Kidney macrophages tap the stream.

Tissue-resident macrophages are essential for maintaining organismal homeostasis, but the precise mechanisms that macrophages use to perform this function are not fully understood. In this issue of Immunity, He et al. demonstrate that renal macrophages surveil and sample urine particles, ensuring optimal collecting duct flow and preventing kidney stone development.

Immunometabolism of Tissue-Resident Macrophages - An Appraisal of the Current Knowledge and Cutting-Edge Methods and Technologies.

Tissue-resident macrophages exist in unique environments, or niches, that inform their identity and function. There is an emerging body of literature suggesting that the qualities of this environment, such as the types of cells and debris they eat, the intercellular interactions they form, and the length of time spent in residence, collectively what we call habitare, directly inform their metabolic state. In turn, a tissue-resident macrophage's metabolic state can inform their function, including whether they resolve inflammation and protect the host from excessive perturbations of homeostasis. In this review, we summarize recent work that seeks to understand the metabolic requirements for tissue-resident macrophage identity and maintenance, for how they respond to inflammatory challenges, and for how they perform homeostatic functions or resolve inflammatory insults. We end with a discussion of the emerging technologies that are enabling, or will enable, in situ study of tissue-resident macrophage metabolism.

IL1ß Promotes Immune Suppression in the Tumor Microenvironment Independent of the Inflammasome and Gasdermin D.

IL1ß is a central mediator of inflammation. Secretion of IL1ß typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1ß in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1ß in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain-like protein in the host were dispensable for the release of intratumoral bioactive IL1ß. Inflammasome-independent IL1ß release promoted systemic neutrophil expansion and fostered accumulation of T-cell-suppressive neutrophils in the tumor. Moreover, IL1ß was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1ß allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1ß-deficient mice, demonstrating a crucial role for CD8+ T-cell-macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1ß through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.

Cryptococcus neoformans Secretes Small Molecules That Inhibit IL-1ß Inflammasome-Dependent Secretion.

Cryptococcus neoformans is an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This ability is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule. Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition of C. neoformans by inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing proper inflammasome function. In this context, we analyzed the impact of molecules secreted by C. neoformans B3501 strain and its acapsular mutant Δcap67 in inflammasome activation in an in vitro model. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome-dependent events (i.e., IL-1ß secretion and LDH release via pyroptosis) more strongly than conditioned media from Δcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens, and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) and DL-p-Hydroxyphenyllactic acid (HPLA) were present in B3501's conditioned media and that ILA alone or with HPLA is involved in the regulation of inflammasome activation by C. neoformans. These results were confirmed by in vivo experiments, where exposure to conditioned media led to higher fungal burdens in Acanthamoeba castellanii culture as well as in higher fungal loads in the lungs of infected mice. Overall, the results presented show that conditioned media from a wild-type strain can inhibit a vital recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survival in vitro and in vivo and suggesting that secretion of aromatic metabolites, such as ILA, during cryptococcal infections fundamentally impacts pathogenesis.

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1ß and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Apoptosis of intestinal epithelial cells restricts Clostridium difficile infection in a model of pseudomembranous colitis.

Clostridium difficile is the leading cause of pseudomembranous colitis in hospitalized patients. C. difficile enterotoxins TcdA and TcdB promote this inflammatory condition via a cytotoxic response on intestinal epithelial cells (IECs), but the underlying mechanisms are incompletely understood. Additionally, TcdA and TcdB engage the Pyrin inflammasome in macrophages, but whether Pyrin modulates CDI pathophysiology is unknown. Here we show that the Pyrin inflammasome is not functional in IECs and that Pyrin signaling is dispensable for CDI-associated IEC death and for in vivo pathogenesis. Instead, our studies establish that C. difficile enterotoxins induce activation of executioner caspases 3/7 via the intrinsic apoptosis pathway, and demonstrate that caspase-3/7-mediated IEC apoptosis is critical for in vivo host defense during early stages of CDI. In conclusion, our findings dismiss a critical role for inflammasomes in CDI pathogenesis, and identify IEC apoptosis as a host defense mechanism that restricts C. difficile infection in vivo.

GSDMD is critical for autoinflammatory pathology in a mouse model of Familial Mediterranean Fever.

Pyroptosis is an inflammasome-induced lytic cell death mode, the physiological role of which in chronic inflammatory diseases is unknown. Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease worldwide, affecting an estimated 150,000 patients. The disease is caused by missense mutations in Mefv that activate the Pyrin inflammasome, but the pathophysiologic mechanisms driving autoinflammation in FMF are incompletely understood. Here, we show that Clostridium difficile infection of FMF knock-in macrophages that express a chimeric FMF-associated MefvV726A Pyrin elicited pyroptosis and gasdermin D (GSDMD)-mediated interleukin (IL)-1ß secretion. Importantly, in vivo GSDMD deletion abolished spontaneous autoinflammatory disease. GSDMD-deficient FMF knock-in mice were fully protected from the runted growth, anemia, systemic inflammatory cytokine production, neutrophilia, and tissue damage that characterize this autoinflammatory disease model. Overall, this work identifies pyroptosis as a critical mechanism of IL-1ß-dependent autoinflammation in FMF and highlights GSDMD inhibition as a potential antiinflammatory strategy in inflammasome-driven diseases.

Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4.

The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1-/- macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1ß (IL-1ß) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.