Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response.

The helicase RIG-I initiates an antiviral immune response after recognition of pathogenic RNA. TRIM25, an E3 ubiquitin ligase, mediates K63-linked ubiquitination of RIG-I, which is crucial for RIG-I downstream signaling and the antiviral innate immune response. The components and mode of the RIG-I-initiated innate signaling remain to be fully understood. Here we identify a novel long noncoding RNA (Lnczc3h7a) that binds to TRIM25 and promotes RIG-I-mediated antiviral innate immune responses. Depletion of Lnczc3h7a impairs RIG-I signaling and the antiviral innate response to RNA viruses in vitro and in vivo. Mechanistically, Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold for stabilization of the RIG-I-TRIM25 complex at the early stage of viral infection. Lnczc3h7a facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and thus promotes downstream signaling transduction. Our findings reveal that host RNAs can enhance the response of innate immune sensors to foreign RNAs, ensuring effective antiviral defense.

Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity.

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity.

Resident macrophage-dependent immune cell scaffolds drive anti-bacterial defense in the peritoneal cavity.

Peritoneal immune cells reside unanchored within the peritoneal fluid in homeostasis. Here, we examined the mechanisms that control bacterial infection in the peritoneum using a mouse model of abdominal sepsis following intraperitoneal Escherichia coli infection. Whole-mount immunofluorescence and confocal microscopy of the peritoneal wall and omentum revealed that large peritoneal macrophages (LPMs) rapidly cleared bacteria and adhered to the mesothelium, forming multilayered cellular aggregates composed by sequentially recruited LPMs, B1 cells, neutrophils, and monocyte-derived cells (moCs). The formation of resident macrophage aggregates (resMφ-aggregates) required LPMs and thrombin-dependent fibrin polymerization. E. coli infection triggered LPM pyroptosis and release of inflammatory mediators. Resolution of these potentially inflammatory aggregates required LPM-mediated recruitment of moCs, which were essential for fibrinolysis-mediated resMφ-aggregate disaggregation and the prevention of peritoneal overt inflammation. Thus, resMφ-aggregates provide a physical scaffold that enables the efficient control of peritoneal infection, with implications for antimicrobial immunity in other body cavities, such as the pleural cavity or brain ventricles.

Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.

TBK1 is essential for interferon-ß (IFN-ß) production and innate antiviral immunity. Here we identified the T cell anergy-related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-ß production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-ß production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component.

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1ß (IL-1ß) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

Cholinergic macrophages promote the resolution of peritoneal inflammation.

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

Gata6+ large peritoneal macrophages: an evolutionarily conserved sentinel and effector system for infection and injury.

There are striking similarities between the sea urchin cavity macrophage-like phagocytes (coelomocytes) and mammalian cavity macrophages in not only their location, but also their behaviors. These cells are crucial for maintaining homeostasis within the cavity following a breach, filling the gap and functioning as a barrier between vital organs and the environment. In this review, we summarize the evolving literature regarding these Gata6+ large peritoneal macrophages (GLPMs), focusing on ontogeny, their responses to perturbations, including their rapid aggregation via coagulation, as well as scavenger receptor cysteine-rich domains and their potential roles in diseases, such as cancer. We challenge the 50-year old phenomenon of the 'macrophage disappearance reaction' (MDR) and propose the new term 'macrophage disturbance of homeostasis reaction' (MDHR), which may better describe this complex phenomenon.

Tissue-resident macrophages actively suppress IL-1beta release via a reactive prostanoid/IL-10 pathway.

The alarm cytokine interleukin-1ß (IL-1ß) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1ß production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1ß; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1ß from its pro-form. However, despite the important role of IL-1ß in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1ß processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1ß processing, providing a previously unrecognized control of IL-1ß in tissue-resident macrophages.

Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses.

Although the mechanisms by which innate pathogen-recognition receptors enhance adaptive immune responses are increasingly well understood, whether signaling events from distinct classes of receptors affect each other in modulating adaptive immunity remains unclear. We found here that the activation of cytosolic RIG-I-like receptors (RLRs) resulted in the selective suppression of transcription of the gene encoding the p40 subunit of interleukin 12 (Il12b) that was effectively induced by the activation of Toll-like receptors (TLRs). The RLR-activated transcription factor IRF3 bound dominantly, relative to IRF5, to the Il12b promoter, where it interfered with the TLR-induced assembly of a productive transcription-factor complex. The activation of RLRs in mice attenuated TLR-induced responses of the T helper type 1 cell (T(H)1 cell) and interleukin 17-producing helper T cell (T(H)17 cell) subset types and, consequently, viral infection of mice caused death at sublethal doses of bacterial infection. The innate immune receptor cross-interference we describe may have implications for infection-associated clinical episodes.

Brains, Blood, and Guts: MeCP2 Regulates Microglia, Monocytes, and Peripheral Macrophages.

Mutations in methyl-CpG-binding protein 2 (MECP2) underlie most cases of Rett Syndrome, a neurodevelopmental disorder with neurological and somatic impairments. In this issue of Immunity, Cronk et al. (2015) find that macrophages in MeCP2-deficient mice are abnormal in number, as well as in glucocorticoid, hypoxia, and inflammatory responses.

POPsicle for Fever! Cooling Down the Inflammasome.

Inhibition of the inflammasome might be beneficial for numerous inflammatory pathologies. In this issue of Immunity, de Almeida et al. (2015) report that the PYRIN domain-only protein (POP1) efficiently inhibits inflammasome activation, identifying it as a pan-inflammasome inhibitor.

The PYRIN Domain-only Protein POP1 Inhibits Inflammasome Assembly and Ameliorates Inflammatory Disease.

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

Methyl-CpG Binding Protein 2 Regulates Microglia and Macrophage Gene Expression in Response to Inflammatory Stimuli.

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis.

INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

Advanced Glycation End Products (AGEs) Inhibit Macrophage Efferocytosis of Apoptotic ß Cells through Binding to the Receptor for AGEs.

Pancreatic ß cell apoptosis is important in the pathogenesis of type 2 diabetes mellitus (T2DM). Generally, apoptotic ß cells are phagocytosed by macrophages in a process known as "efferocytosis." Efferocytosis is critical to the resolution of inflammation and is impaired in T2DM. Advanced glycation end products (AGEs), which are increased in T2DM, are known to suppress phagocytosis function in macrophages. In this study, we found that AGEs inhibited efferocytosis of apoptotic ß cells by primary peritoneal macrophages in C57BL/6J mice or mouse macrophage cell line Raw264.7. Mechanistically, AGEs inhibit efferocytosis by blocking Ras-related C3 botulinum toxin substrate 1 activity and cytoskeletal rearrangement through receptor for advanced glycation end products/ras homolog family member A/Rho kinase signaling in macrophages. Furthermore, it was observed that AGEs decreased the secretion of anti-inflammatory factors and promoted the proinflammatory ones to modulate the inflammation function of efferocytosis. Taken together, our results indicate that AGEs inhibit efferocytosis through binding to receptor for advanced glycation end products and activating ras homolog family member A/Rho kinase signaling, thereby inhibiting the anti-inflammatory function of efferocytosis.

BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses.

BRD4 is a BET family protein that binds acetylated histones and regulates transcription. BET/BRD4 inhibitors block blood cancer growth and inflammation and serve as a new therapeutic strategy. However, the biological role of BRD4 in normal hematopoiesis and inflammation is not fully understood. Analysis of Brd4 conditional knockout (KO) mice showed that BRD4 is required for hematopoietic stem cell expansion and progenitor development. Nevertheless, BRD4 played limited roles in macrophage development and inflammatory response to LPS ChIP-seq analysis showed that despite its limited importance, BRD4 broadly occupied the macrophage genome and participated in super-enhancer (SE) formation. Although BRD4 is critical for SE formation in cancer, BRD4 was not required for macrophage SEs, as KO macrophages created alternate, BRD4-less SEs that compensated BRD4 loss. This and additional mechanisms led to the retention of inflammatory responses in macrophages. Our results illustrate a context-dependent role of BRD4 and plasticity of epigenetic regulation.

Network Analysis Reveals a Distinct Axis of Macrophage Activation in Response to Conflicting Inflammatory Cues.

Macrophages are subject to a wide range of cytokine and pathogen signals in vivo, which contribute to differential activation and modulation of inflammation. Understanding the response to multiple, often-conflicting cues that macrophages experience requires a network perspective. In this study, we integrate data from literature curation and mRNA expression profiles obtained from wild type C57/BL6J mice macrophages to develop a large-scale computational model of the macrophage signaling network. In response to stimulation across all pairs of nine cytokine inputs, the model predicted activation along the classic M1-M2 polarization axis but also a second axis of macrophage activation that distinguishes unstimulated macrophages from a mixed phenotype induced by conflicting cues. Along this second axis, combinations of conflicting stimuli, IL-4 with LPS, IFN-γ, IFN-ß, or TNF-α, produced mutual inhibition of several signaling pathways, e.g., NF-κB and STAT6, but also mutual activation of the PI3K signaling module. In response to combined IFN-γ and IL-4, the model predicted genes whose expression was mutually inhibited, e.g., iNOS or Nos2 and Arg1, or mutually enhanced, e.g., Il4rα and Socs1, validated by independent experimental data. Knockdown simulations further predicted network mechanisms underlying functional cross-talk, such as mutual STAT3/STAT6-mediated enhancement of Il4rα expression. In summary, the computational model predicts that network cross-talk mediates a broadened spectrum of macrophage activation in response to mixed pro- and anti-inflammatory cytokine cues, making it useful for modeling in vivo scenarios.