Crystal Formation in Inflammation.

The formation and accumulation of crystalline material in tissues is a hallmark of many metabolic and inflammatory conditions. The discovery that the phase transition of physiologically soluble substances to their crystalline forms can be detected by the immune system and activate innate immune pathways has revolutionized our understanding of how crystals cause inflammation. It is now appreciated that crystals are part of the pathogenesis of numerous diseases, including gout, silicosis, asbestosis, and atherosclerosis. In this review we discuss current knowledge of the complex mechanisms of crystal formation in diseased tissues and their interplay with the nutrients, metabolites, and immune cells that account for crystal-induced inflammation.

Mitochondrial damage activates the NLRP10 inflammasome.

Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase.

Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.

The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation.

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.

Cutting Edge: STING Induces ACLY Activation and Metabolic Adaptations in Human Macrophages through TBK1.

The 2'3'-cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) pathway can sense infection and cellular stress by detecting cytosolic DNA. Upon ligand binding, cGAS produces the cyclic dinucleotide messenger cGAMP, which triggers its receptor STING. Active STING initiates gene transcription through the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB and induces autophagy, but whether STING can cause changes in the metabolism of macrophages is unknown. In this study, we report that STING signaling activates ATP-citrate lyase (ACLY) by phosphorylation in human macrophages. Using genetic and pharmacologic perturbation, we show that STING targets ACLY via its prime downstream signaling effector TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1). We further identify that TBK1 alters cellular metabolism upon cGAMP treatment. Our results suggest that STING-mediated metabolic reprogramming adjusts the cellular response to DNA sensing in addition to transcription factor activation and autophagy induction.

Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Impairment of lysosomal function by Clostridioides difficile TcdB.

TcdB is a potent cytotoxin produced by pathogenic Clostridioides difficile that inhibits Rho GTPases by mono-glucosylation. TcdB enters cells via receptor-mediated endocytosis. The pathogenic glucosyltransferase domain (GTD) egresses endosomes by pH-mediated conformational changes, and is subsequently released in an autoproteolytic manner. We here investigated the uptake, localization and degradation of TcdB. TcdB colocalized with lysosomal marker protein LAMP1, verifying the endosomal-lysosomal route of the toxin. In pulse assays endocytosed TcdB declined to a limit of detection within 2 hr, whereas the released GTD accumulated for up to 8 hr. We observed that autoproteolytic deficient TcdB NXN C698S was degraded significantly faster than wildtype TcdB, suggesting interference of TcdB with lysosomal degradation process. In fact, TcdB reduced lysosomal degradation of endosome cargo as tested with DQ-Green BSA. Lysosomal dysfunction was accompanied by perinuclear accumulation of LAMP1 and a weaker detection in immunoblots. Galectin-8 or galectin-3 was not recruited to lysosomes speaking against lysosome membrane damage. Changes in the autophagosomal marker LC3B suggested additional indirect effect of lysosomal dysfunction on the autophagic flux. In contrast to necrotic signaling induced in by TcdB, lysosomal dysfunction was not abolished by calcium channel blocker nifedipin, indicating separate cytopathogenic effects induced by TcdB during endo-lysosomal trafficking.

NLRC3 puts the brakes on STING.

NOD-like receptors (NLRs) are increasingly implicated in regulating pathogen-sensing pathways. In this issue of Immunity, Zhang et al. (2014) describe a role for NLRC3 in regulating STING and the inflammatory response to cytosolic DNA.

Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation.

A critical component of innate immune response to infection and tissue damage is the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, and this pathway and its activation products have been implicated in the pathophysiology of a variety of diseases. NLRP3 inflammasome activation leads to the cleavage of pro-IL-1ß and pro-IL-18, as well as the subsequent release of biologically active IL-1ß, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome-selective, IL-1ß processing inhibitor CP-456,773 (also known as MCC950), and we demonstrate its efficacy in two in vivo models of inflammation. Specifically, we show that in human and mouse innate immune cells CP-456,773 is an inhibitor of the cellular release of IL-1ß, IL-1α, and IL-18, that CP-456,773 prevents inflammasome activation induced by disease-relevant soluble and crystalline NLRP3 stimuli, and that CP-456,773 inhibits R848- and imiquimod-induced IL-1ß release. In mice, CP-456,773 demonstrates potent inhibition of the release of proinflammatory cytokines following acute i.p. challenge with LPS plus ATP in a manner that is proportional to the free/unbound concentrations of the drug, thereby establishing an in vivo pharmacokinetic/pharmacodynamic model for CP-456,773. Furthermore, CP-456,773 reduces ear swelling in an imiquimod cream-induced mouse model of skin inflammation, and it reduces airway inflammation in mice following acute challenge with house dust mite extract. These data implicate the NLRP3 inflammasome in the pathogenesis of dermal and airway inflammation, and they highlight the utility of CP-456,773 for interrogating the contribution of the NLRP3 inflammasome and its outputs in preclinical models of inflammation and disease.

A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP.

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1' in rodents and P1'-P2' in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3' reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5' residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 2: Assessing Inflammasome Activation.

Inflammasomes are macromolecular complexes that assemble upon the detection of cytoplasmic pathogen-associated or danger-associated signals and induce a necrotic type of cell death termed pyroptosis, facilitating pro-inflammatory cytokine release. Inflammasomes play a critical role in innate immunity and inflammatory response; however, they have also been associated with multiple diseases, including autoinflammatory and neurodegenerative conditions. In the following chapter, we describe methods to detect inflammasome activation and its downstream effects, including detection of ASC oligomerization, detection of activated caspase-1 and cleaved IL-1ß, as well as read-outs for inflammasome-mediated cell death.

Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 1: Cell Preparation and Inflammasome Stimulation.

Inflammasomes are intracellular, multiprotein supercomplexes that mediate a post-translational inflammatory response to both pathogen and endogenous danger signals. They consist of a sensor, the adapter ASC, and the protease caspase 1 and, following their activation, lead to cl1ß, as well as lytic cell death. Due to this potent inflammatory capacity, understanding inflammasome biology is important in many pathological conditions. It is increasingly clear that inflammasomes are particularly relevant in macrophages, which express a diverse range of inflammasome sensors. In these two chapters, we detail methods to isolate and differentiate human macrophages, murine bone marrow-derived macrophages, and murine microglia and stimulate the inflammasomes known to be expressed in macrophages, including the AIM2, NLRP3, NLRC4, NLRP1, and non-canonical inflammasomes. Furthermore, we describe the methodology required to measure the various results of inflammasome activation including ASC speck formation, monitoring lytic cell death and cytokine release, as well as caspase-1 activation.

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins.

TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-ß/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1-153 is reported. The 2.22 Šresolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTDA66M/L113M). The structure consists of eight antiparallel helices that can be divided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-ß promoter, as well as NF-κB-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation.

Serpinb9 (Spi6)-deficient mice are impaired in dendritic cell-mediated antigen cross-presentation.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (GrB) that protects activated cytotoxic lymphocytes from apoptosis. We show here that the CD8(+) subset of splenic dendritic cells (DC), specialized in major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation), produce high levels of Sb9. Mice deficient in Sb9 are unable to generate a cytotoxic T-cell response against cell-associated antigen by cross-presentation, but maintain normal MHC-II presentation to helper T cells. This impaired cross-priming ability is autonomous to DC and is evident in animals deficient in both Sb9 and GrB, indicating that this role of Sb9 in DC is GrB-independent. In Sb9-deficient mice, CD8(+) DC develop normally, survive as well as wild-type DC after antigenic challenge, and exhibit unimpaired capacity to take up antigen. Although the core processing machinery is unaffected, Sb9-deficient DC appear to process antigen faster. Our results point to a novel, GrB-independent role for Sb9 in DC cross-priming.

How location and cellular signaling combine to activate the NLRP3 inflammasome.

NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) is a cytosolic innate immune sensor of cellular stress signals, triggered by infection and sterile inflammation. Upon detection of an activating stimulus, NLRP3 transitions from an inactive homo-oligomeric multimer into an active multimeric inflammasome, which promotes the helical oligomeric assembly of the adaptor molecule ASC. ASC oligomers provide a platform for caspase-1 activation, leading to the proteolytic cleavage and activation of proinflammatory cytokines in the IL-1 family and gasdermin D, which can induce a lytic form of cell death. Recent studies investigating both the cellular requirement for NLRP3 activation and the structure of NLRP3 have revealed the complex regulation of NLRP3 and the multiple steps involved in its activation. This review presents a perspective on the biochemical and cellular processes controlling the assembly of the NLRP3 inflammasome with particular emphasis on structural regulation and the role of organelles. We also highlight the latest research on metabolic control of this inflammatory pathway and discuss promising clinical targets for intervention.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

1-Deoxysphingolipids cause autophagosome and lysosome accumulation and trigger NLRP3 inflammasome activation.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1ß release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.