Journal of Experimental Medicine

Gain-of-function mutations in STimulator of INterferon Genes (STING) cause STING-Associated Vasculopathy with Onset in Infancy (SAVI), a rare autoinflammatory disease characterized by debilitating inflammatory lung disease and hallmark skin manifestations, such as chilblains and progressive, non-healing ulcers. Mice expressing the most common SAVI-associated variant STINGV154M (VM) recapitulate many clinical features of SAVI, including inflammatory lung disease, but do not develop spontaneous skin lesions. In this study, we show that a single low dose of ultraviolet B (UVB) irradiation, which induces only transient skin inflammation in wild-type (WT) mice, causes severe and progressive skin injury in VM mice. Notably, this phenotype persisted in VM mice depleted of hematopoietic cells and reconstituted with WT bone marrow, demonstrating that STINGV154M expression in non-hematopoietic cells is sufficient to drive persistent skin inflammation. Further analysis identified endothelial cells expressing STINGV154M as the primary driver of the cutaneous phenotype. Flow cytometry and bulk RNA sequencing showed that VM mice exhibited reduced early skin infiltration of macrophages and dendritic cells after UVB exposure. These findings establish a critical link between endothelial STING activation, impaired recruitment of skin myeloid cells, and defective resolution of acute inflammation, offering new insights into the pathogenesis of SAVI-associated skin disease.

Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in Infancy (SAVI). SAVI patients develop interstitial lung disease (ILD) and commonly produce anti-nuclear antibodies (ANAs), indicative of concomitant autoimmunity. Mice heterozygous for the most common SAVI mutation, V154M (VM), also develop ILD, triggered by nonhematopoietic VM cells, but exhibit severe peripheral lymphopenia, low serum Ig titers and fail to produce autoantibodies. In contrast, we now show that lethally irradiated VM mice reconstituted with WT stem cells (WT[->]VM chimeras) develop ANAs and lung-reactive autoantibodies associated with accumulation of activated lymphocytes and formation of germinal centers in lung tissues. Moreover, when splenocytes from WT[->]VM chimeras were adoptively transferred into unmanipulated Rag1-/- mice, donor T cells accumulated in the lung. Overall, these findings demonstrate that expression of the VM mutation in non-hematopoietic cells can promote the activation of immunocompetent autoreactive lymphocytes. SummaryChimeric mice expressing STING only in non-hematopoietic cells develop systemic and lung directed autoimmunity which recapitulates what is seen in pediatric patients with SAVI disease.

Dendritic cell (DC) homeostasis is maintained in secondary lymphoid organs (SLOs) by Fms-like tyrosine kinase 3 ligand (FLT3L). The specific niche providing this DC growth factor within human and mouse SLOs is unclear. Here, we show that Gremlin1 (Grem1)-expressing lymph node fibroblastic reticular cells (FRCs) support DC homeostasis via provision of FLT3L. Grem1 FRCs co-localize with DCs and express FLT3L in human and mouse lymph nodes. Using a new genetic model, we provide evidence that FLT3L produced by GREM1 FRCs maintains lymph node preDCs, cDCs, and plasmacytoid DCs (pDCs). Spatial transcriptomics and cytofluorometry reveal that Grem1 FRC-derived FLT3L supports not only proliferation, but also survival of lymph node cDCs within the interfollicular zone (IFZ). Functionally, loss of Grem1 FRC-derived FLT3L impairs cDC priming of antigen-specific T cell responses. These findings provide key mechanistic insights underlying stromal cell support of DC homeostasis and function.

Lethal COVID-19 outcomes are most often attributed to classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes which significantly correlated with COVID-19 viral load, include many canonical innate immune genes. However, 22 much less studied "non-canonical" genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, with a tightly associated mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies validate the transcriptomic findings showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap findings in SARS-CoV-2 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early variants and later SARS-CoV-2 strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.

Most of the mast cells (MCs) in connective tissues, such as skin, are long-lived embryonic-derived immune cells that play important roles in host defense and various immunological diseases, including allergies. Their embryonic origin and ontogeny remain to be fully studied since several overlapping waves of embryonic hematopoiesis have been shown to give rise to these cells. Here, combining single-cell RNA sequencing and new genetic fate mapping models, we identified a Cpa3-expressing population sequentially appearing in the yolk sac, fetal liver, and peripheral tissues which gives rise to dermal MCs during embryonic days 11.5 to 14.5. Using in vitro differentiation and in vivo transplant methods, we identified a Ter119-F4/80- CD45+CD117+CD16/32+CD135-CD115-Ly6C-CD34lo population as potential fetal liver MC precursors. Fate mapping with Cpa3CreERT2and Zbtb16CreERT2 models, as well as the granulocyte-monocyte progenitor (GMP) models Ms4a3Cre and ElaneCre, demonstrated that MCs arise from Cpa3+ progenitors rather than Ms4a3+ or Elane+ GMPs. A corresponding population with a similar developmental trajectory was also identified in human early yolk sac and fetal liver, suggesting a conserved MC developmental program across species. These findings delineate the detailed developmental path of MCs in embryos, permitting future functional studies in immunity and development. HighlightsO_LICpa3-expressing cells in the yolk sac and fetal liver contain mast cell precursors C_LIO_LICpa3CreERT2 labels embryonic mast cells and their precursors C_LIO_LIEmbryonic mast cells arise through Cpa3+ mast cell precursors, but not Elane+/Ms4a3+ GMPs C_LIO_LIThe seeding of MCPs to embryonic skin slows down after E14.5 C_LI

ELF4 is an ETS family transcription factor involved in immune regulation, and germline loss-of-function mutations in ELF4 have been known as deficiency in ELF4, X-linked (DEX). To date, ELF4-related disease has been exclusively associated with germline mutations. Here, we report a pediatric patient with recurrent mucocutaneous inflammation and periodic fever caused by a somatic truncating mutation in ELF4. By directly comparing ELF4-mutant and wild-type immune cells within the same individual using full-length single-cell RNA sequencing, we identified mutation-associated transcriptional alterations across multiple immune cell types. Pathway analyses revealed cell type-specific immune alterations, characterized by reduced antiviral and interferon-related signaling in NK cells and enhanced inflammatory pathways related to Th17 differentiation and inflammatory bowel disease in CD16 monocytes. This study expands the disease spectrum of ELF4 deficiency by identifying somatic truncation of ELF4 as a genetic mechanism underlying autoinflammatory diseases and biased immune programs.

Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV (PLWH) despite treatment with combined anti-retroviral therapy (cART), but the underlying immune mechanisms are poorly understood. Here we report an altered immune landscape involving upregulation of TLR- and inflammasome signaling, localized CD4+ T cell hyperactivation, and counterintuitively, an enrichment of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in the oral mucosa of HIV+ patients on therapy. Using human oral tonsil cultures, we found that HIV infection causes an increase in a unique population of FOXP3+ cells expressing PD-1, IFN-{gamma}, Amphiregulin (AREG), and IL-10. These cells persisted even in the presence of the anti-retroviral drug and underwent further expansion driven by TLR-2 ligands and IL-1{beta}. IL-1{beta} also promoted PD-1 upregulation in AKT1 dependent manner. PD-1 stabilized FOXP3 and AREG expression in these cells through a mechanism requiring the activation of Asparaginyl Endopeptidase (AEP). Importantly, these FOXP3+ cells were incapable of suppressing CD4+ T cells in vitro. Concurrently, HIV+ patients harbored higher levels of PD-1, IFN-{gamma}, Amphiregulin (AREG), and IL-10 expressing FOXP3+ cells, which strongly correlated with CD4+ T cell hyperactivation, suggesting an absence of CD4+ T cell regulation in the oral mucosa. Taken together, this study provides insights into a novel mechanism of FOXP3+ cell dysregulation and reveals a critical link in the positive feedback loop of oral mucosal immune activation events in HIV+ patients on therapy. One Sentence SummaryHIV-induced immune dysfunction in lymphoid and mucosal tissues

ADAR1 edits adenosine to inosine (A-to-I) in double-stranded RNA to prevent MDA5 sensing of cellular transcripts, its key physiological role. However, editing-independent functions of ADAR1 remain poorly understood. Using a series of Adar1 mutant mice rescued by loss of MDA5 and PKR, we investigated isoform-specific, editing-independent roles of ADAR1 in vivo. We found that the cytoplasmic ADAR1p150 isoform is essential for maintaining peripheral T cell numbers and differentiation of hematopoietic stem and progenitor cells (HSPCs). In bone marrow transplants, ADAR1p150 protein, but not its editing activity, was crucial for T cell regeneration and HSPC repopulation, demonstrating a cell-intrinsic function in hematopoiesis. Experiments with IFN{beta}-treatment of purified HSPCs in vitro and in vivo IFNAR1 neutralization revealed hypersensitivity to tonic type I interferon (IFN) in the absence of ADAR1p150. Using cell lines, we demonstrate that type I IFN activates the OAS-RNaseL pathway, leading to cell death. This study shows that tonic type I IFN induces immunogenic cellular RNAs in sterile conditions. ADAR1p150 suppresses immune sensing of self-dsRNAs through both editing-dependent (MDA5) and editing-independent (PKR, OAS-RNaseL) mechanisms. Thus, ADAR1p150 protein levels and activity combine to set the threshold for tolerance to self-derived dsRNAs.

Donor CD4 T cell priming is a pivotal determinant of acute graft-versus-host disease (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). While professional hematopoietic antigen-presenting cells (APCs) have long been implicated in the pathogenesis of aGvHD, the contribution of non-hematopoietic APCs has remained unclear. Here, we show that naive alloreactive CD4 T cells initially localize and activate specifically within secondary lymphoid organs (SLOs) before infiltrating target tissues. Using genetic models to selectively ablate MHC class II on endothelial cells (ECs) or hematopoietic cells, we demonstrate that blood endothelial cells (BECs) in SLOs function as APCs, efficiently processing and presenting antigen to prime donor CD4 T cells. Deletion of MHC class II (MHCII) specifically in ECs substantially attenuates T cell activation and protects mice from lethal aGvHD, whereas selective deletion of MHCII in lymphatic ECs has no effect. Likewise, selective deletion of MHCII in hematopoietic cells also protects mice against aGvHD, suggesting that both cell types contribute to pathogenic allogeneic T cells activation after allo-HCT. Mechanistically, IL-12/IFN{gamma} signaling upregulates MHC class II expression on BECs. These findings identify BECs in SLOs as initiators of alloreactive CD4 T cell responses and highlight a potential target for preventing aGvHD. HighlightsO_LIBlood endothelial cells in secondary lymphoid organs prime naive CD4 T cells to trigger acute GvHD. C_LIO_LIT cell activation occurs exclusively in secondary lymphoid organs before tissue infiltration. C_LIO_LIExpression of MHC class II only on endothelial cells is sufficient to drive lethal GvHD, independent of other antigen presenting cells. C_LIO_LIRegulation of MHC class II expression in blood endothelial cells by IL-12/IFN{gamma} offers the potential for new therapeutic targets and corroborates findings for existing therapeutics. C_LI

Hematopoietic stem cells (HSCs) are localized within specialized niches of the bone marrow (BM). However, during hematological disorders or infections, the functionality of HSCs in the BM is compromised, leading to extramedullary hematopoiesis (EMH). Chronic inflammation drives EMH, yet its impact on HSCs outside the BM is poorly understood. Using a mouse model of chronic autoinflammatory disease, we demonstrated the presence of extramedullary HSCs in blood, spleen, and inflamed tails and paws. Single-cell transcriptomics revealed a unique expression profile in extramedullary HSCs, with significant upregulation of Cd53, MHCII-associated, and immunosuppressive genes. We further demonstrated that extramedullary CD53+ HSCs act as antigen-presenting cells, promoting the development of regulatory T cells (Tregs) to control chronic inflammation at extramedullary sites. Conversely, Tregs exert a protective role on extramedullary HSCs. Altogether, our findings revealed a mutually supportive relationship between a unique subset of HSCs and T cells in inflamed tissues during chronic inflammation.

Acquired mutations in the UBA1 gene, occurring in myeloid cells and resulting in expression of a catalytically impaired isoform of the enzyme E1, were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). The precise physiological and clinical impact of these mutations remains poorly defined. Here, we studied a unique prospective cohort of individuals with severe autoinflammatory disease with (VEXAS) or without (VEXAS-like) UBA1 somatic mutations and compared with low-risk myelodysplastic syndromes (MDS) and aged gender-matched healthy controls. We performed an integrated immune analysis including multiparameter phenotyping of peripheral blood leukocytes, cytokines profiling, bulk and single-cell gene expression analyses and skin tissue imaging mass cytometry. Focusing on myeloid cells, we show that monocytes from UBA1-mutated individuals were quantitatively and qualitatively impaired and displayed features of exhaustion with aberrant expression of chemokine receptors. Within affected tissues, pathological skin biopsies from VEXAS patients showed an abundant enrichment of CD16+ CD163+ monocytes adjacent to blood vessels and M1 macrophages, possibly promoting local inflammation in part through STAT3 activation. In peripheral blood from VEXAS patients, we identified a significant increase in circulating levels of many proinflammatory cytokines, including IL-1{beta} and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirmed the role of circulating cells in the IL-1{beta} and IL-18 dysregulation in VEXAS patients and revealed a significant enrichment of TNF- and NF{kappa}B signaling pathways that could mediate cell death and inflammation. Single-cell analysis confirmed the inflammatory state of monocytes from VEXAS patients and allowed us to identify specific molecular pathways that could explain monocytopenia, especially the activation of PANoptosis and a deficiency in the TYROBP/DAP12 axis and {beta}-catenin signaling pathway. Together, these findings on monocytes from patients with UBA1 mutations provide important insights into the molecular mechanisms involving the mature myeloid commitment in VEXAS syndrome and suggest that the control of the undescribed inflammasome activation and PANoptosis could be novel therapeutic targets in this condition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/22281005v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@10a14e4org.highwire.dtl.DTLVardef@e1b88eorg.highwire.dtl.DTLVardef@11bb0a2org.highwire.dtl.DTLVardef@e971ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Innate immune cellular effectors are actively consumed during systemic inflammation but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF{kappa}B independent, Traf6/I{kappa}B-kinase/SNAP23 activation which is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 (cDC2) and upregulation of BM myeloid progenitor Ccr7 signaling. The consequence of this progenitor traffic is anti-inflammatory with promotion of early survival and initiation of replenishment of lymph node cDC.

The detection of cytosolic dsDNA is tightly regulated to avoid pathological inflammatory responses. A major pathway involved in their detection relies on the cyclic GMP-AMP synthase (cGAS) that triggers activation of the Stimulator of interferon genes (STING) which subsequently drives the expression of inflammatory genes and type I Interferons (IFNs). Here, we show that the methyl-CpG-binding protein 2 (MECP2), a major transcriptional regulator, controls dsDNA-associated inflammatory responses. We show that the presence of cytosolic dsDNA promotes MECP2 export from the nucleus to the cytosol where it interacts with dsDNA, dampening cGAS activation. Our data also indicate that MECP2 export from the nucleus partially phenocopies MECP2 deficiency, leading to the expression of inflammatory and interferon stimulated genes, enforcing an antiviral state. Finally, we also show that MECP2 displacement from the nucleus following dsDNA stimulation is sufficient to disrupt its canonical function, leading to the reactivation of otherwise repressed genes, such endogenous retroelements of the Long interspersed nuclear element-1 (LINE-1) family. Re-expression of the latter led to the accumulation of DNA species feeding cGAS-dependent signaling and can be dampened by reverse transcriptase inhibitors. We thus establish a previously unforeseen direct role of MECP2 in the regulation of the breadth and nature of dsDNA-associated inflammatory responses. Furthermore, our results suggest that targeting dsDNA-associated pathways or pharmacological inhibition of LINE-1 may bear therapeutic hopes for Rett syndrome (RTT) patients that present with MECP2 deficiency.

Autoimmunity develops as a result of a breakdown in immune tolerance and activation of autoreactive immune cells. Most of the common autoimmune diseases are polygenic1 suggesting dysregulation in multiple signalling pathways. By contrast, in monogenic Inborn Errors of Immunity (IEI), which also can result in autoimmunity, the disease is triggered by a single genetic defect. Therefore, the discovery of causative mutations in IEI allows tracing the molecular mechanisms leading to autoimmunity in humans from a defect in the function of a specific gene to patients clinical and immunological phenotype. Here, we discovered an IEI patient with systemic autoimmunity caused by a private homozygous protein-truncating mutation in gene ZC3H12A leading to deficiency of Regnase-1, a regulatory RNase2-5. Flow cytometry, bulk T cell transcriptome analysis and single-cell RNA sequencing demonstrated expansion of {gamma}{delta} T cells expressing VCAM-1 and IFN{gamma} genes. We show that Regnase-1 directly targets 3UTR of VCAM1 and the coding sequence of IFNG mRNAs. These findings highlight a new autoimmunity mechanism in humans, where Regnase-1 deficiency causes expansion of VCAM1+IFNG+ T cells and their interaction with integrin 4{beta}1-expressing B cells, which showed upregulation of IFN-response genes and activation, leading to systemic autoimmunity. Furthermore, we show that VCAM1+ T cells are present in organs of donors and are expanded in the blood of patients with systemic lupus erythematosus, a common autoimmune disease characterised by systemic autoimmunity.

The blood-thymus-barrier (BTB) is essential for regulating the entry of thymic-seeding progenitors into the thymus, an organ lacking self-renewing T cell progenitors. Here, we identify Early B-cell factor 1 (Ebf1), a transcription factor with established roles in hematopoiesis, as specifically expressed in thymic endothelial cells. Tie2-Cre-mediated deletion of Ebf1 results in a significant reduction of early thymic progenitors (ETPs), despite unaltered bone marrow lymphoid progenitor populations, unaltered thymic stromal subset frequencies, and intact downstream thymocyte differentiation. Transcriptome analysis of Ebf1-deficient thymic endothelial cells reveals increased expression of tight-junction-associated genes, in particular Cldn5, a key tight-junction component that restricts the BTB. Consistent with reduced BTB permeability, ETP subset analysis demonstrates a proportional reduction in the ETP1 fraction, the progenitor population that resides in closest proximity to thymic endothelial cells at the cortico-medullary junction. Together, our study implicates Ebf1 in the regulation of the BTB permeability, through modulation of tight-junction programs, thereby enabling thymus-seeding progenitor entry.

Persistent production of type I interferons (IFN-Is) is a hallmark of cutaneous lupus erythematosus (CLE). Ultraviolet (UV) light stimulates IFN-I response in the skin and exacerbates CLE. Here, we identify V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) as a negative regulator of both basal and UV-induced IFN-I responses in the skin and show that VISTA limits skin photosensitivity in an IFN-I- dependent manner, in part through Stimulator of Interferon Genes (STING). Furthermore, we demonstrate a novel role for VISTA in keratinocytes both at steady state and in response to UV light. Conditional deletion of VISTA in epidermal keratinocytes results in >10-fold increase in the basal skin IFN-I score and a heightened UV-induced skin injury score, which is dependent on IFN-I signaling. VISTA-targeting monoclonal antibodies suppress UV-induced IFN-I response in human keratinocytes and in mice expressing human VISTA in vivo, which also reduces UV-induced skin injury score. Our findings highlight VISTA as a potential therapeutic target for suppressing IFN-I responses in cutaneous lupus patients who suffer from UV-induced skin inflammation.

High levels of extracellular adenosine, highly abundant in the tumor microenvironment, promote immune suppression mainly through the A2AR expressed by tumor-infiltrating immune cells. Here we show that plasma cells are the most negatively affected by adenosine among the immune cells present in the tumor microenvironment. Furthermore, both tonsillar and tumor-associated B cells, including germinal center (GC)-like B cells, plasma cells and plasma blasts (PCs and PBs, respectively, and collectively referred to as antibody-secreting cells (ASCs), express high levels of A2AR. Given the importance of tumor-infiltrating B and PCs in antitumor responses, we investigated how adenosine impairs their numbers and function. Triggering of A2AR inhibited B cell maturation into ASCs and immunoglobulin production in vitro, and impaired upregulation of PC genes upon stimulation. These effects were restored by inupadenant (EOS100850), a potent and highly selective small molecule A2AR antagonist. Spatial transcriptomics analysis of tumor biopsies from patients treated with inupadenant revealed that ASCs specifically increased in tertiary lymphoid structures. Altogether, these data demonstrate that A2AR plays a key role in adenosine-mediated inhibition of B cell maturation toward ASCs through a B cell-intrinsic mechanism, and that this effect is fully reverted by inupadenant.

The NLRP3 (NACHT-, leucine-rich repeat [LRR]- and pyrin domain [PYD]- containing protein 3) inflammasome is a cytoplasmic signaling complex that promotes inflammation in response to signals from infection and cellular damage. Increased activation of the NLRP3 inflammasome is linked to numerous diseases including gout, osteoarthritis, cardiovascular disease, neuroinflammatory diseases and cancer, which has prompted the search for therapeutics that can inhibit the NLRP3 pathway. Recent work suggested that NIMA-related kinase 7 (NEK7) may be required for proper assembly and activation of the NLRP3 inflammasome independent of its kinase activity, and that hence reduction of NEK7 protein levels may block NLRP3 activation. Since NEK7 contains a glycine beta hairpin loop structural degron found in many targets of cereblon (CRBN) molecular glue degraders, we used this strategy to degrade NEK7 and inhibit the NLRP3 inflammasome. We identified NK7-902, a CRBN glue degrader of NEK7. NK7-902 potently and selectively degraded NEK7 in human primary monocytes, peripheral blood mononuclear cells (PBMCs) and whole blood. Unexpectedly, full NEK7 degradation led to only partial blockade of NLRP3-dependent interleukin-1{beta} (IL-1{beta}) release in these cells under different stimulation conditions, with the extent of IL-1{beta} inhibition varying greatly across donors. Unlike most CRBN glue degraders, NK7-902 degraded NEK7 in mouse cells and efficiently inhibited NLRP3-dependent IL-1{beta} release in a mouse cryopyrin-associated syndrome (CAPS) model. By contrast, oral administration of NK7-902 in non-human primates led to profound and long lasting NEK7 degradation but only transiently blocked IL-1{beta} production in blood. Collectively, our data suggest that NEK7 is involved but may not be strictly required for NLRP3 activation in primates and humans. HighlightsO_LIIdentification of NK7-902, a CRBN molecular glue degrader of NEK7 C_LIO_LINK7-902 fully degrades NEK7 in human primary monocytes and whole blood but only partially inhibits NLRP3-dependent IL-1{beta} production C_LIO_LIUnlike most CRBN glue degraders, NK7-902 shows activity in murine systems C_LIO_LIIn vivo oral administration of NK7-902 in non-human primates leads to profound and long-lasting NEK7 degradation but only transiently blocks NLRP3 inflammasome activation C_LI

Homozygous OTU deubiquitinase with linear linkage specificity (OTULIN) variants cause OTULIN-Related Autoinflammatory Syndrome (ORAS). This disease is characterized by early-onset autoinflammation, fever, panniculitis, diarrhea, and arthritis. In contrast, heterozygous and compound-heterozygous OTULIN variants have been associated with a phenotype defined by abscess development in different organs. Whether homozygous OTULIN variants can cause abscessing in affected patients is currently unknown. Here, we report a juvenile female patient harboring a novel homozygous OTULIN variant (Chr5:14687605G>T, p.V185F; referred to as OTULINV185F), presenting with autoinflammation and sterile abscesses in lung and skin. Through in silico analysis and functional assays, we show that OTULINV185F impairs OTULIN function, leading to compromised degradation of linear ubiquitin linkages. Notably, the patient clinically improved on anti-TNF therapy. Our findings underscore the diverse clinical manifestations of OTULIN dysfunction and call for a new classification of the disease that includes abscess formation as potential ORAS symptom.

The D301N NLRP3 mutation in mice (D303N in humans) causes severe multi-organ damage and early death driven by the constitutively activated NLRP3 (NLRP3ca) inflammasome. Triggered inflammasomes activate caspase-1 to process IL-1 family cytokines and gasdermin D (GSDMD), generating N-terminal fragments, which oligomerize within the plasma membrane to form pores, which cause inflammatory cell death (pyroptosis) and through which IL-1{beta} and IL-18 are secreted. GSDMD activation is central to disease symptoms since spontaneous inflammation in Nlrp3ca;Gsdmd-/- mice is negligible. Unexpectedly, when Nlrp3ca;Gsdmd-/- mice were challenged with LPS or TNF-, they secreted high amounts of IL-1{beta} and IL-18, suggesting an alternative GSDMD-independent inflammatory pathway. Here we show that GSDMD deficient macrophages subjected to inflammatory stimuli activate caspase-8, -3 and GSDME-dependent cytokine release and pyroptosis. Caspase-8, -3 and GSDME also activated pyroptosis when NLRP3 was stimulated in caspase-1 deficient macrophages. Thus, a salvage caspase-8, -3-GSDME inflammatory pathway is activated following NLRP3 activation when the canonical NLRP3-caspase-1-GSDMD is blocked. Surprisingly, the active metabolite of the GSDMD-inhibitor disulfiram, inhibited not only GSDMD but also GSDME-mediated inflammation in vitro and suppressed severe inflammatory disease symptoms in Nlrp3ca mice, a model for severe neonatal multisystem inflammatory disease. Although disulfiram did not directly inhibit GSDME, it suppressed inflammasome activation in GSDMD-deficient cells. Thus, the combination of inflammatory signals and NLRP3ca overwhelmed the protection provided by GSDMD deficiency, rewiring signaling cascades through caspase-8, -3 and GSDME to propagate inflammation. This functional redundancy suggests that concomitant inhibition of GSDMD and GSDME may be necessary to suppress disease in inflammasomopathy patients.