Mediator Of Sterile Inflammation Research Articles

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia.

Objective Cold-inducible RNA-binding protein (CIRBP) has been shown to be involved not only in cooling-induced cellular protection but also as a mediator of sterile inflammation, a critical mechanism of the innate immune response in ischemia/reperfusion (I/R) injury. The role of microglia and its activation in cerebral I/R injury warrants further investigation as both detrimental and regenerative properties have been described. Therefore, we investigated the effects of cooling, specifically viability, activation, and release of damage associated molecular patterns (DAMPs) on oxygen glucose deprivation/reperfusion- (OGD/R-) induced injury in murine BV-2 microglial cells. Methods Murine BV-2 microglial cells were exposed to 2 to 6 h OGD (0.2% O2 in glucose- and serum-free medium) followed by up to 19 h of reperfusion, simulated by restoration of oxygen (21% O2) and nutrients. Cells were maintained at either normothermia (37°C) or cooled to 33.5°C, 1 h after experimental start. Cultured supernatants were harvested after exposure to OGD for analysis of DAMP secretions, including high-mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and CIRBP, and cytotoxicity was assessed by lactate dehydrogenase releases after exposure to OGD and reperfusion. Intracellular cold-shock proteins CIRBP and RNA-binding motif 3 (RBM3) as well as caspases 9, 8, and 3 were also analyzed via Western blot analysis. Furthermore, inducible nitric oxide synthase (iNOS), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-1α (IL-1α), monocyte chemotactic protein 1 (MCP-1), transforming growth factor β (TGFβ), CIRBP, and RBM3 gene expressions were assessed via reverse transcription polymerase chain reaction, and TNF-α, IL-6, and IL-1β releases into the cultured supernatants were assessed via enzyme-linked immunosorbent assays (ELISA). Results Prolonged exposure to OGD resulted in increased BV-2 necrotic cell death, which was attenuated by cooling. Cooling also significantly induced cold-shock proteins CIRBP and RBM3 gene expressions, with CIRBP expression more rapidly regulated than RBM3 and translatable to significantly increased protein expression. DAMPs including HMGB-1, HSP70, and CIRBP could be detected in cultured supernatants after 6 h of OGD with CIRBP release being significantly attenuated by cooling. Exposure to OGD suppressed cytokine gene expressions of IL-1β, TNF-α, MCP-1, and TGFβ independently of temperature management, whereas cooling led to a significant increase in IL-1α gene expression after 6 h of OGD. In the reperfusion phase, TNF-α and MCP-1 gene expressions were increased, and cooling was associated with significantly lower TGFβ gene expression. Interestingly, cooled Normoxia groups had significant upregulations of microglial activation marker, Iba1, IL-1β, and TNF-α gene expressions. Conclusion BV-2 microglial cells undergo necrotic cell death resulting in DAMP release due to OGD/R-induced injury. Cooling conveyed neuroprotection in OGD/R-injury as observable in increased cell viability as well as induced gene expressions of cold shock proteins. As cooling alone resulted in both upregulation of microglial activation, expression of proinflammatory cytokines, and cold shock protein transcript and protein expression, temperature management might have ambiguous effects in sterile inflammation. However, cooling resulted in a significant decrease of extracellular CIRBP, which has recently been characterized as a novel DAMP and a potent initiator and mediator of inflammation.

Sterile inflammation in the pathogenesis of maturation failure of arteriovenous fistula.

Chronic kidney disease is a widespread terminal illness that afflicts millions of people across the world. Hemodialysis is the predominant therapeutic management strategy for kidney failure and involves the external filtration of metabolic waste within the circulation. This process requires an arteriovenous fistula (AVF) for vascular access. However, AVF maturation failures are significant obstacles in establishing long-term vascular access for hemodialysis. Appropriate stimulation, activation, and proliferation of smooth muscle cells, proper endothelial cell orientation, adequate structural changes in the ECM, and the release of anti-inflammatory markers are associated with maturation. AVFs often fail to mature due to inadequate tissue repair and remodeling, leading to neointimal hyperplasia lesions. The transdifferentiation of myofibroblasts and sterile inflammation are possibly involved in AVF maturation failures; however, limited data is available in this regard. The present article critically reviews the interplay of various damage-associated molecular patterns (DAMPs) and the downstream sterile inflammatory signaling with a focus on the NLRP3 inflammasome. Improved knowledge concerning AVF maturation pathways can be unveiled by investigating the novel DAMPs and the mediators of sterile inflammation in vascular remodeling that would open improved therapeutic opportunities in the management of AVF maturation failures and its associated complications.

The circadian clock protects against ferroptosis-induced sterile inflammation

The circadian clock, a biochemical oscillator, plays a fundamental role in health and diseases. Ferroptosis, a type of regulated cell death driven by oxidative stress, is a prominent feature in iron-induced tissue injury. However, whether an impaired circadian clock contributes to ferroptosis-induced sterile inflammation remains unknown. Here, we show that the circadian transcription factor ARNTL (also known as BMAL1) protects against experimental acute pancreatitis through blocking the ferroptosis-mediated release of HMGB1, a mediator of sterile inflammation. We utilized a Cre/LoxP system to generate mice with a specific depletion of Arntl in the pancreas (Pdx1-Cre;Arntlflox/flox). These Arntl-deficient mice developed l-arginine–induced acute pancreatitis more rapidly than controls, with increased mortality, tissue injury, neutrophil infiltration, and HMGB1 release. In contrast, the administration of liproxstatin-1 (a ferroptosis inhibitor) or anti-HMGB1 neutralizing antibody attenuated the development of acute pancreatitis in the Arntl-deficient mice. Mechanistically, pancreatic ARNTL is a key regulator of the expression of multiple antioxidant or membrane repair systems (e.g., SLC7A11, GPX4, SOD1, TXN, NFE2L2, and CHMP5) to suppress ferroptotic tissue injury. Collectively, these findings uncover a novel link between the circadian clock and ferroptotic response in inflammation and pancreatic injury.

Activation of Macrophages by P2X7-Induced Microvesicles from Myeloid Cells Is Mediated by Phospholipids and Is Partially Dependent on TLR4

ATP-mediated activation of the purinergic receptor P2X(7) elicits morphological changes and proinflammatory responses in macrophages. These changes include rapid shedding of microvesicles (MV) and the nonconventional secretion of cytokines, such as IL-1beta and IL-18 following priming. In this study, we demonstrate the activation potential of P2X(7)-induced MV isolated from nonprimed murine macrophages. Cotreatment of nonprimed macrophages with ATP and calcium ionophore induced a rapid release of MV that were predominantly 0.5-1 microm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-alpha secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-kappaB dependent, and partially dependent on TLR4 activity, but was high-mobility group box 1 independent. Biochemical fractionation of the MV demonstrated that the phospholipid fraction, not the protein fraction, mediated macrophage activation through a TLR4-dependent process. P2X(7) activation is known to induce calcium-independent phospholipase A(2), calcium-dependent phospholipase A(2), and phospholipase D activities, but inhibition of these enzymes did not inhibit MV generation or shedding. However, blocking phospholipase D activity resulted in release of MV incapable of activating recipient macrophages. These data demonstrate a novel mechanism of macrophage activation resulting from exposure to MV from nonprimed macrophages, and identifies phospholipids in these MV as the biologically active component. We suggest that phospholipids delivered by MV may be mediators of sterile inflammation in a number of diseases.