Loss of IRF2BP2 in Microglia Increases Inflammation and Functional Deficits after Focal Ischemic Brain Injury.

Ischemic stroke causes neuronal cell death and triggers a cascade of inflammatory signals that contribute to secondary brain damage. Microglia, the brain-resident macrophages that remove dead neurons, play a critical role in the brain's response to ischemic injury. Our previous studies showed that IRF2 binding protein 2 (IRF2BP2) regulates peripheral macrophage polarization, limits their inflammatory response and reduces susceptibility to atherosclerosis. Here, we show that loss of IRF2BP2 in microglia leads to increased inflammatory cytokine expression in response to lipopolysaccharide challenge and impaired activation of anti-inflammatory markers in response to interleukin-4 (IL4) stimulation. Focal ischemic brain injury of the sensorimotor cortex induced by photothrombosis caused more severe functional deficits in mice with IRF2BP2 ablated in macrophages/microglia, associated with elevated expression of inflammatory cytokines in the brain. These mutant mice had larger infarctions 4 days after stroke associated with fewer anti-inflammatory M2 microglia/macrophages recruited to the peri-infarct area, suggesting an impaired clearance of injured tissues. Since IRF2BP2 modulates interferon signaling, and interferon beta (IFNß) has been reported to be anti-inflammatory and reduce ischemic brain injury, we asked whether loss of IRF2BP2 in macrophages/microglia would affect the response to IFNß in our stroke model. IFNß suppressed inflammatory cytokine production of macrophages and reduced infarct volumes at 4 days after photothrombosis in wild type mice. The anti-inflammatory effect of IFNß was lost in IRF2BP2-deficient macrophages and IFNß failed to protect mice lacking IRF2BP2 in macrophages/microglia from ischemic injury. In summary, IRF2BP2 expression in macrophages/microglia is important to limit inflammation and stroke injury, in part by mediating the beneficial effect of IFNß.

IRF2BP2-deficient microglia block the anxiolytic effect of enhanced postnatal care.

Enhanced postnatal care (EPC) increases resilience to adversity in adulthood. Since microglia participate in shaping neural circuits, we asked how ablation of an inflammation-suppressing factor IRF2BP2 (Interferon Regulatory Factor 2 Binding Protein 2) in microglia would affect the responses to EPC. Mice lacking IRF2BP2 in microglia (KO) and littermate controls (WT) were subjected to EPC during the first 3 weeks after birth. EPC reduced anxiety in WT but not KO mice. This was associated with reduced inflammatory cytokine expression in the hypothalamus. Whole genome RNAseq profiling of the hypothalamus identified 101 genes whose expression was altered by EPC: 95 in WT, 11 in KO, with 5 in common that changed in opposite directions. Proteoglycan 4 (Prg4), prostaglandin D2 synthase (Ptgds) and extracellular matrix protease inhibitor Itih2 were suppressed by EPC in WT but elevated in KO mice. On the other hand, the glutamate transporter VGLUT1 (Slc17a7) was increased by EPC in WT but not KO mice. Prostaglandin D2 (PGD2) is known to enhance microglial inflammation and promote Gfap expression. ELISA confirmed reduced PGD2 in the hypothalamus of WT mice after EPC, associated with reduced Gfap expression. Our study suggests that the anxiety-reducing effect of EPC operates by suppressing microglial inflammation, likely by reducing neuronal prostaglandin D2 production.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner.

IRF2BP2 Reduces Macrophage Inflammation and Susceptibility to Atherosclerosis.

RATIONALE: Inflammation impairs macrophage cholesterol clearance from vascular tissues and promotes atherosclerosis. Inflammatory macrophages suppress expression of the transcription cofactor interferon regulatory factor 2-binding protein 2 (IRF2BP2), and genetic variants near IRF2BP2 associate with ischemic heart disease progression in humans. OBJECTIVES: To test whether IRF2BP2 in macrophages affects atherosclerosis in mice and humans. METHODS AND RESULTS: We generated mice that delete IRF2BP2 in macrophages. IRF2BP2-deficient macrophages worsened atherosclerosis in irradiated low-density lipoprotein receptor null-recipient mice and in apolipoprotein E null mice. IRF2BP2-deficient macrophages were inflammatory and had impaired cholesterol efflux because of their inability to activate the cholesterol transporter ABCA1 in response to cholesterol loading. Their expression of the anti-inflammatory transcription factor Krüppel-like factor 2 was markedly reduced. Promoter studies revealed that IRF2BP2 is required for MEF2-dependent activation of Krüppel-like factor 2. Importantly, restoring Krüppel-like factor 2 in IRF2BP2-deficient macrophages attenuated M1 inflammatory and rescued M2 anti-inflammatory gene activation and improved the cholesterol efflux deficit by restoring ABCA1 activation in response to cholesterol loading. In a cohort of 1066 angiographic cases and 1011 controls, homozygous carriers of a deletion polymorphism (rs3045215) in the 3' untranslated region sequence of human IRF2BP2 mRNA had a higher risk of coronary artery disease (recessive model, odds ratio [95% confidence interval]=1.560 [1.179-2.065], P=1.73E-03) and had lower IRF2BP2 (and Krüppel-like factor 2) protein levels in peripheral blood mononuclear cells. The effect of this deletion polymorphism to suppress protein expression was confirmed in luciferase reporter studies. CONCLUSION: Ablation of IRF2BP2 in macrophages worsens atherosclerosis in mice, and a deletion variant that lowers IRF2BP2 expression predisposes to coronary artery disease in humans.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

Redirecting soluble antigen for MHC class I cross-presentation during phagocytosis.

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.

Activation of NLRP3 inflammasome by crystalline structures via cell surface contact.

Crystalline structures activate the NLRP3 inflammasome, leading to the production of IL-1ß, however, the molecular interactions responsible for NLRP3 activation are not fully understood. Cathepsin B release from the ruptured phagolysosome and potassium ion efflux have been suggested to be critical for this activation. Here, we report that Cathepsin B redistribution was not a crucial event in crystal-induced IL-1ß production. Silica and monosodium urate crystal-treated macrophages with undisturbed lysosomes demonstrated strong co-localization of ASC and Caspase-1, indicative of NLRP3 inflammasome activation. Importantly, we provided evidence to suggest that macrophage cell membrane binding to immobilized crystals was sufficient to induce IL-1ß release, and this activation of the NLRP3 inflammasome was inhibited by blocking potassium efflux. Therefore, this work reveals additional complexity in crystalline structure-mediated NLRP3 inflammasome regulations.

Inflammatory Flt3l is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection.

Innate sensing mechanisms trigger a variety of humoral and cellular events that are essential to adaptive immune responses. Here we describe an innate sensing pathway triggered by Plasmodium infection that regulates dendritic cell homeostasis and adaptive immunity through Flt3 ligand (Flt3l) release. Plasmodium-induced Flt3l release in mice requires Toll-like receptor (TLR) activation and type I interferon (IFN) production. We found that type I IFN supports the upregulation of xanthine dehydrogenase, which metabolizes the xanthine accumulating in infected erythrocytes to uric acid. Uric acid crystals trigger mast cells to release soluble Flt3l from a pre-synthesized membrane-associated precursor. During infection, Flt3l preferentially stimulates expansion of the CD8-α(+) dendritic cell subset or its BDCA3(+) human dendritic cell equivalent and has a substantial impact on the magnitude of T cell activation, mostly in the CD8(+) compartment. Our findings highlight a new mechanism that regulates dendritic cell homeostasis and T cell responses to infection.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Toll-like receptors: role in dermatological disease.

Toll-like receptors (TLRs) are a class of conserved receptors that recognize pathogen-associated molecular patterns (PAMPs) present in microbes. In humans, at least ten TLRs have been identified, and their recognition targets range from bacterial endotoxins to lipopeptides, DNA, dsRNA, ssRNA, fungal products, and several host factors. Of dermatological interest, these receptors are expressed on several skin cells including keratinocytes, melanocytes, and Langerhans cells. TLRs are essential in identifying microbial products and are known to link the innate and adaptive immune systems. Over the years, there have been significant advances in our understanding of TLRs in skin inflammation, cutaneous malignancies, and defence mechanisms. In this paper, we will describe the association between TLRs and various skin pathologies and discuss proposed TLR therapeutics.