon arrival, animals were acclimatized for 20 days prior to any experimentation. At the onset of the study, rats were two months of age. They were group-housed (maximum of three per cage) in standard ventilated cages with corn cob bedding, maintained in a climate-controlled vivarium under a 12-hour light/dark cycle (lights on 07:00\u0026ndash;19:00), with ambient temperature at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity at 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, and a controlled light intensity of 20\u0026ndash;22 lux. Standard rodent diet and filtered water were provided ad libitum. All animal procedures strictly adhered to the 3Rs principles (Replacement, Reduction, Refinement), and efforts were made to utilize in vitro approaches to complement and inform the \u003cem\u003ein vivo\u003c/em\u003e work.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eknockdown of\u003c/b\u003e \u003cb\u003eTrim21\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn Vitro knockdown\u003c/em\u003e: A pool of four siRNA sequences targeting \u003cem\u003eTrim21\u003c/em\u003e (Rat Gene ID: 308901; Eurofins Genomics, Germany) and a corresponding non-targeting scrambled siRNA pool (control) were first validated for knockdown efficiency in rat peripheral blood mononuclear cells (PBMCs). Rat PBMCs or rat brain microvascular endothelial cells were transfected with 300 pmol of the siRNA pool using the Amaxa Nucleofector system (Lonza, Basel, Switzerland), following the manufacturer's protocol and established methods [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Knockdown efficiency was assessed by measuring TRIM21 protein levels 96 hours post-transfection. The sequences for all siRNA constructs are listed in the Supplementary Materials.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIn Vivo knockdown\u003c/strong\u003e \u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e studies, to enable non-invasive delivery of siRNA across the BBB to target central expression of TRIM21, an intranasal delivery route was employed to administer the siRNA pool to the brain of two-month-old rats, as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, the pool of four \u003cem\u003eTrim21\u003c/em\u003e siRNAs or the scrambled control was complexed with the \u003cem\u003ein vivo\u003c/em\u003e-jetPEI cationic polymer transfection reagent (Sartorius, Germany). The siRNA-\u003cem\u003ein vivo\u003c/em\u003e-jetPEI complex, prepared in a 10% glucose solution, was administered dropwise (15 \u0026micro;g siRNA per nostril; total 30 \u0026micro;g/rat/day) into each nostril using a Hamilton syringe. To ensure sustained and efficient knockdown, this intranasal administration was performed once daily for seven consecutive days.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRepeated social defeat stress\u003c/h3\u003e\n\u003cp\u003eThe repeated social defeat (RSD) paradigm was employed to induce psychosocial stress in a same-strain intruder-aggressor paradigm, as previously validated [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To minimize physical injury, we used SD rats for both resident aggressors and intruder test subjects, a strategy shown to reduce the severity of attacks compared to more aggressive strains [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Resident aggressors (5-month-old male SD rats) were pre-selected for consistent aggressive behavior, i.e. attacks and victories in screening sessions. For stress induction, 2-month-old test rats (intruders) were introduced into the aggressor's home cage for 10 minutes, allowing for physical confrontation characterized by pursuits, sideways threats, and bites. To maintain psychosocial stress while preventing injury, intruders were then housed within a protective wire mesh enclosure inside the aggressor's cage for an additional 20 minutes. This 30-minute procedure was repeated daily for 7 consecutive days, with intruders facing a novel aggressor each day to prevent habituation. Only intruders displaying consistent submissive postures were included in the study. Control animals were handled but not subjected to defeat stress.\u003c/p\u003e\n\u003ch3\u003ePharmacological interventions and behavioral testing\u003c/h3\u003e\n\u003cp\u003eDuring the 7-day repeated social defeat paradigm, rats concurrently received daily intranasal administration of \u003cem\u003eTrim21\u003c/em\u003e siRNA or intraperitoneal injections of disulfiram (50 mg/kg). Behavioral assessments commenced 72 hours after the final stress session to evaluate anxiety-like behavior, social interaction, and working memory. All tests were conducted during the dark (active) phase of the light cycle (19:00\u0026ndash;23:30) to align with the rodents' natural behavioral state. Apparatuses were thoroughly cleaned with 70% ethanol between trials to remove olfactory cues.\u003c/p\u003e\n\u003ch3\u003eOpen field test (OFT)\u003c/h3\u003e\n\u003cp\u003eAnxiety-like behavior was assessed in an open-air black polycarbonate arena (80 \u0026times; 80 \u0026times; 60 cm) as previously described [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Individual rats were placed in a corner and allowed to explore freely for 5 minutes. The time spent in the central zone (40 \u0026times; 40 cm) was quantified, with less time indicating higher anxiety-like behavior.\u003c/p\u003e\n\u003ch3\u003eElevated plus maze test (EPM)\u003c/h3\u003e\n\u003cp\u003eAnxiety-like behavior was further assessed using an elevated plus maze, consisting of two open arms and two enclosed arms (each 60 \u0026times; 15 cm) elevated 100 cm above the floor. Rats were placed in the central platform and allowed to explore the maze for 5 minutes under 100 lux illumination. Anxiety levels were quantified as the percentage of time spent in the open arms, with less time indicating higher anxiety [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLight-dark test (LDT)\u003c/h2\u003e \u003cp\u003eThe innate conflict between rodent exploratory drive and aversion to brightly lit areas was assessed using a two-chamber apparatus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The box featured identical compartments (25 \u0026times; 25 \u0026times; 45 cm), one dark (opaque) and one illuminated (transparent), connected by a small doorway (10 \u0026times; 10 cm). Rats were allowed to explore freely for 10 minutes, and anxiety-like behavior was quantified as the ratio of time spent in the dark versus light chamber.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThree-chamber social interaction test\u003c/h3\u003e\n\u003cp\u003eSocial behavior was assessed using a three-chamber polycarbonate apparatus. Following a 10-minute habituation, test rats could interact for 10 minutes with a familiar conspecific (Intruder 1) in one chamber and a novel conspecific (Intruder 2) in the opposite chamber. A social preference index was calculated as the ratio of time spent investigating the novel versus the familiar rat, with investigation defined as nose-point proximity within ~\u0026thinsp;1 cm.\u003c/p\u003e\n\u003ch3\u003eNovel object recognition test (NOR)\u003c/h3\u003e\n\u003cp\u003eWorking memory was assessed using a novel object recognition paradigm [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. After habituation, rats were exposed to two identical objects for 10 minutes (familiarization). Following a 20-minute retention interval, one object was replaced with a novel one. Rats were given 5 minutes to explore, and working memory was quantified as the percentage of time spent investigating the novel object.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eY-maze test\u003c/h2\u003e \u003cp\u003eSpatial working memory was assessed using a Y-maze as described earlier [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. During a 15-minute acquisition trial, one arm was blocked. After a 30-minute inter-trial interval, rats were allowed to explore all three arms for 15 minutes. Memory was quantified by the number of entries into the novel arm versus total arm entries, with reduced novel arm exploration indicating stress-induced memory impairment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eblood-brain barrier permeability assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBBB integrity was assessed using two tracer molecules with distinct molecular weights based on an earlier protocol [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Control and RSD-stressed female rats were administered sodium fluorescein (376 Da) and Evans blue (which binds serum albumin to form a\u0026thinsp;~\u0026thinsp;68 kDa complex) intraperitoneally. After 30 minutes, rats were transcardially perfused with PBS to remove intravascular dye. For sodium fluorescein quantification, a low-mass tracer of BBB integrity, brain homogenate supernatants were analyzed fluorometrically against a standard curve. For the Evans Blue-albumin complex, a high-molecular-weight tracer indicating severe leakage, dye was extracted from homogenates with trichloroacetic acid and quantified spectrophotometrically at 610 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection\u003c/h2\u003e \u003cp\u003eOn day 14 post-stress, rats were anesthetized (ketamine/xylazine, 80/10 mg/kg). Cerebrospinal fluid (150 \u0026micro;l) was collected from the cisterna magna. Blood was obtained via cardiac puncture and centrifuged to isolate plasma. Following transcardial perfusion with ice-cold PBS, brains were rapidly extracted. The hippocampus and amygdala were micro-dissected on ice and snap-frozen for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGolgi-Cox staining and dendritic spine analysis\u003c/h2\u003e \u003cp\u003eHippocampal dendritic spine density and morphology in the CA3 region were analyzed using Golgi-Cox staining as described earlier [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In brief, brains were impregnated for 40 days, sectioned at 100 \u0026micro;m, and processed for color development. Apical dendrites were imaged at 100x magnification. Spine density was quantified per 10 \u0026micro;m of dendrite using ImageJ by an investigator blinded to experimental groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePBMC isolation and culture\u003c/h2\u003e \u003cp\u003ePBMCs from experimental rats were isolated from heparinized blood by density gradient centrifugation using OptiPrep. Cells (\u0026ge;\u0026thinsp;99% pure) were cultured in DMEM/10% FBS and primed with LPS (100 ng/ml, Sigma-Aldrich) and nigericin (10 \u0026micro;M, Sigma-Aldrich). Where indicated, cells were pre-treated with \u003cem\u003eTrim21\u003c/em\u003e siRNA or GSDMD inhibitor disulfiram (100 nM). Supernatants and cell lysates were collected for analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eblood-brain barrier modelling\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRat brain microvascular endothelial cells (RBECs) were isolated from SD rats brain microvessels by collagenase type 2 digestion and Percoll density gradient centrifugation, as adapted from established protocol [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To model stress-induced neuroinflammation, RBECs were cultured to confluency on Transwell inserts. The luminal side was then exposed to PBMCs (2x10⁵) and 2% sera, collected from either control or repeated social defeat (RSD) rats, mimicking the pro-inflammatory peripheral milieu. After 72 hours of co-culture, RBECs and media were collected for analysis.\u003c/p\u003e \u003cp\u003eFor functional BBB assessment, a co-culture model was employed where RBECs on Transwell inserts were cultured above a layer of primary rat astrocytes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. After 72-hour exposure to control or RSD PBMCs/sera, barrier integrity was quantified by measuring the apical-to-basolateral flux of 70 kDa FITC-dextran [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Please see \u0026lsquo;Supplementary Materials\u0026rsquo; for the detailed protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and membrane permeabilization assays\u003c/h2\u003e \u003cp\u003ePBMC viability was assessed using a WST-8 assay, where metabolic reduction of the tetrazolium salt to formazan was quantified by absorbance at 460 nm. Plasma membrane integrity was evaluated using the impermeant nucleic acid stain SYTOX Green (2.5 \u0026micro;M, Sigma-Aldrich). Dye influx, indicating pore formation, was measured by an increase in fluorescence (485/528 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eProtein lysates from brain tissues, PBMCs, and RBECs were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors. Total protein concentration was determined by BCA assay. Equal amounts of protein were resolved by SDS-PAGE on 4\u0026ndash;12% Bis-Tris gels and transferred to PVDF membranes. After blocking with 5% BSA, membranes were probed overnight at 4\u0026deg;C with primary antibodies against targets of interest, followed by incubation with appropriate HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence. A complete list of antibodies and their catalogue identifiers is provided in the Supplementary Materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from tissues and cells using a RNeasy kit (Qiagen) with on-column DNase digestion. RNA integrity was verified by spectrophotometry (A260/A280 and A260/A230\u0026thinsp;\u0026gt;\u0026thinsp;1.7). cDNA was synthesized from 1 \u0026micro;g RNA using SuperScript III reverse transcriptase and oligo(dT) primers. Quantitative PCR was performed using SYBR Green qPCR master mix on an Applied Biosystems instrument. Gene expression was normalized to GAPDH and calculated via the ΔΔCT method. Primer sequences are listed in the Supplementary Materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStructural prediction and interface analysis\u003c/h2\u003e \u003cp\u003eTo generate testable hypotheses for potential molecular interactions, we used AlphaFold 3 to predict the structures of rat GSDMD-TRIM21 and p65-TRIM21 complexes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. From the five generated models, one representative model was selected for each complex. It is important to note that these models had low confidence scores (GSDMD-TRIM21, model M2: ipTM\u0026thinsp;=\u0026thinsp;0.13, pTM\u0026thinsp;=\u0026thinsp;0.43; p65-TRIM21, model M0: ipTM\u0026thinsp;=\u0026thinsp;0.17, pTM\u0026thinsp;=\u0026thinsp;0.34), where scores below 0.5 are generally considered low-confidence. These \u003cem\u003ein silico\u003c/em\u003e predictions, though not confirmatory, provided a rationale for experimentally testing these interactions via co-immunoprecipitation. The predicted structures were visualized and analyzed in UCSF ChimeraX [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Putative intermolecular interfaces were characterized by identifying residue pairs with atomic distances\u0026thinsp;\u0026lt;\u0026thinsp;3.5 \u0026Aring;, which were visualized and labelled. Domains involved in the predicted interactions were highlighted for documentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eProtein-protein interactions were analyzed by co-immunoprecipitation. Hippocampal lysates from RSD rats were prepared and pre-cleared by centrifugation. Pre-cleared lysates were incubated overnight at 4\u0026deg;C with a TRIM21-specific antibody or with a species-matched non-specific IgG (control). Immune complexes were captured using Protein G-Sepharose beads, eluted, and subsequently analyzed by immunoblotting for TRIM21, GSDMD, and NF-κB p65.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assays (ELISA)\u003c/h2\u003e \u003cp\u003ePlasma corticosterone (Arbor Assays, K014-H1), IL-1β in brain homogenates/plasma/PBMC lysates (R\u0026amp;D Systems, RLB00), and MCP-1 in RBEC conditioned media (R\u0026amp;D Systems, DY3144-05) were quantified using commercial ELISA kits per manufacturer protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-1 activity assay\u003c/h2\u003e \u003cp\u003eCaspase-1 enzymatic activity in tissue and PBMC lysates was determined by a colorimetric assay (Merck Millipore) as per manufacturer\u0026rsquo;s instructions. Lysates were incubated with the substrate Ac-YVAD-pNA for 2 hours at 37\u0026deg;C. Cleavage of the p-nitroaniline (p-NA) moiety was quantified by measuring absorbance at 405 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using JAMOVI (v2.7.6) with factorial ANOVA. Significant interaction effects were prioritized, with effect sizes reported as partial eta-squared (ηp\u0026sup2;). Where no significant interaction was found, main effects are reported. All post-hoc comparisons for significant effects were conducted using Bonferroni-corrected t-tests, with t-statistics and p-values reported.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eRSD induces a sexually dimorphic anxiety phenotype and upregulation of TRIM21 and inflammasome signaling\u003c/h2\u003e \u003cp\u003eWe established a repetitive social defeat model in SD rats, validating its anxiogenic effect using the elevated plus maze and open field test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). RSD significantly reduced time spent in the EPM open arms and OFT center (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). A two-way ANOVA of EPM data showed a significant main effect of stress (F(1, 40)\u0026thinsp;=\u0026thinsp;126.85, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a stress \u0026times; sex interaction (F(1, 40)\u0026thinsp;=\u0026thinsp;11.13, p\u0026thinsp;=\u0026thinsp;0.002). Post-hoc analysis revealed that RSD provoked a more severe anxiety-like response in females, which spent significantly less time in the EPM open arms than stressed males (mean difference = \u0026minus;\u0026thinsp;7.02, SE\u0026thinsp;=\u0026thinsp;1.88, t(40) = \u0026minus;\u0026thinsp;3.73, p\u0026thinsp;=\u0026thinsp;0.004). This finding was corroborated in the OFT, where stressed females spent less time in the center than males (mean difference\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;5.02, SE\u0026thinsp;=\u0026thinsp;1.18, t(40)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.26, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Corticosterone levels were also significantly more elevated in RSD females versus males (Supplementary Fig.\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eMolecular analysis revealed a parallel sexual dimorphism. A two-way ANOVA of TRIM21 protein levels showed significant main effects of stress (F(1, 28)\u0026thinsp;=\u0026thinsp;294.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.913) and sex (F(1, 28)\u0026thinsp;=\u0026thinsp;11.3, p\u0026thinsp;=\u0026thinsp;0.002, ηp\u0026sup2; = 0.288), and a significant stress \u0026times; sex interaction (F(1, 28)\u0026thinsp;=\u0026thinsp;15.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.394). Post-hoc tests confirmed RSD increased TRIM21 in both sexes (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with levels higher in stressed females than males (mean difference\u0026thinsp;=\u0026thinsp;0.87, SE\u0026thinsp;=\u0026thinsp;0.17, t(28)\u0026thinsp;=\u0026thinsp;5.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Post-hoc tests confirmed that RSD females exhibited higher levels of GSDMD-N (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), NLRP3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and caspase-1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to RSD males (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G; Supplementary Fig.\u0026nbsp;1B). Furthermore, qRT-PCR confirmed greater \u003cem\u003eTrim21\u003c/em\u003e mRNA upregulation in RSD females versus males in both amygdala (mean difference\u0026thinsp;=\u0026thinsp;0.733, SE\u0026thinsp;=\u0026thinsp;0.135, t(40)\u0026thinsp;=\u0026thinsp;5.44, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and hippocampus (mean difference\u0026thinsp;=\u0026thinsp;0.740, SE\u0026thinsp;=\u0026thinsp;0.128, t(40)\u0026thinsp;=\u0026thinsp;5.77, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Notably, elevated \u003cem\u003eTrim21\u003c/em\u003e mRNA was significantly correlated with increased anxiety-like behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J), demonstrating negative associations with time spent in the EPM open arms (males: r = -0.656, p\u0026thinsp;=\u0026thinsp;0.028; females: r = -0.672, p\u0026thinsp;=\u0026thinsp;0.017) and OFT center (males: r = -0.629, p\u0026thinsp;=\u0026thinsp;0.038; females: r = -0.644, p\u0026thinsp;=\u0026thinsp;0.024).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eTRIM21 knockdown attenuates RSD-induced GSDMD cleavage and synaptic deficits\u003c/h2\u003e \u003cp\u003eTo investigate the causal role of TRIM21 in RSD pathogenesis, we knocked down \u003cem\u003eTrim21\u003c/em\u003e in rats using siRNA for one week (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which was confirmed by immunoblot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Critically, RSD-exposed rats with TRIM21 knockdown exhibited significantly lower levels of cleaved GSDMD compared to scrambled siRNA controls (mean difference\u0026thinsp;=\u0026thinsp;1.60, SE\u0026thinsp;=\u0026thinsp;0.24, t(28)\u0026thinsp;=\u0026thinsp;6.74, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003eThis attenuation extended to downstream inflammatory markers. In the hippocampus of RSD rats, TRIM21 knockdown significantly reduced IL-1β protein levels (mean difference\u0026thinsp;=\u0026thinsp;230.1, SE\u0026thinsp;=\u0026thinsp;25.2, t(68)\u0026thinsp;=\u0026thinsp;9.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), IL-1β levels in the CSF (mean difference\u0026thinsp;=\u0026thinsp;4.98, SE\u0026thinsp;=\u0026thinsp;0.36, t(68)\u0026thinsp;=\u0026thinsp;13.86, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), and caspase-1 activity (mean difference\u0026thinsp;=\u0026thinsp;1.18, SE\u0026thinsp;=\u0026thinsp;0.14, t(68)\u0026thinsp;=\u0026thinsp;8.23, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Crucially, the functional consequences of RSD were also rescued by TRIM21 knockdown. Golgi staining demonstrated that the loss of dendritic spine density following RSD was significantly attenuated in TRIM21-knockdown rats (mean difference\u0026thinsp;=\u0026thinsp;3.07, SE\u0026thinsp;=\u0026thinsp;0.49, t(68)\u0026thinsp;=\u0026thinsp;6.22, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Furthermore, the RSD-induced phospho-p65 (NF-κB) levels in the hippocampus was also reduced following TRIM21 silencing (mean difference\u0026thinsp;=\u0026thinsp;2.07, SE\u0026thinsp;=\u0026thinsp;0.37, t(24)\u0026thinsp;=\u0026thinsp;5.61, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eTRIM21 knockdown rescues RSD-Induced behavioral deficits\u003c/h2\u003e \u003cp\u003eWe next assessed its effect on RSD-induced behavioral deficits. TRIM21 knockdown significantly attenuated anxiety-like behaviours in the RSD model. Compared to RSD rats treated with scrambled siRNA, TRIM21-knockdown rats spent more time in the open arms of the EPM (mean difference\u0026thinsp;=\u0026thinsp;9.02, SE\u0026thinsp;=\u0026thinsp;1.34, t(76)\u0026thinsp;=\u0026thinsp;6.71, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), exhibited a lower\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eD/L ratio in the LDT (mean difference\u0026thinsp;=\u0026thinsp;1.71, SE\u0026thinsp;=\u0026thinsp;0.20, t(76)\u0026thinsp;=\u0026thinsp;8.69, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and spent more time in the center of the OFT (mean difference\u0026thinsp;=\u0026thinsp;8.86, SE\u0026thinsp;=\u0026thinsp;1.46, t(76)\u0026thinsp;=\u0026thinsp;6.08, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTRIM21 knockdown produced a comprehensive rescue of RSD-induced behavioral deficits, restoring function in both cognitive and social domains. Silencing TRIM21 robustly improved working memory, as evidenced by significantly enhanced performance on two independent tests: the NOL (F(1, 76)\u0026thinsp;=\u0026thinsp;104.20, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.578; Post-hoc: RSD\u0026thinsp;+\u0026thinsp;knockdown vs. RSD\u0026thinsp;+\u0026thinsp;scrambled, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the Y-maze (F(1, 76)\u0026thinsp;=\u0026thinsp;30.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.289; Post-hoc: RSD\u0026thinsp;+\u0026thinsp;knockdown vs. RSD\u0026thinsp;+\u0026thinsp;scrambled, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). This cognitive recovery was accompanied by a significant improvement in social interaction, with knockdown animals spending significantly more time interacting with a novel conspecific (F(1, 76)\u0026thinsp;=\u0026thinsp;34.73, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.314; Post-hoc: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Together, these behavioral data demonstrate that TRIM21 silencing produces a comprehensive rescue, normalizing anxiety-like behavior, working memory, and social interaction in RSD-exposed animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eTRIM21 knockdown attenuates RSD-induced BBB dysfunction\u003c/h2\u003e \u003cp\u003eWe next investigated whether TRIM21 contributes to RSD-induced BBB disruption. Analysis of isolated hippocampal microvessels revealed that RSD significantly increased both TRIM21 (F(1, 30)\u0026thinsp;=\u0026thinsp;104.67, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.777) and cleaved GSDMD (F(1, 30)\u0026thinsp;=\u0026thinsp;134.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = 0.817) protein levels, localizing this pathway to the cerebral vasculature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eTo define the functional role of vascular TRIM21 in RSD, we performed targeted knockdown in female rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Supplementary Fig.\u0026nbsp;2A, B). TRIM21 silencing significantly protected BBB integrity, as shown by markedly reduced sodium fluorescein extravasation (mean difference\u0026thinsp;=\u0026thinsp;4396.75, SE\u0026thinsp;=\u0026thinsp;665, t(74)\u0026thinsp;=\u0026thinsp;6.61, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F) and Evans blue leakage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) compared to RSD controls. The functional relevance of this TRIM21-GSDMD axis was confirmed using the GSDMD inhibitor disulfiram, which phenocopied the protective effect of TRIM21 knockdown by similarly reducing both sodium fluorescein and Evans blue leakage (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Mechanistically, both interventions significantly restored claudin-5 protein levels compared to RSD controls (post-hoc: both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with no statistical difference between their efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results define a novel pathway in which RSD-upregulated TRIM21 promotes GSDMD-mediated cleavage of tight junctions, directly compromising the BBB. This pathology is tractable, as demonstrated by its rescue through inhibition at either the level of TRIM21 or its effector, GSDMD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eTRIM21 forms complexes with GSDMD and NF-κB p65\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanism by which TRIM21 coordinates inflammasome and NF-κB signaling, we investigated its potential physical interactions with GSDMD and p65. We first employed AlphaFold 3 multimer for structural prediction, which generated low-confidence models (ipTM\u0026thinsp;\u0026lt;\u0026thinsp;0.2) suggesting plausible complexes. These \u003cem\u003ein silico\u003c/em\u003e data proposed a broad interaction interface between the TRIM21 PRY-SPRY domain and GSDMD, and a more localized binding mode with p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). While not confirmatory, these predictions provided a rationale to experimentally test for these interactions.\u003c/p\u003e \u003cp\u003eWe therefore performed co-immunoprecipitation from hippocampal lysates of RSD-stressed rats. Immunoprecipitation with a TRIM21-specific antibody, but not with a control IgG, successfully co-precipitated both cleaved GSDMD and the p65 subunit of NF-κB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), demonstrating a specific physical interaction. This result provides direct experimental evidence that TRIM21 physically interacts with both GSDMD and p65 \u003cem\u003ein vivo\u003c/em\u003e, confirming the central role of TRIM21 as a molecular hub in the stress-induced inflammatory cascade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eTRIM21 knockdown in PBMC attenuates RSD-induced GSDMD pore formation and efflux of IL-1β\u003c/h2\u003e \u003cp\u003eWe first investigated the systemic role of TRIM21 by measuring plasma IL-1β in RSD-exposed rats. TRIM21 knockdown significantly attenuated the RSD-induced increase in plasma IL-1β in both females (mean difference\u0026thinsp;=\u0026thinsp;14.53, t(79)\u0026thinsp;=\u0026thinsp;7.71, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and males (mean difference\u0026thinsp;=\u0026thinsp;8.28, t(79)\u0026thinsp;=\u0026thinsp;4.40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), indicating a key role for TRIM21 in stress-induced systemic inflammation.\u003c/p\u003e \u003cp\u003eSince peripheral immune cells are a primary source of plasma IL-1β, we analyzed PBMCs. Immunoblot and qPCR analyses revealed that RSD stress significantly induced TRIM21 and GSDMD expression in PBMCs, with females exhibiting markedly higher levels of both TRIM21 protein (mean difference\u0026thinsp;=\u0026thinsp;1.134, t(28)\u0026thinsp;=\u0026thinsp;5.40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C) and GSDMD protein (mean difference\u0026thinsp;=\u0026thinsp;1.243, t(28)\u0026thinsp;=\u0026thinsp;5.51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), as well as \u003cem\u003eTrim21\u003c/em\u003e mRNA (mean difference\u0026thinsp;=\u0026thinsp;0.814, t(40)\u0026thinsp;=\u0026thinsp;5.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) than males. Critically, elevated \u003cem\u003eTrim21\u003c/em\u003e mRNA expression correlated strongly with increased anxiety-like behavior in the EPM test (males: r(9)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.64, p\u0026thinsp;=\u0026thinsp;0.034; females: r(10)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.65, p\u0026thinsp;=\u0026thinsp;0.022; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), linking PBMC TRIM21 to a core behavioral outcome.\u003c/p\u003e \u003cp\u003eTo establish the mechanistic role of TRIM21 in peripheral inflammation, we performed \u003cem\u003ein vitro\u003c/em\u003e knockdown in PBMCs from RSD rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, H). TRIM21 silencing significantly suppressed the NLRP3 inflammasome pathway, reducing both caspase-1 activation (mean difference\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2.92, t(66)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;7.06, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI) and IL-1β release (mean difference = -208.3, t(67)\u0026thinsp;=\u0026thinsp;7.44, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). The NLRP3 inhibitor disulfiram phenocopied this effect (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001),\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003epositioning TRIM21 upstream of inflammasome activation in immune cells. We then asked if TRIM21 facilitates IL-1β release via GSDMD pore formation. A SYTOX green uptake assay confirmed that PBMCs from RSD rats exhibited significantly increased plasma membrane permeability (t(25)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;9.78, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). This pore formation was abolished by both TRIM21 knockdown and disulfiram treatment (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The absence of cell death (Supplementary Fig.\u0026nbsp;3A) confirms that these were sub-lytic pores, sufficient for the efflux of IL-1β without resulting in pyroptosis.\u003c/p\u003e \u003cp\u003eOur findings establish TRIM21 as a critical regulator of the RSD stress response, where it drives GSDMD-mediated, sub-lytic pore formation in PBMCs to facilitate IL-1β release, thereby linking peripheral immune activation to systemic inflammation and anxiety-like behavior.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTRIM21 is a critical mediator of RSD-induced BBB dysfunction\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of TRIM21 in RSD-induced BBB impairment, we employed \u003cem\u003ean in vitro\u003c/em\u003e BBB model where rat brain endothelial cell monolayers were exposed to PBMCs and sera from RSD rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This exposure robustly induced TRIM21 expression in RBECs [F(1, 24)\u0026thinsp;=\u0026thinsp;342.9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ηp\u0026sup2; = .935], with females exhibiting significantly higher TRIM21 levels than males (mean difference\u0026thinsp;=\u0026thinsp;1.41, t(24)\u0026thinsp;=\u0026thinsp;5.60, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). qRT-PCR confirmed this induction, showing a significant upregulation of \u003cem\u003eTrim21\u003c/em\u003e mRNA (Supplementary Fig.\u0026nbsp;4A). This was accompanied by a significant female-predominant increase in NF-κB activation, as measured by p-p65 (mean difference\u0026thinsp;=\u0026thinsp;1.25, t(24)\u0026thinsp;=\u0026thinsp;3.61, p\u0026thinsp;=\u0026thinsp;0.008; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Cell viability remained unaffected across all groups (Supplementary Fig.\u0026nbsp;4B), indicating that the effects were not due to cytotoxicity.\u003c/p\u003e \u003cp\u003eStrikingly, TRIM21 knockdown in RBECs dramatically attenuated the pathogenic cascade triggered by RSD exposure. It significantly reduced the cleavage of GSDMD (mean difference = -2.43, t(24)\u0026thinsp;=\u0026thinsp;7.48, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F), indicating suppression of downstream signaling.\u003c/p\u003e \u003cp\u003eFurthermore, TRIM21 depletion protected BBB integrity and suppressed the neurovascular inflammatory response. This was evidenced by the restoration of key endothelial proteins, including a decrease in the adhesion molecule ICAM-1 (mean difference = -2.15, t(24)\u0026thinsp;=\u0026thinsp;5.38, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG) and an increase in the tight junction protein ZO-1 (mean difference\u0026thinsp;=\u0026thinsp;0.39, t(24) = -3.19, p\u0026thinsp;=\u0026thinsp;0.024; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Concomitantly, TRIM21 knockdown attenuated endothelial activation, significantly reducing levels of the chemokine MCP-1 (mean difference = -179.3, t(24)\u0026thinsp;=\u0026thinsp;3.56, p\u0026thinsp;=\u0026thinsp;0.010; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI) and downregulating the expression of MMP9 and E-selectin (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Fig.\u0026nbsp;4C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCritically, in a functional BBB permeability assay, exposure to RSD components caused a profound increase in FITC-dextran leakage (mean difference\u0026thinsp;=\u0026thinsp;58.30, t(56) = -11.65, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). TRIM21 knockdown effectively rescued this barrier impairment, significantly reducing dextran leakage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), thereby demonstrating that TRIM21 is a central driver of RSD-induced vascular hyperpermeability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study identifies TRIM21 as a key molecular regulator that transduces psychosocial stress into a coordinated multi-system pathology, bridging peripheral inflammation, BBB dysfunction, and behavioral impairment. We delineate a previously unrecognized signaling axis\u0026mdash;TRIM21-GSDMD\u0026mdash;that operates as a critical amplifier of neuroinflammation, functioning both in parallel to and upstream of inflammasome pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The discovery that an E3 ubiquitin ligase directly facilitates GSDMD-mediated IL-1β release provides a mechanistic explanation for how a psychological stimulus is converted into a sustained inflammatory response, positioning TRIM21 as a nodal point for therapeutic intervention.\u003c/p\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eTRIM21 as a key inflammatory hub in the stressed brain\u003c/h2\u003e \u003cp\u003eOur data demonstrate that RSD induces a robust upregulation of TRIM21 across a distributed network, including emotion-regulating brain regions (hippocampus, amygdala), the cerebral vasculature, and peripheral blood mononuclear cells (PBMCs). This widespread induction suggests TRIM21 is a core component of the stress-responsive transcriptome. While the upstream signals driving \u003cem\u003eTrim21\u003c/em\u003e transcription following stress remain to be fully elucidated, its promoter contains binding sites for stress-responsive transcription factors like NF-κB and AP-1 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], potentially creating a feed-forward loop that is initiated by the primary stress response and amplified by subsequent inflammation.\u003c/p\u003e \u003cp\u003eOur most significant mechanistic insight is that TRIM21's role in facilitating GSDMD activation, previously defined in peripheral immunity, is a central driver of pathology in the stressed brain. While it was previously shown that TRIM21 binds the GSDMD C-terminal domain via its PRY-SPRY region to promote N-terminal oligomerization in macrophages during bacterial infection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], its function in the brain under psychological stress was unknown. Our co-immunoprecipitation data demonstrates that this interaction occurs \u003cem\u003ein vivo\u003c/em\u003e in the brain following RSD, a finding supported by our AlphaFold structural predictions. The multi-domain interaction we observed suggests a model where TRIM21 binding may not only promote oligomerization but also potentially displace the autoinhibitory C-terminal domain, lowering the activation threshold for GSDMD [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, our finding that TRIM21 interacts with the Rel Homology Domain (RHD) of the p65 subunit of NF-κB positions it as a potent inflammatory amplifier. This interaction suggests TRIM21 is poised to influence NF-κB DNA-binding capacity and transcriptional activity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This interaction could stabilize p65 or promote its ubiquitination in a manner that enhances its transcriptional activity, as suggested in other systems [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This creates a powerful, self-reinforcing inflammatory circuit: TRIM21-driven IL-1β release activates NF-κB, which in turn can further upregulate TRIM21, pro-IL-1β, and NLRP3, perpetuating a chronic inflammatory state characteristic of stress-related psychiatric disorders [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eSublytic pore formation: a mechanism for chronic neuroinflammation\u003c/h2\u003e \u003cp\u003eA pivotal finding is the demonstration that TRIM21 activation leads to sublytic GSDMD pore formation. The significant SYTOX Green uptake in the absence of cell death indicates a state of controlled membrane permeabilization. This sublytic signaling is emerging as a crucial mode of inflammatory signaling, allowing for the sustained release of IL-1β and other alarmins without committing the cell to pyroptotic death [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. It provides a mechanistic basis for the chronic neuroinflammation observed in major depressive disorder and PTSD, where a continuous, low-grade release of inflammatory mediators from glial and endothelial cells could disrupt neural circuitry without causing overt cell loss [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eSexual dimorphism: a molecular basis for differential vulnerability\u003c/h2\u003e \u003cp\u003eThe pronounced sexual dimorphism in the TRIM21 response is a finding with profound implications. The consistently higher upregulation of TRIM21, cleaved GSDMD, and IL-1β in female rats provides a plausible molecular substrate for the well-documented increased prevalence of anxiety and depressive disorders in women [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This dimorphism may be driven by interactions between the TRIM21 pathway and sex hormones. This aligns with foundational research demonstrating that susceptibility to chronic stress is associated with profoundly sex-specific transcriptomic signatures in key limbic brain regions [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Our data extend this principle by identifying a specific, inducible signaling node (the TRIM21-GSDMD axis) within the neuroinflammatory cascade that exhibits a female-predominant activation pattern. For instance, estradiol has been shown to potentiate NF-κB signaling in certain contexts [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], which could synergize with the TRIM21-p65 interaction we identified to create a more robust inflammatory response in females.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eFrom synapses to behavior: preserving circuit integrity\u003c/h3\u003e\n\u003cp\u003eThe behavioral rescue achieved through \u003cem\u003ein vivo\u003c/em\u003e TRIM21 knockdown underscores its central role in the pathophysiology of stress-induced behavioral deficits. The restoration of hippocampal dendritic spine density provides a structural correlate for the recovery of cognitive function [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Critically, these structural changes reflect the potential restoration of functional synaptic plasticity, the cellular basis of learning and memory, which is fundamentally governed by the dynamic regulation of AMPA receptor trafficking at the postsynaptic density [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The well-established ability of IL-1β to disrupt long-term potentiation and promote long-term depression and spine loss [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] strongly supports a model where TRIM21 knockdown preserves synaptic integrity and function primarily by dampening this key inflammatory mediator. This is consistent with foundational evidence demonstrating that stress-induced NF-κB activation within limbic brain circuits is a critical driver of synaptic remodeling and maladaptive behavior [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Therefore, by inhibiting the TRIM21-GSDMD-IL-1β axis, we likely restore the molecular environment necessary for normal synaptic plasticity, learning, and emotional regulation.\u003c/p\u003e\n\u003ch3\u003eTRIM21 as a mediator of BBB dysfunction\u003c/h3\u003e\n\u003cp\u003ePerhaps our most significant advance is in elucidating the active role of TRIM21 in breaching the BBB. We move beyond correlation to demonstrate causality, showing that TRIM21 within brain endothelial cells is both necessary and sufficient for stress-induced barrier breakdown. The discovery of elevated TRIM21 and cleaved GSDMD in isolated brain microvessels localizes this pathway directly to the BBB. Our \u003cem\u003ein vitro\u003c/em\u003e BBB model mechanistically dissects this process: inflammatory signals from the periphery require endothelial TRIM21 to initiate a cascade of tight junction loss (ZO-1, claudin-5), adhesion molecule induction (ICAM-1, VCAM-1), chemokine production (MCP-1), and MMP9 activation. Consequently, TRIM21 emerges as a novel upstream regulator of a pathogenic cascade whose endpoint\u0026mdash;BBB failure\u0026mdash;is a recognized therapeutic target across neurological disorders [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe protection afforded by TRIM21 knockdown, which was comparable to direct GSDMD inhibition with disulfiram, underscores that GSDMD pore formation is a critical, and likely the predominant, downstream executor of TRIM21 signaling in endothelial dysfunction. The subsequent upregulation of ICAM-1 and MCP-1 creates a chemotactic gradient and adhesion platform, facilitating the firm adhesion of peripheral immune cells to the vessel wall, a critical step in their transmigration into the brain parenchyma, as observed in stress models [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This positions TRIM21 as the key mediator of a pathway that directly links psychological stress to the recruitment of peripheral immune cells into the brain.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eImplications and future directions\u003c/h2\u003e \u003cp\u003eIn summary, we propose a unified model wherein psychosocial stress upregulates TRIM21, which then acts as a proteostatic hub, directly promoting GSDMD activation and NF-κB signaling to drive a self-amplifying inflammatory loop that disrupts the BBB, compromises synaptic integrity, and manifests as behavioral pathology. From a clinical standpoint, TRIM21 represents a uniquely promising target. Its upstream position means its inhibition could simultaneously dampen multiple inflammatory pathways (IL-1β release, NF-κB signaling, NLRP3 expression) and preserve BBB integrity, offering a more comprehensive strategy.\u003c/p\u003e \u003cp\u003eFuture work should focus on several key areas: First, identifying the precise upstream initiators of TRIM21 induction by stress, potentially involving glucocorticoid receptor signaling or catecholamine surges. Second, developing and testing specific pharmacological TRIM21 inhibitors in chronic stress models to validate its therapeutic potential. Third, the pronounced sex differences demand that these future strategies be evaluated and optimized with biological sex as a critical variable. Fourth, investigating cell-type-specific functions of TRIM21 (e.g., in microglia, neurons, endothelia) using conditional knockout models. Finally, longitudinal studies are needed to determine if TRIM21 inhibition can promote stress resilience or reverse established pathology.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings fundamentally advance the understanding of neuroimmune mechanisms in stress-induced neuropsychological disorders by identifying TRIM21 as the critical missing link that translates psychological stress into a structured biological response. By orchestrating GSDMD-mediated release of IL-1β and downstream neurovascular dysfunction, the TRIM21 pathway integrates multiple pathological hallmarks of stress-related disorders. This work establishes a new mechanistic framework for stress-induced inflammation and provides a compelling impetus for developing therapeutics that intercept this stress-induced pathological cascade at its origin.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their gratitude to the institutional core facilities that supported this work, specifically the Ashoka-ZEISS Core Imaging Facility at Ashoka University and the University Science Instrumentation Centre (USIC) at the University of Delhi. This research was made possible through funding from Ashoka University (S.G. and Z.M.), the Brain Aneurysm Foundation (I.S.), a DBT-Ramalingaswamy Fellowship (I.S.), and the Savrdh Foundation (S.T.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Ashoka University (S.G. and Z.M.), the Brain Aneurysm Foundation (I.S.), a DBT-Ramalingaswamy Fellowship (I.S.), and the Savrdh Foundation (S.T.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.T., Z.M., E.Y., S.K., and S.R.A. conducted the animal experiments and behavioral testing. S.T. carried out the \u003cem\u003ein vitro\u003c/em\u003e studies and histological staining. S.R.A. performed the \u003cem\u003ein-silico\u003c/em\u003e interaction studies. A.T. conducted data quantification and formal statistical analysis. S.T., Z.M., S.R.A., A.T., A.Y., S.K., A.C., I.S., and S.G. were involved in writing – original draft preparation, review, and editing. I.S. and S.R.A. prepared the schematic figures. S.G. conceived the study, designed the methodology, and provided overall supervision. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo large datasets were generated or analysed during the current study. All data supporting the findings of this study are available within the paper and its ‘Supplementary Material’. Additional data related to this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the Institutional Animal Ethics Committees (IAEC) of the University of Delhi and Maharshi Dayanand University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e(2022) Global, regional, and national burden of 12 mental disorders in 204 countries and territories, 1990\u0026ndash;2019: a systematic analysis for the Global Burden of Disease Study 2019. 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Nat Rev Neurol 6:393\u0026ndash;403. https://doi.org/10.1038/nrneurol.2010.74\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Stress, anxiety, inflammasomes, TRIM21, Gasdermin D","lastPublishedDoi":"10.21203/rs.3.rs-8260120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8260120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe upstream mechanism that transduces psychosocial stress into the release of the pivotal pro-inflammatory cytokine IL-1β has remained a fundamental gap in understanding neuropsychiatric disorders. Here, we identify tripartite motif-containing protein 21 (TRIM21) as the critical trigger. In a rat model of repeated social defeat, TRIM21 was upregulated in the hippocampus, brain microvessels, and peripheral blood mononuclear cells (PBMCs), correlating with anxiety-like behavior and memory deficits, with females exhibiting a more pronounced pathophysiology. Mechanistically, TRIM21 directly binds to and promotes the cleavage of gasdermin D, facilitating the formation of membrane pores for IL-1β release. This pathway was active in PBMCs, where TRIM21 drove sub-lytic pore formation and IL-1β release, linking peripheral inflammation to behavioral deficits. Critically, \u003cem\u003ein vivo\u003c/em\u003e TRIM21 knockdown abrogated this pathway systemically, reducing IL-1β release and NF-κB signaling, which in turn rescued blood-brain barrier integrity, restored synaptic density, and normalized behavior. Using an \u003cem\u003ein vitro \u003c/em\u003eblood-brain barrier model, we pinpointed this effect to a TRIM21-IL-1β-p65 axis. We therefore define TRIM21 as the critical node in a stress-responsive circuit, bridging peripheral immune activation to GSDMD-dependent neuroinflammation and blood-brain barrier disruption, thereby providing a mechanistic framework that redefines the pathophysiology of stress-related neuropsychiatric disorders and reveals new avenues for their treatment.\u003c/p\u003e","manuscriptTitle":"TRIM21 drives stress-induced neuroinflammation and behavioral deficits via gasdermin D- dependent IL-1β release in a rat model of repeated social defeat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 06:35:53","doi":"10.21203/rs.3.rs-8260120/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4c34d21-a278-423e-abc1-8631a12a98d7","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-14T22:08:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-12 06:35:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8260120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8260120","identity":"rs-8260120","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}