The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2.

BACKGROUND: C1q has been reported to reveal complement-independent roles in immune and non-immune cells. C1q binds to its specific receptors to regulate distinct functions that rely on the environment and cell types. Discoidin domain receptor 2 (DDR2) is activated by collagen and functions in wound healing by controlling matrix metalloproteinase (MMP) expression. Since C1q exhibits a collagen-like structure, we hypothesized that C1q might engage DDR2 to regulate wound healing and extracellular matrix (ECM) remodeling. METHODS: Cell-based assay, proximity ligation assay, ELISA, and surface plasmon analysis were utilized to investigate DDR2 and C1q binding. We also investigate the C1q-mediated in vitro wound healing ability using the human fibrosarcoma cell line, HT1080. RESULTS: C1q induced the phosphorylation of DDR2, p38 kinase, and ERK1/2. C1q and DDR2 binding improved cell migration and induced MMP2 and MMP9 expression. DDR2-specific shRNA reduced C1q-mediated cell migration for wound healing. CONCLUSIONS: C1q is a new DDR2 ligand that promotes wound healing. These findings have therapeutic implications in wound healing-related diseases.

Identification of Pyrido[3,4-d]pyrimidine derivatives as RIPK3-Mediated necroptosis inhibitors.

Necroptosis executed by RIPK3-mediated phosphorylation of MLKL is a programmed necrotic cell death and implicated with various diseases such as sterile inflammation. We designed and synthesized pyrido[3,4-d]pyrimidine derivatives as novel necroptosis inhibitors capable of suppressing the phosphorylation of MLKL. Our SAR studies reveal that 20 possesses comparable inhibitory activity against RIPK3-mediated pMLKL in HT-29 cells relative to GSK872 (2), a representative selective RIPK3 inhibitor. Based on biochemical kinase assay results, 20 is comparable to GSK872 (2) with regard to activity against RIPK3 and less potent against RIPK1 than GSK872, indicating selectivity of 20 towards RIPK3 over RIPK1 is higher than that of GSK872. In HT-29 cells, 20 inhibits necroptosis via MLKL oligomerization impediment. Moreover, 20 suppresses migration and invasion of AsPC-1 cells by necroptosis induced- CXCL5 secretion downregulation. Significantly, 20 could relieve the TNFα-induced systemic inflammatory response syndrome in vivo. Taken together, this study would provide a useful insight into the design of novel necroptosis inhibitors possessing RIPK3-mediated pMLKL inhibitory activity.

Canagliflozin protects against cisplatin-induced acute kidney injury by AMPK-mediated autophagy in renal proximal tubular cells.

Sodium-glucose cotransporter 2 inhibitors, which are recently introduced as glucose-lowering agents, improve cardiovascular and renal outcomes in patients with diabetes mellitus. These drugs also have beneficial effects in various kidney disease models. However, the effect of SGLT2 inhibitors on cisplatin-induced acute kidney injury (AKI) and their mechanism of action need to be elucidated. In this study, we investigated whether canagliflozin protects against cisplatin-induced AKI, depending on adenosine monophosphate-activated protein kinase (AMPK) activation and following induction of autophagy. In the experiments using the HK-2 cell line, cell viability assay and molecular analysis revealed that canagliflozin protected renal proximal tubular cells from cisplatin, whereas addition of chloroquine or compound C abolished the protective effect of canagliflozin. In the mouse model of cisplatin-induced AKI, canagliflozin protected mice from cisplatin-induced AKI. However, treatment with chloroquine or compound C in addition to administration of cisplatin and canagliflozin eliminated the protective effect of canagliflozin. Collectively, these findings indicate that canagliflozin protects against cisplatin-induced AKI by activating AMPK and autophagy in renal proximal tubular cells.

Sulfatide Inhibits HMGB1 Secretion by Hindering Toll-Like Receptor 4 Localization Within Lipid Rafts.

The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis, secreted by multiple stimuli, involving pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, in contrast, is a sphingolipid commonly found in myelin sheets with a disputed immunological role. We sought to determine the immunological characteristics of sulfatide in the periphery by analyzing the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after pre-treatment with sulfatide before LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, increased survivability, and reduced pathological severity.

Electrical antimicrobial susceptibility testing based on aptamer-functionalized capacitance sensor array for clinical isolates.

To prescribe effective antibiotics to patients with bacterial infections in a timely manner and to avoid the misuse of antibiotics, a rapid antimicrobial susceptibility test (AST) is essential. However, conventional AST methods require more than 16 h to provide results; thus, we developed an electrical AST (e-AST) system, which provides results within 6 h. The proposed e-AST is based on an array of 60 aptamer-functionalized capacitance sensors that are comparable to currently available AST panels and a pattern-matching algorithm. The performance of the e-AST was evaluated in comparison with that of broth microdilution as the reference test for clinical strains isolated from septic patients. A total of 4,554 tests using e-AST showed a categorical agreement of 97% with a minor error of 2.2%, major error of 0.38%, and very major error of 0.38%. We expect that the proposed e-AST could potentially aid antimicrobial stewardship efforts and lead to improved patient outcomes.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia-reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.