Avril A. B. Robertson

A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.

The NOD-like receptor (NLR) family, pyrin domain–containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimers disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1β (IL-1β) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle–Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.

Clonal hematopoiesis associated with Tet2 deficiency accelerates atherosclerosis development in mice

Faulty blood cells and heart disease Recent studies have shown that elderly peoples blood cells often harbor mutations in genes encoding certain epigenetic regulators. These mutations can lead to clonal expansion of the mutant blood cells, which increases the risk of blood cancers and cardiovascular disease. Fuster et al. generated a mouse model to investigate how one of these genes, Tet2, affects atherosclerosis development (see the Perspective by Zhu et al.). They found that the disease progressed more rapidly in mice transplanted with Tet2-deficient bone marrow cells. This was due to increased secretion of interleukin-1β by Tet2-deficient macrophages in a process that depended on the action of inflammasomes. Science, this issue p. 842; see also p. 798 Bone marrow deficient in a gene frequently mutated in blood cells of elderly humans promotes atherosclerosis in mice. Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr–/–) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome–mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis.

T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

Innate immune crosstalk in T cells The classical view of immune activation is that innate immune cells, such as macrophages and dendritic cells, recognize invading microbes and then alert adaptive immune cells, such as T cells, to respond. Arbore et al. now show that innate and adaptive immunity converge in human and mouse T cells. Activated T cells express components of the complement cascade, which in turn leads to the assembly of NLRP3 inflammasomes—both critical components of innate immunity that help hosts detect and eliminate microbes. In T cells, complement and inflammasomes work together to push T cells to differentiate into a specialized subset of T cells important for eliminating intracellular bacteria. Science, this issue p. 10.1126/science.aad1210 Complement and NLRP3 inflammasomes work together to promote T helper 1 cell differentiation. INTRODUCTION The inflammasomes and the complement system are traditionally viewed as quintessential components of innate immunity required for the detection and elimination of pathogens. Assembly of the NLRP3 inflammasome in innate immune cells controls the maturation of interleukin (IL)–1β, a proinflammatory cytokine critical to host defense, whereas activation of the liver-derived complement key components C3 and C5 in serum leads to opsonization and removal of microbes and induction of the inflammatory reaction. Recent studies, however, have highlighted an unanticipated direct role for complement C3 also in human T cell immunity: The anaphylatoxin C3a receptor (C3aR) and the complement regulator CD46 (which binds C3b) are critical checkpoints in human T cell lineage commitment, and they control initiation and resolution of T helper 1 (TH1) responses in an autocrine fashion via T cell–derived and intracellularly activated C3. We explored a novel functional cross-talk of complement with the NLRP3 inflammasome within CD4+ T cells and determined how the cooperation between these two “classically” innate systems directly affects interferon-γ (IFN-γ) production by adaptive immune cells. RATIONALE Given the critical role of intracellular C3 activation in human TH1 responses and the importance of C5 activation products in inflammation, we investigated whether human CD4+ T cells also harbor an “intracellular C5 activation system” and by what means this system may contribute to effector responses by using C5aR1 and C5aR2 agonists and antagonists, T cells from patients with cryopyrin-associated periodic syndromes (CAPS), and mouse models of infection and autoimmunity. RESULTS Human CD4+ T cells expressed C5 and generated increased intracellular C5a upon T cell receptor activation and CD46 autocrine costimulation. Subsequent engagement of the intracellular C5aR1 by C5a induced the generation of reactive oxygen species (ROS) and the unexpected assembly of a functional NLRP3 inflammasome in CD4+ T cells, whereas the surface-expressed C5aR2 negatively controlled this process. NLRP3 inflammasome–dependent autocrine IL-1β secretion and activity were required for optimal IFN-γ production by T cells; consequently, dysregulation of NLRP3 function in these cells affected their normal effector responses. For example, mutated, constitutively active NLRP3 in T cells from patients with CAPS induced hyperactive TH1 responses that could be normalized with a NLRP3 inhibitor. The in vivo importance of a T cell–intrinsic NLRP3 inflammasome was further supported by the finding that IFN-γ production by Nlrp3–/– CD4+ T cells was significantly reduced during viral infections in mice and that diminished TH1 induction due to lack of NLRP3 function in a CD4+ T cell transfer model of colitis led to uncontrolled TH17 infiltration and/or expansion in the intestine and aggravated disease. CONCLUSION Our results demonstrate that the regulated cross-talk between intracellularly activated complement components (the “complosome”) and the NLRP3 inflammasome is fundamental to human TH1 induction and regulation. The finding that established innate immune pathways are also operative in adaptive immune cells and orchestrate immunological responses contributes to our understanding of immunobiology and immune system evolution. In addition, the results suggest that the complement-NLRP3 axis in T cells represents a novel therapeutic target for the modulation of TH1 activity in autoimmunity and infection. An intrinsic complement-NLRP3 axis regulates human TH1 responses. T cell receptor activation and CD46 costimulation trigger NLRP3 expression and intracellular C5a generation. Subsequent intracellular C5aR1 engagement induces ROS production (and possibly IL1B gene transcription) and NLRP3 assembly, which in turn mediates IL-1β maturation. Autocrine IL-1β promotes TH1 induction (IFN-γ production) but restricts TH1 contraction (IL-10 coexpression). C5aR2 cell surface activation by secreted C5a negatively controls these events via undefined mechanisms. Dysfunction of this system contributes to impaired TH1 responses in infection or increased TH17 responses during intestinal inflammation. The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4+ T cells and initiates caspase-1–dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (TH1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to “innate immune cells” but is an integral component of normal adaptive TH1 responses.

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

BACKGROUND & AIMS NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223-/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1&bgr; was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1&bgr; or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3′ untranslated region, phenocopied the characteristics of miR-223-/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1&bgr; release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation.

The selective NLRP3-inflammasome inhibitor MCC950 reduces infarct size and preserves cardiac function in a pig model of myocardial infarction.

Aims Myocardial infarction (MI) triggers an intense inflammatory response that is associated with infarct expansion and is detrimental for cardiac function. Interleukin (IL)-1β and IL-18 are key players in this response and are controlled by the NLRP3-inflammasome. In the current study, we therefore hypothesized that selective inhibition of the NLRP3-inflammasome reduces infarct size and preserves cardiac function in a porcine MI model. Methods and results Thirty female landrace pigs were subjected to 75 min transluminal balloon occlusion and treated with the NLRP3-inflammasome inhibitor MCC950 (6 or 3 mg/kg) or placebo for 7 days in a randomized, blinded fashion. After 7 days, 3D-echocardiography was performed to assess cardiac function and Evans blue/TTC double staining was executed to assess the area at risk (AAR) and infarct size (IS). The IS/AAR was lower in the 6 mg/kg group (64.6 ± 8.8%, P = 0.004) and 3 mg/kg group (69.7 ± 7.2%, P = 0.038) compared with the control group (77.5 ± 6.3%). MCC950 treatment markedly preserved left ventricular ejection fraction in treated animals (6 mg/kg 47 ± 8%, P = 0.001; 3 mg/kg 45 ± 7%, P = 0.031; control 37 ± 6%). Myocardial neutrophil influx was attenuated in treated compared with non-treated animals (6 mg/kg 132 ± 72 neutrophils/mm2, P = 0.035; 3 mg/kg 207 ± 210 neutrophils/mm2, P = 0.5; control 266 ± 158 neutrophils/mm2). Myocardial IL-1β levels were dose-dependently reduced in treated animals. Conclusions NLRP3-inflammasome inhibition reduces infarct size and preserves cardiac function in a randomized, blinded translational large animal MI model. Hence, NLRP3-inflammasome inhibition may have therapeutic potential in acute MI patients.

Role for NLRP3 Inflammasome-mediated, IL-1β-dependent Responses in Severe, Steroid-resistant Asthma.

&NA; Rationale: Severe, steroid‐resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. Excessive nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing 3 (NLRP3) inflammasome and concomitant IL‐1&bgr; responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid‐resistant asthma. Objectives: To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL‐1&bgr; in severe, steroid‐resistant asthma. Methods: We developed mouse models of Chlamydia and Haemophilus respiratory infection‐mediated, ovalbumin‐induced severe, steroid‐resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL‐1&bgr; responses. The roles and potential for targeting of NLRP3 inflammasome, caspase‐1, and IL‐1&bgr; responses in experimental severe, steroid‐resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase‐1 inhibitor Ac‐YVAD‐cho; and neutralizing anti‐IL‐1&bgr; antibody. Roles for IL‐1&bgr;‐induced neutrophilic inflammation were examined using IL‐1&bgr; and anti‐Ly6G. Measurements and Main Results: Chlamydia and Haemophilus infections increase NLRP3, caspase‐1, IL‐1&bgr; responses that drive steroid‐resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL‐1&bgr; expression. Treatment with anti‐IL‐1&bgr;, Ac‐YVAD‐cho, and MCC950 suppressed IL‐1&bgr; responses and the important steroid‐resistant features of disease in mice, whereas IL‐1&bgr; administration recapitulated these features. Neutrophil depletion suppressed IL‐1&bgr;‐induced steroid‐resistant airway hyperresponsiveness. Conclusions: NLRP3 inflammasome responses drive experimental severe, steroid‐resistant asthma and are potential therapeutic targets in this disease.

The NLRP3 Inflammasome functions as a driver of the myelodysplastic syndrome phenotype.

Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and β-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention.

Evaluation of liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry for the identification and quantification of desulphoglucosinolates

The use of liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry has been investigated as a potential method for both the identification and quantification of desulphoglucosinolates. The results indicate that this methodology is particularly useful as a rapid technique for the confirmation of the identity of desulphoglucosinolates and has, in full scan mode, limits of detection at least an order of magnitude lower than is normally achieved by conventional high pressure liquid chromatography with UV detection. It has been demonstrated that, using a per-deuterated desulphoglucosinolate as an internal standard, quantification could be achieved, but for accurate analysis at low concentrations it would be necessary to produce independent calibration curves for each desulphoglucosinolate present in the sample. Copyright