Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis.

Inflammasomes are multiprotein complexes that function as sensors of endogenous or exogenous damage-associated molecular patterns. Here, we show that deficiency of NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. NLRP6 inflammasome-deficient mice were characterized by spontaneous intestinal hyperplasia, inflammatory cell recruitment, and exacerbation of chemical colitis induced by exposure to dextran sodium sulfate (DSS). Cross-fostering and cohousing experiments revealed that the colitogenic activity of this microbiota is transferable to neonatal or adult wild-type mice, leading to exacerbation of DSS colitis via induction of the cytokine, CCL5. Antibiotic treatment and electron microscopy studies further supported the role of Prevotellaceae as a key representative of this microbiota-associated phenotype. Altogether, perturbations in this inflammasome pathway, including NLRP6, ASC, caspase-1, and IL-18, may constitute a predisposing or initiating event in some cases of human IBD.

Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2.

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.

Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

NF-κB-Independent Role of IKKα/IKKß in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling.

TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKß or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

MALT1 Protease Plays a Dual Role in the Allergic Response by Acting in Both Mast Cells and Endothelial Cells.

The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

The NLRP12 inflammasome recognizes Yersinia pestis.

Yersinia pestis, the causative agent of plague, is able to suppress production of inflammatory cytokines IL-18 and IL-1ß, which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. Here, we sought to elucidate the role of NLRs and IL-18 during plague. Lack of IL-18 signaling led to increased susceptibility to Y. pestis, producing tetra-acylated lipid A, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. We found that the NLRP12 inflammasome was an important regulator controlling IL-18 and IL-1ß production after Y. pestis infection, and NLRP12-deficient mice were more susceptible to bacterial challenge. NLRP12 also directed interferon-γ production via induction of IL-18, but had minimal effect on signaling to the transcription factor NF-κB. These studies reveal a role for NLRP12 in host resistance against pathogens. Minimizing NLRP12 inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis.

Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells.

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-ß Synthesis Induced by Lipopolysaccharide.

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-ß. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-ß production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-ß synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-ß production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-ß production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-ß is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-ß during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.

S100A12 and S100A8/9 proteins are biomarkers of articular disease activity in Blau syndrome.

OBJECTIVE: To identify biomarkers of articular and ocular disease activity in patients with Blau syndrome (BS). METHODS: Multiplex plasma protein arrays were performed in five BS patients and eight normal healthy volunteers (NHVs). Plasma S100A12 and S100A8/9 were subsequently measured by ELISA at baseline and 1-year follow-up in all patients from a prospective multicentre cohort study. CRP was measured using Meso Scale Discovery immunoassay. Active joint counts, standardization uveitis nomenclature for anterior uveitis cells and vitreous haze by Nussenblatt scale were the clinical parameters. RESULTS: Multiplex Luminex arrays identified S100A12 as the most significantly elevated protein in five selected BS vs eight NHVs and this was confirmed by ELISA on additional samples from the same five BS patients. In the patient cohort, S100A12 (n = 39) and S100A8/9 (n = 33) were significantly higher compared with NHVs (n = 44 for S100A12, n = 40 for S100A8/9) (P = 0.0000004 and P = 0.0003, respectively). Positive correlations between active joint counts and S100 levels were significant for S100A12 (P = 0.0008) and S100A8/9 (P = 0.015). CRP levels did not correlate with active joint count. Subgroup analysis showed significant association of S100 proteins with active arthritis (S100A12 P = 0.01, S100A8/9 P = 0.008). Active uveitis was not associated with increased S100 levels. CONCLUSION: S100 proteins are biomarkers of articular disease activity in BS and potential outcome measures in future clinical trials. As secreted neutrophil and macrophage products, S100 proteins may reflect the burden of granulomatous tissue in BS.