Loss of T follicular regulatory cell-derived IL-1R2 augments germinal center reactions via increased IL-1.

Inappropriate immune activity is key in the pathogenesis of multiple diseases, and it is typically driven by excess inflammation and/or autoimmunity. IL-1 is often the effector owing to its powerful role in both innate and adaptive immunity, and, thus, it is tightly controlled at multiple levels. IL-1R2 antagonizes IL-1, but effects of losing this regulation are unknown. We found that IL-1R2 resolves inflammation by rapidly scavenging free IL-1. Specific IL-1R2 loss in germinal center (GC) T follicular regulatory (Tfr) cells increased the GC response after a first, but not booster, immunization, with an increase in T follicular helper (Tfh) cells, GC B cells, and antigen-specific antibodies, which was reversed upon IL-1 blockade. However, IL-1 signaling is not obligate for GC reactions, as WT and Il1r1-/- mice showed equivalent phenotypes, suggesting that GC IL-1 is normally restrained by IL-1R2. Fascinatingly, germline Il1r2-/- mice did not show this phenotype, but conditional Il1r2 deletion in adulthood recapitulated it, implying that compensation during development counteracts IL-1R2 loss. Finally, patients with ulcerative colitis or Crohn's disease had lower serum IL-1R2. All together, we show that IL-1R2 controls important aspects of innate and adaptive immunity and that IL-1R2 level may contribute to human disease propensity and/or progression.

Human vascular smooth muscle cells utilise chymase for the atypical cleavage and activation of Interleukin-1ß.

BACKGROUND AND AIMS: Atherosclerosis and other cardiovascular diseases (CVD) are well established to be both instigated and worsened by inflammation. Indeed, CANTOS formally proved that targeting the inflammatory cytokine IL-1ß only could reduce both cardiovascular events and death. However, due to the central role of IL-1ß in host defence, blockade increased fatal infections, suggesting targeting key immune mediators over the long natural history of CVD is unsuitable. Thus, discovering alternative mechanisms that generate vascular inflammation may identify more actionable targets. METHODS: We used primary human VSMCs and a combination of biochemical, pharmacological and molecular biological techniques to generate the data. Human carotid atherosclerotic plaques were also assessed histologically. RESULTS: We showed that VSMCs expressed and efficiently processed pro-IL-1ß to the active form after receiving a single stimulus via IL-1R1 or TLR4. Importantly, pro-IL-1ß processing did not utilise inflammasomes or caspases. Unusually, we found that cathepsin C-activated chymase was responsible for cleaving IL-1ß in VSMCs, and provided evidence for chymase expression in cultured VSMCs and in the fibrous cap of human plaques. Chymase also efficiently cleaved and activated recombinant pro-IL-1ß. CONCLUSIONS: Thus, VSMCs are efficient activators of IL-1ß that do not use canonical inflammasomes or caspases. Hence, this alternative pathway could be targeted for long-term treatment of CVDs, as it is not central to everyday host defence.

Thrombin-activated interleukin-1α drives atherogenesis, but also promotes vascular smooth muscle cell proliferation and collagen production.

AIMS: Atherosclerosis is driven by multiple processes across multiple body systems. For example, the innate immune system drives both atherogenesis and plaque rupture via inflammation, while coronary artery-occluding thrombi formed by the coagulation system cause myocardial infarction and death. However, the interplay between these systems during atherogenesis is understudied. We recently showed that coagulation and immunity are fundamentally linked by the activation of interleukin-1α (IL-1α) by thrombin, and generated a novel knock-in mouse in which thrombin cannot activate endogenous IL-1α [IL-1α thrombin mutant (IL-1αTM)]. METHODS AND RESULTS: Here, we show significantly reduced atherosclerotic plaque formation in IL-1αTM/Apoe-/- mice compared with Apoe-/- and reduced T-cell infiltration. However, IL-1αTM/Apoe-/- plaques have reduced vascular smooth muscle cells, collagen, and fibrous caps, indicative of a more unstable phenotype. Interestingly, the reduced atherogenesis seen with thrombin inhibition was absent in IL-1αTM/Apoe-/- mice, suggesting that thrombin inhibitors can affect atherosclerosis via reduced IL-1α activation. Finally, bone marrow chimeras show that thrombin-activated IL-1α is derived from both vessel wall and myeloid cells. CONCLUSIONS: Together, we reveal that the atherogenic effect of ongoing coagulation is, in part, mediated via thrombin cleavage of IL-1α. This not only highlights the importance of interplay between systems during disease and the potential for therapeutically targeting IL-1α and/or thrombin, but also forewarns that IL-1 may have a role in plaque stabilization.

The common IL1A single nucleotide polymorphism rs17561 is a hypomorphic mutation that significantly reduces interleukin-1α release from human blood cells.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that drives inflammation and modulates adaptive immunity. Due to these powerful effects, IL-1α is controlled at multiple levels from transcription to cleavage and release from the cell. Genome-wide association studies can identify loci that drive important diseases, although often the functional effect of the variant on phenotype remains unknown or small, with most risk variants in non-coding regions. We find that the common variant rs17561 changes a conserved amino acid in the central region of IL-1α linking the pro piece to the cytokine domain. Using a recall-by-genotype study and whole blood stimulation, we find that minor allele homozygotes release ~50% less IL-1α than the major allele, with IL-1ß release equivalent. IL-1α transcript level was identical between groups, implying a post-transcriptional effect, whilst cleavage of recombinant pro-IL-1α by multiple proteases was also equivalent for both forms. Importantly, transfected macrophages also release less minor allele IL-1α upon inflammasome activation, revealing that reduced secretion is directly caused by the missense amino acid substitution and more minor allele IL-1α was retained within the cell. Thus, rs17561 represents a very common hypomorphic mutation in IL-1α. We believe this novel data will be important for determining the potential contribution of IL-1α to disease and/or physiological processes, for example, by Mendelian randomisation, and may aid patient stratification when considering anti-IL-1 therapies.

Cell surface IL-1α trafficking is specifically inhibited by interferon-γ, and associates with the membrane via IL-1R2 and GPI anchors.

IL-1 is a powerful cytokine that drives inflammation and modulates adaptive immunity. Both IL-1α and IL-1ß are translated as proforms that require cleavage for full cytokine activity and release, while IL-1α is reported to occur as an alternative plasma membrane-associated form on many cell types. However, the existence of cell surface IL-1α (csIL-1α) is contested, how IL-1α tethers to the membrane is unknown, and signaling pathways controlling trafficking are not specified. Using a robust and fully validated system, we show that macrophages present bona fide csIL-1α after ligation of TLRs. Pro-IL-1α tethers to the plasma membrane in part through IL-1R2 or via association with a glycosylphosphatidylinositol-anchored protein, and can be cleaved, activated, and released by proteases. csIL-1α requires de novo protein synthesis and its trafficking to the plasma membrane is exquisitely sensitive to inhibition by IFN-γ, independent of expression level. We also reveal how prior csIL-1α detection could occur through inadvertent cell permeabilisation, and that senescent cells do not drive the senescent-associated secretory phenotype via csIL-1α, but rather via soluble IL-1α. We believe these data are important for determining the local or systemic context in which IL-1α can contribute to disease and/or physiological processes.

IL-1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence-associated secretory phenotype.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1ß release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION.

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.

Senescent Vascular Smooth Muscle Cells Drive Inflammation Through an Interleukin-1α-Dependent Senescence-Associated Secretory Phenotype.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown. APPROACH AND RESULTS: We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization. CONCLUSIONS: Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α-driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1ß.

Intracellular interleukin-1 receptor 2 binding prevents cleavage and activity of interleukin-1α, controlling necrosis-induced sterile inflammation.

Necrosis can induce profound inflammation or be clinically silent. However, the mechanisms underlying such tissue specificity are unknown. Interleukin-1α (IL-1α) is a key danger signal released upon necrosis that exerts effects on both innate and adaptive immunity and is considered to be constitutively active. In contrast, we have shown that necrosis-induced IL-1α activity is tightly controlled in a cell type-specific manner. Most cell types examined expressed a cytosolic IL-1 receptor 2 (IL-1R2) whose binding to pro-IL-1α inhibited its cytokine activity. In cell types exhibiting a silent necrotic phenotype, IL-1R2 remained associated with pro-IL-1α. Cell types possessing inflammatory necrotic phenotypes either lacked IL-1R2 or had activated caspase-1 before necrosis, which degraded and dissociated IL-1R2 from pro-IL-1α. Full IL-1α activity required cleavage by calpain after necrosis, which increased its affinity for IL-1 receptor 1. Thus, we report a cell type-dependent process that fundamentally governs IL-1α activity postnecrosis and the mechanism allowing conditional release of this blockade.

The plastidic bile acid transporter 5 is required for the biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana.

Aliphatic glucosinolate biosynthesis is highly compartmentalized, requiring import of 2-keto acids or amino acids into chloroplasts for side chain elongation and export of the resulting compounds into the cytosol for conversion into glucosinolate. Aliphatic glucosinolate biosynthesis in Arabidopsis thaliana is regulated by three R2R3-MYB transcription factors, the major player being High Aliphatic Glucosinolate 1 (HAG1/MYB28). Here, we show that BAT5, which belongs to the putative bile acid transporter family, is the only member of this family that is transactivated by HAG1/MYB28, HAG2/MYB76, and HAG3/MYB29. Furthermore, two isopropylmalate isomerases genes, IPMI1 and IPMI2, and the isopropylmalate dehydrogenase gene, IPMDH1, were identified as targets of HAG1/MYB28 and the corresponding proteins localized to plastids, suggesting a role in plastidic chain elongation reactions. The BAT proteins also localized to plastids; however, only mutants defective in BAT5 function contained strongly reduced levels of aliphatic glucosinolates. The bat5 mutant chemotype was rescued by induced overexpression of BAT5. Feeding experiments using 2-keto acids and amino acids of different chain length suggest that BAT5 is a plastidic transporter of (chain-elongated) 2-keto acids. Mechanical stimuli and methyl jasmonate transiently induced BAT5 expression in inflorescences and leaves. Thus, BAT5 was identified as the first transporter component of the aliphatic glucosinolate biosynthetic pathway.