Background: Cardiovascular disease (CVD) and cancer are the leading causes of global mortality, and emerging evidence indicates a complex, bidirectional relationship between these conditions. Our epidemiologic analyses link cancer diagnosis to elevated atherosclerotic risk, yet direct evidence and underlying mechanisms remain poorly defined. Hypothesis: Cancer directly promotes atherosclerosis progression. Methods: We performed RNA sequencing on aortic tissues from atheroprone mice injected subcutaneously with colorectal cancer cells or control buffer, examining effects across diverse genetic backgrounds, diets, and ages. Apoe-deficient mice underwent carotid ligation followed by tumor inoculation; plaques were assessed histologically. Serum biomarkers were identified using O-link proteomics. Human endothelial cells were treated with tumor-conditioned media and evaluated by RT-qPCR and angiogenesis assays. Therapeutically, tumor-bearing mice received systemic TNF-neutralizing antibody or vascular-targeted AAV-mediated LRG1 knockdown. Human Biobank vascular tissues and clinical databases were utilized to confirm translational relevance. Results: LRG1 was consistently elevated in the aortas of tumor-bearing mice across multiple experimental models. Tumor-bearing mice exhibited significantly increased plaque burden, intraplaque neovascularization, and hemorrhage compared to controls. TNF levels were elevated in tumor-bearing mice. Conditioned media from cancer cells robustly induced endothelial LRG1 expression and promoted endothelial tube formation, effects effectively reversed by TNF blockade. In vivo, treatment with either TNF-neutralizing antibody or endothelial-targeted LRG1 knockdown markedly attenuated tumor-induced plaque neovascularization and intraplaque hemorrhage. Analysis of human vascular tissues further confirmed elevated vascular TNF-LRG1 signaling in cancer patients. Conclusion: Our findings demonstrate that cancer accelerates atherosclerosis via a TNF-driven LRG1-dependent mechanism, promoting intraplaque neovascularization and instability. Targeting this inflammatory-angiogenic pathway effectively mitigates tumor-induced vascular remodeling, highlighting novel therapeutic strategies to manage cardiovascular risk among cancer patients.

Inflammasomes, particularly NLRC4, play crucial roles in immune responses to intracellular bacterial infections. However, gain-of-function mutations in NLRC4 are linked to severe autoinflammatory diseases, including autoinflammation with infantile enterocolitis (AIFEC). AIFEC patients who survive infancy typically have no further intestinal symptoms but retain susceptibility to macrophage activation syndrome (MAS). However, existing mouse models do not adequately replicate the inflammation observed in AIFEC patients. To better understand this, we developed a mouse model capable of conditional expression of the activating V341A mutation in NLRC4 (NLRC4-V341A KI). Global conversion to NLRC4-V341A at the germline resulted in symptoms closely mirroring those of human AIFEC, including severe infantile enterocolitis characterized by heightened intestinal inflammation, disrupted gut epithelium, compromised intestinal barrier integrity, severe diarrhea, and mortality within 10 days post-natally. Additionally, they displayed systemic autoinflammation marked by elevated levels of IL-1β, IL-18, and IL-6, alongside cytopenia and hemophagocytosis. In contrast, conditional conversion to NLRC4-V341A in adulthood caused systemic autoinflammation with only mild enterocolitis, mirroring AIFEC patients. Using this model, we demonstrated that IL-18 and TNF blockade effectively ameliorated AIFEC disease symptoms. Unexpectedly, glucose supplementation has emerged as a promising therapeutic strategy. These findings advance our understanding of AIFEC and illuminate the ways in which inflammasome activation contributes to very early onset inflammatory bowel disease (VEO-IBD) in the developing gut.

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a range of affinities for tumor antigens while suppressing regulatory T cells. Computational protein design has enabled the precise tailoring of proteins to meet specific needs. Here, we report a de novo designed IL-21 mimic, 21h10, with high stability and signaling potency in humans and mice. In murine and ex vivo human organotypic tumor models, 21h10 showed robust antitumor activity, with more prolonged signaling and stronger antitumor activity than native IL-21. 21h10 induced pancreatitis that could be mitigated by TNF blockade without compromising antitumor efficacy. Although antidrug antibodies to 21h10 formed, they were not neutralizing. 21h10 induced highly cytotoxic T cells with a range of affinities, robustly expanding intratumoral low-affinity cytotoxic T cells and driving high expression of IFN-γ and granzyme B compared with native IL-21, while increasing the frequency of IFN-γ+ T helper 1 cells and reducing regulatory T cells. The full human-mouse cross-reactivity, high stability and potency, and low-affinity antitumor responses support the translational potential of 21h10.

Abstract Background: Trastuzumab deruxtecan (T-DXd) is indicated for the treatment of adult patients with unresectable or metastatic HER2-positive (HER2+) breast cancer who have received a prior anti-HER2-based regimen. Results from DESTINY-Breast03 demonstrated that the median overall survival in this setting was 52.6 months, showing a need for novel biomarkers and efficient agents that may improve T-DXd outcome. Furthermore, it causes adverse effects that lead to dose reduction or treatment discontinuation. We have demonstrated that mucin 4 (MUC4) is an independent biomarker of poor disease-free survival in early HER2+ breast cancer patients treated with trastuzumab and reflects an immunosuppressive tumor microenvironment (TME). In addition, we have shown that TNF induces MUC4 expression and that soluble TNF (sTNF) neutralization with a dominant negative protein to TNF, INB03 (DN), downregulates MUC4 and overcomes trastuzumab resistance in preclinical models. Previously, we observed that administration of 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg T-DXd in nude mice reduce JIMT-1 tumor growth by 37%, 61% and 83%, respectively. Administration of 1.25mg/kg + DN induces tumor reduction that mimics the effect of the 5 mg/kg dose. In this work, we explored the precise contribution of TNF- induced MUC4 expression on T-DXd antitumor effect. Methods: The HER2+, trastuzumab-resistant JIMT-1 cells were engineered to express a doxycycline (Dox)-inducible shRNA targeting human MUC4, rendering JIMT-shMUC4 cells. These cells were injected in nude mice and, once tumors were established, animals were administered with Dox (Dox+) or not (-Dox), and treated with IgG 2.5 mg/kg, T-DXd 2.5 mg/kg (T-DXd 2.5), or 1.25 mg/kg (T-DXd 1.25), DN 10 mg/kg or the combined therapies (n=6-8/group). T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored and differences among groups were analyzed using two-way ANOVA coupled with a Bonferroni post hoc test. Results: In JIMT-1-shMUC4 Dox- tumors, T-DXd 1.25 inhibited tumor growth by 55% vs IgG (p<0.0001) at day 21. Addition of DN significantly enhanced this effect (73%, p<0.05 vs T-DXd 1.25). T-DXd 2.5 decreased growth by 72% (p<0.0001) and T-DXd 2.5 + DN by 74% (p<0.00001) vs IgG. Of notice, T-DXd 1.25+DN inhibits tumor growth to a similar extent to T-DXd 2.5 alone. In Dox+ tumors, with silenced MUC4 expression (30-50%), T-DXd 1.25 inhibited tumor growth by 61% and T-DXd 2.5 by60% (p<0.0001 each, vs IgG). Addition of DN did not further increase these effects. Conclusion: Our results suggest that MUC4 interferes with T-DXd antitumor effect when this drug is administered at a low dose (1.25 mg/kg). This detrimental effect can be tackled by blocking sTNF, which downregulates MUC4 expression and enhances-DXd therapeutic effect. MUC4 expression seems not to affect T-DXd effect when administered at a higher dose (2.5 mg/kg). As bioavailability of anti-cancer drugs can be affected by the TME, we propose that MUC4 would be a suitable biomarker to evaluate T-DXd resistance in HER2+ breast cancer, Therefore, addition of sTNF blocking agents would be an option to reinstate tumor sensitivity to T-DXd. In addition, dose reduction of T-DXd in combination with DN could be a feasible therapeutic option to reduce adverse effects. Citation Format: Sofía Bruni, Florencia Mauro, Camila Jencquel, María Florencia Mercogliano, Roxana Schillaci. MUC4 downregulation by TNF blockade enhances trastuzumab deruxtecan antitumor effect in a HER2-positive breast cancer model [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-05-30.

Pyoderma gangrenosum (PG) is an understudied inflammatory skin condition that causes significant morbidity and rarely mortality. Treatment options for PG are limited due to our relatively poor understanding of its molecular mechanisms. Prior studies have suggested a genetic basis for PG, but causal genes have remained elusive. Through the study of siblings with severe recurrent PG in childhood, we identified a novel mutation in OTULIN. OTULIN is a linear deubiquitylase that regulates several immune signaling pathways in ways that are cell type specific. Interestingly, this mutation affects the PIM domain, responsible for protein–protein interactions, but does not impair catalytic functions of the OTU domain, thus its mechanism of action is distinct from previously reported OTULIN mutations. Through a combination of in vitro mechanistic studies that isolate the functional impact of the variant, together with single cell analyses of patient and healthy control PBMC samples, we demonstrate this novel OTULIN mutation causes PG via i) hyperactivation of the NLRP3 inflammasome and elevated IL-1b secretion from patients’ myeloid cells and ii) heightened keratinocyte sensitivity to TNF-driven cell death. The patients responded well to anti-TNF therapy and are now free of PG flares. This study establishes the first monogenic etiology of isolated PG of childhood, identifies the molecular mechanisms of disease, and suggests that TNF and/or IL-1b blockade may be effective therapeutic options.

Since DESTINY-Breast03 results where trastuzumab-deruxtecan (T-DXd) showed remarkable overall survival improvement, this drug is used to treat patients with unresectable or metastatic HER2-positive (HER2+) breast cancer who have received prior anti-HER2-based regimen. One antitumor mechanism of T-DXd is attributed to DXd inhibition of topoisomerase I in the tumor cells that internalized the drug, and to the bystander effect of DXd on neighbor tumor cells. Reports describe that topoisomerase I inhibitors produce DNA fragmentation, stimulating cGAS/STING pathway and producing type-I IFN, a key mediator of the innate immune response against tumors. Previously, we reported that soluble TNF (sTNF) overproduction by HER2+ tumors generates resistance to trastuzumab-based therapies. Blocking sTNF with INB03 (DN), a dominant-negative molecule, sensitized tumors to therapy and improved the antitumoral immune response. Here, we explore if blocking sTNF can also improve T-DXd antitumor effect, and its impact on the innate immune response involving cGAS/type-I IFN, in a model with resistance to multiple HER2-targeted therapies. Female nude mice bearing JIMT-1 tumors were treated i.p. twice/week with 5 mg/kg of IgG or 10 mg/kg DN, or with 5, 2.5 or 1.25 mg/kg of T-DXd i.v. on days 0, 7 and 14, or the combination therapies. Tumor volume was measured twice/week for 21 days (n=6-8). Tumor-infiltrating macrophages and NK cells were studied by flow cytometry. ISG15, a downstream mediator of type-I IFN pathway, and cGAS were evaluated by western blot on tumor extracts.The dose-response curves showed a tumor growth inhibition of 83% (T-DXd 5 mg/kg), 61% (T-DXd 2.5 mg/kg) and 37% (T-DXd 1.25 mg/kg) vs. IgG (p<0.0001, 0.0001 and 0.05, respectively). Adding DN enhanced this inhibition to 98% (5 mg/kg), 81% (2.5 mg/kg) and 73% (1.25 mg/kg), p<0.0001 vs. IgG. T-DXd 1.25 mg/kg +DN achieved an antitumor effect that resembles T-DXd 5 mg/kg alone. Furthermore, addition of DN to T-DXd 1.25 and 5 mg/kg increased macrophage infiltration and polarization to the antitumoral M1-like phenotype. T-DXd 1.25 mg/kg +DN treatment mimics the increase of infiltrating NK cells observed in the higher doses of T-DXd alone. Finally, we observed an increase in cGAS and ISG15 expression in tumor extracts form T-DXd 1.25 mg/kg +DN, compared to T-DXd 1.25 mg/kg alone.Our results suggest that sTNF blockade with DN enhances T-DXd antitumor effect, inducing a reinforced innate immune response with increased infiltration of M1-like macrophages and NK cells, and activation of cGAS and type-I IFN pathway, measured by its surrogate ISG15. Adding DN allows to de-escalate T-DXd dose without impairing the antitumor effect. Neutralizing sTNF could improve the outcome of patients treated with T-DXd by enhancing the antitumor immune response. Citation Format: Sofia Bruni, Camila Jencquel, Martin A. Rivas, Roxana Schillaci, Maria F. Mercogliano. Soluble TNF blockade enhances trastuzumab-deruxtecan innate antitumor immune response and type-I IFN signalling in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 882.

Mucin 4 expression is associated with metastasis in triple-negative breast cancer and can be tackled by soluble TNF blockade, improving immunotherapy outcome.

Immune checkpoint inhibitors (ICI) represent new anticancer agents and have been used worldwide. However, ICI can potentially induce life-threatening severe cutaneous adverse reaction (SCAR), such as Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), hindering continuous ICI therapy. We examine 6 cohorts including 25 ICI-induced SJS/TEN patients and conduct single-cell RNA sequencing (scRNA-seq) analysis, which shows overexpression of macrophage-derived CXCL10 that recruits CXCR3+ cytotoxic T lymphocytes (CTL) in blister cells from ICI-SJS/TEN skin lesions. ScRNA expression profiles and ex vivo blocking studies further identify TNF signaling as a pathway responsible for macrophage-derived CXCL10 and CTL activation. Based on the trajectory analysis, ICI-activated T cells from whole blood are proposed to serve as the initial cells involved in inflammation, that lead to monocytes differentiating into macrophages and increasing their susceptibility to migrate to the lesion sites. Compared with systemic corticosteroids treatment, ICI-induced SJS/TEN patients treated with biologic TNF blockade showed a significantly rapid recovery and no recurrence of SCAR with continuous ICI therapy. Our findings identify that macrophage-eliciting CTL contribute to the pathogenesis of ICI-induced epidermal necrolysis and provide potential therapeutic targets for the management and prevention of SCAR induced by ICI therapy.

Therapeutic single-cell landscape: methotrexate exacerbates interstitial lung disease by compromising the stemness of alveolar epithelial cells under systemic inflammation

IL-12-mediated toxicity from localized oncolytic virotherapy can be reduced using systemic TNF blockade

Inflammatory disease status and response to TNF blockade are associated with mechanisms of endotoxin tolerance

Recently, it has been questioned whether vaccination of patients with inflammatory (auto)immune diseases under anti-tumor necrosis factor (TNF) treatment leads to impaired vaccine-induced immune responses and protection against breakthrough infections. However, the effects of TNF blockade on short- and long-term immune responses after repeated vaccination remain unclear. Vaccination studies have shown that initial short-term IgG antibodies (Abs) carry highly galactosylated and sialylated Fc glycans, whilst long-term IgG Abs have low levels of galactosylation and sialylation and are most likely generated by long-lived plasma cells (PCs) derived primarily from the germinal center (GC) response. Thus, IgG Fc glycosylation patterns may be applicable to distinguish short- and long-term vaccine responses after repeated vaccination under the influence of anti-TNF treatment. We used COVID-19 vaccination as a model to investigate vaccine-induced IgG subclass levels and Fc glycosylation patterns, B cell subsets, and effector functions of short- and long-term Ab responses after up to three vaccinations in patients on anti-TNF or other immunosuppressive treatments and in healthy individuals. Using TriNetX, a global healthcare database, we determined the risk of SARS-CoV-2 breakthrough infections in vaccinated patients treated with anti-TNF or other immunosuppressive drugs. Anti-TNF treatment reduced the long-term abundance of all anti-S IgG subclasses with low levels of galactosylation and sialylation. Re-activation of potential memory B cells initially generated highly galactosylated and sialylated IgG antibodies, which were progressively reduced after each booster dose in anti-TNF-treated patients, especially in the elderly. The reduced short- and long-term IgG (1) levels in anti-TNF-treated patients correlated with diminished functional activity and an increased risk for the development of COVID-19. The data suggest that anti-TNF treatment reduces both GC-dependent long-lived PCs and GC-dependent memory B cell-derived short-lived PCs, hence both the long- and short-term IgG subclass responses, respectively, after repeated vaccination. We propose that anti-TNF therapy, especially in the elderly, reduces the benefit of booster vaccination.

The observation that certain therapeutic strategies for targeting inflammation benefit patients with distinct immune-mediated inflammatory diseases (IMIDs) is exemplified by the success of TNF blockade in conditions including rheumatoid arthritis, ulcerative colitis, and skin psoriasis, albeit only for subsets of individuals with each condition. This suggests intersecting "nodes" in inflammatory networks at a molecular and cellular level may drive and/or maintain IMIDs, being "shared" between traditionally distinct diagnoses without mapping neatly to a single clinical phenotype. In line with this proposition, integrative tumour tissue analyses in oncology have highlighted novel cell states acting across diverse cancers, with important implications for precision medicine. Drawing upon advances in the oncology field, this narrative review will first summarise learnings from the Human Cell Atlas in health as a platform for interrogating IMID tissues. It will then review cross-disease studies to date that inform this endeavour before considering future directions in the field.

Abstract Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and has the worst survival rate. Immunotherapy is a new treatment option but resistance is frequent. We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein involved in metastasis (MTS) dissemination. Here, we evaluated the impact of the axis TNF/MUC4 on the invasive capacity of TNBC cell lines and in a preclinical model of spontaneous MTS. Finally, we assessed the clinical impact of MUC4 expression in TNBC patients. TNF blockade was achieved with etanercept (E), which blocks the soluble (sTNF) and transmembrane isoform of TNF, or with the dominant negative protein INB03 (DN) which neutralizes only sTNF. BT-549 and MDA-MB-231 human TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. To assess the impact of TNF blockade on tumor cells and its effect on the tumor microenvironment, we collected conditioned media (CM) from MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 and MDA-MB-231 was impaired with CM-Eta (p≤0.01) and CM-DN (p≤0.01 and p≤0.05, respectively). Female BALB/c mice bearing the TNBC LMM3 tumors were treated with IgG, DN, anti-PD-1 antibodies or DN+anti-PD-1 for two weeks. After the treatments we performed surgery to remove the primary tumor and the animals were sacrificed 2 weeks later. No effect on tumor growth was observed. However, the DN+anti-PD-1 group was the only one with less of 3 MTS per lung (p≤0.05). We obtained tumor extracts and found that the tumors of the animals treated with DN or DN+anti-PD-1 had decreased MUC4 expression measured by Western blot. We determined the presence of TILs by H & E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 56 early TNBC patients. MUC4 expression was inversely correlated with TILs presence (p=0.0003). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM and MDA-MB-231-CM on T cell migration. CM-DN from both cell lines increased T cell migration compared to control CM (p≤0.01). MUC4 expression was inversely correlated with Ki67 (≥30%, p=0.036), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher MTS risk (p=0.005). MUC4 is associated with poorly-infiltrated TNBC, and sTNF blockade downregulates its expression decreasing MTS when combined with anti-PD-1. We propose the TNF as a new target for the treatment of TNBC, and MUC4 as a predictive biomarker to guide a combined treatment of TNF blockers with immunotherapy. Citation Format: Florencia Luciana Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, Maria Florencia Mercogliano, Roxana Schillaci. Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2753.

Necroptosis, a potent host defense mechanism, limits viral replication and pathogenesis through three distinct initiation pathways. Toll-like receptor 3 (TLR3) via TIR-domain-containing adapter-inducing interferon-β (TRIF), Z-DNA-binding protein 1 (ZBP1) and tumor necrosis factor (TNF)α mediate necroptosis, with ZBP1 and TNF playing pivotal roles in controlling viral infections, with the role of TLR3-TRIF being less clear. ZBP1-mediated necroptosis is initiated when host ZBP1 senses viral Z-form double stranded RNA and recruits receptor-interacting serine/threonine-protein kinase 3 (RIPK3), driving a mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis pathway, whereas TNF-mediated necroptosis is initiated by TNF signaling, which drives a RIPK1-RIPK3-MLKL pathway, resulting in necroptosis. Certain viruses (cytomegalovirus, herpes simplex virus and vaccinia) have evolved to produce proteins that compete with host defense systems, preventing programmed cell death pathways from being initiated. Two engineered viruses deficient of active forms of these proteins, murine cytomegalovirus M45mutRHIM and vaccinia virus E3∆Zα, trigger ZBP1-dependent necroptosis in mouse embryonic fibroblasts. By contrast, when bone-marrow-derived macrophages are infected with the viruses, necroptosis is initiated predominantly through the TNF-mediated pathway. However, when the TNF pathway is blocked by RIPK1 inhibitors or a TNF blockade, ZBP1-mediated necroptosis becomes the prominent pathway in bone-marrow-derived macrophages. Overall, these data implicate a cell-type preference for either TNF-mediated or ZBP1-mediated necroptosis pathways in host responses to viral infections. These preferences are important to consider when evaluating disease models that incorporate necroptosis because they may contribute to tissue-specific reactions that could alter the balance of inflammation versus control of virus, impacting the organism as a whole.

Sarcoidosis is a chronic granulomatous disorder characterized by unknown etiology, undetermined mechanisms, and non-specific therapies except TNF blockade. To improve our understanding of the pathogenicity and to predict the outcomes of the disease, the identification of new biomarkers and molecular endotypes is sorely needed. In this study, we systematically evaluate the biomarkers identified through Omics and non-Omics approaches in sarcoidosis. Most of the currently documented biomarkers for sarcoidosis are mainly identified through conventional "one-for-all" non-Omics targeted studies. Although the application of machine learning algorithms to identify biomarkers and endotypes from unbiased comprehensive Omics studies is still in its infancy, a series of biomarkers, overwhelmingly for diagnosis to differentiate sarcoidosis from healthy controls have been reported. In view of the fact that current biomarker profiles in sarcoidosis are scarce, fragmented and mostly not validated, there is an urgent need to identify novel sarcoidosis biomarkers and molecular endotypes using more advanced Omics approaches to facilitate disease diagnosis and prognosis, resolve disease heterogeneity, and facilitate personalized medicine.

Chronic TNF in the Aging Microenvironment Exacerbates TET2-loss-of-Function Myeloid Expansion

Patients with spondyloarthritis (SpA) often present with microscopic signs of gut inflammation, a risk factor for progressive disease. We investigated whether mucosal innate-like T cells are involved in dysregulated interleukin-23 (IL-23)/IL-17 responses in the gut-joint axis in SpA. Ileal and colonic intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), and paired peripheral blood mononuclear cells (PBMCs) were isolated from treatment-naive patients with nonradiographic axial SpA with (n=11) and without (n=14) microscopic gut inflammation and healthy controls (n=15) undergoing ileocolonoscopy. The presence of gut inflammation was assessed histopathologically. Immunophenotyping of innate-like T cells and conventional T cells was performed using intracellular flow cytometry. Unsupervised clustering analysis was done by FlowSOM technology. Serum IL-17A levels were measured via Luminex. Microscopic gut inflammation in nonradiographic axial SpA was characterized by increased ileal intraepithelial γδ-hi T cells, a γδ-T cell subset with elevated γδ-T cell receptor expression. γδ-hi T cellswere also increased in PBMCs of patients with nonradiographic axial SpA versus healthy controls and were strongly associated with Ankylosing Spondylitis Disease Activity Score. The abundance of mucosal-associated invariant T cells and invariant natural killer T cells was unaltered. Innate-like T cells in the inflamed gut showed increased RORγt, IL-17A, and IL-22 levels with loss of T-bet, a signature that was less pronounced in conventional T cells. Presence of gut inflammation was associated with higher serum IL-17A levels. In patients treated with tumor necrosis factor blockade, the proportion of γδ-hi cells and RORγt expression in blood was completely restored. Intestinal innate-like T cells display marked type 17 skewing in the inflamed gut mucosa of patients with nonradiographic axial SpA. γδ-hi T cells are linked to intestinal inflammation and disease activity in SpA.

FADD- and RIPK3-Mediated Cell Death Ensures Clearance of Ly6Chigh Wound Macrophages from Damaged Tissue

Synovial fibroblasts (SFs) are key pathogenic drivers in rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models, and TNF blockade proved efficacious for a high percentage of patients with RA albeit coinducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF-transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely amisulpride, which reduced SFs' inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that amisulpride function was neither through its known targets dopamine receptors D2 and D3 and serotonin receptor 7 nor through TNF-TNF receptor I binding inhibition. Through a click chemistry approach, potentially novel targets of amisulpride were identified, which were further validated to repress hTNFtg SFs' inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, amisulpride could prove beneficial to patients experiencing RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its antidepressive activity, serving further as a "lead" compound for the development of novel therapeutics against fibroblast activation.