The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia.

Inflammation is closely related to the extent of damage following cerebral ischaemia, and the targeting of this inflammation has emerged as a promising therapeutic strategy. Here, we present that hypoxia-induced glial T-cell immunoglobulin and mucin domain protein (TIM)-3 can function as a modulator that links inflammation and subsequent brain damage after ischaemia. We find that TIM-3 is highly expressed in hypoxic brain regions of a mouse cerebral hypoxia-ischaemia (H/I) model. TIM-3 is distinctively upregulated in activated microglia and astrocytes, brain resident immune cells, in a hypoxia-inducible factor (HIF)-1-dependent manner. Notably, blockade of TIM-3 markedly reduces infarct size, neuronal cell death, oedema formation and neutrophil infiltration in H/I mice. Hypoxia-triggered neutrophil migration and infarction are also decreased in HIF-1α-deficient mice. Moreover, functional neurological deficits after H/I are significantly improved in both anti-TIM-3-treated mice and myeloid-specific HIF-1α-deficient mice. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against hypoxia-associated brain diseases

A novel sphingosylphosphorylcholine and sphingosine-1-phosphate receptor 1 antagonist, KRO-105714, for alleviating atopic dermatitis

Background Atopic dermatitis (eczema) is a type of inflammation of the skin, which presents with itchy, red, swollen, and cracked skin. The high global incidence of atopic dermatitis makes it one of the major skin diseases threatening public health. Sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) act as pro-inflammatory mediators, as an angiogenesis factor and a mitogen in skin fibroblasts, respectively, both of which are important biological responses to atopic dermatitis. The SPC level is known to be elevated in atopic dermatitis, resulting from abnormal expression of sphingomyelin (SM) deacylase, accompanied by a deficiency in ceramide. Also, S1P and its receptor, sphingosine-1-phosphate receptor 1 (S1P1) are important targets in treating atopic dermatitis. Results In this study, we found a novel antagonist of SPC and S1P1, KRO-105714, by screening 10,000 compounds. To screen the compounds, we used an SPC-induced cell proliferation assay based on a high-throughput screening (HTS) system and a human S1P1 protein-based [35S]-GTPγS binding assay. In addition, we confirmed the inhibitory effects of KRO-105714 on atopic dermatitis through related cell-based assays, including a tube formation assay, a cell migration assay, and an ELISA assay on inflammatory cytokines. Finally, we confirmed that KRO-105714 alleviates atopic dermatitis symptoms in a series of mouse models. Conclusions Taken together, our data suggest that SPC and S1P1 antagonist KRO-105714 has the potential to alleviate atopic dermatitis.This work was supported by a grant from the Korea Research Council for Industrial Science and Technology (KK-1933-20) to HC, under the industrial infrastructure program for fundamental technologies and Korea Institute for Advancement of Technology through the Inter-ER Cooperation Projects (R0002017) which are funded by the Ministry of Trade, Industry & Energy, Korea to YDG

Regulation of microglia activity by glaucocalyxin-A: attenuation of lipopolysaccharide-stimulated neuroinflammation through NF-κB and p38 MAPK signaling pathways.

Microglial cells are the resident macrophages and intrinsic arm of the central nervous system innate immune defense. Microglial cells become activated in response to injury, infection, environmental toxins, and other stimuli that threaten neuronal survival. Therefore, regulating microglial activation may have therapeutic benefits that lead to alleviating the progression of inflammatory-mediated neurodegeneration. In the present study, we investigated the effect of glaucocalyxin A (GLA) isolated from Rabdosia japonica on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated primary microglia and BV-2 cells. GLA significantly inhibited LPS-induced production of nitric oxide and reversed the morphological changes in primary microglia. Further, GLA suppressed expression of inducible nitric oxide synthase and cyclooxygenase-2 dose-dependently at the mRNA and protein levels. The production of proinflammatory cytokines such as tumor necrosis factor-α, interleukin-1β (IL)-1β, and IL-6 were inhibited by suppressing their transcriptional activity. Furthermore, GLA suppressed nuclear factor-κB activation by blocking degradation of IκB-α and inhibited the induction of lipocalin-2 expression in LPS-stimulated BV-2 cells. Mechanistic study revealed that the inhibitory effects of GLA were accompanied by blocking the p38 mitogen activated protein kinase signaling pathway in activated microglia. In conclusion, given that microglial activation contributes to the pathogenesis of neurodegenerative diseases, GLA could be developed as a potential therapeutic agent for treating microglia-mediated neuroinflammatory diseases

Inhibitory effect of glaucocalyxin-A (GLA) on IκB-α phosphorylation and degradation in lipopolysaccharide (LPS)-stimulated microglia cells.

<p>Cells were treated with the indicated dose of GLA for 1 h before LPS (100 ng/mL) treatment for 30 min. A: Primary Microglia, B: BV-2 cells C: BV-2 cells were pre-treated with 20 µM PDTC for 2 h before 5 µM GLA was added for 1 h and before incubating with LPS (100 ng/mL) for 30 min. Lysates were analyzed by immunoblotting with an anti p-IκB-α/IκB-α antibody. Representative densitometric analyses was shown in the lower panels. Results are expressed as a ratio of p-IκB-α to IκB-α. D: BV-2 microglial cells were pre-treated with 20 µM PDTC for 2 h before 5 µM GLA was added and 1 h before incubating with LPS (100 ng/mL) for 24 h. Data are mean ± S.E.M. (n = 3) for three independent experiments. ^#P<0.001 compared with control group; *P<0.05, **P<0.01, and ***P<0.001 compared with LPS-treated group by One-Way analysis of variance (ANOVA) followed by <i>Bonferroni’s</i> multiple comparison test.</p

Inhibitory effect of glaucocalyxin-A (GLA) on nuclear factor (NF)-κB activity in lipopolysaccharide (LPS)-stimulated BV-2 microglia.

<p>A: BV-2 microglia cells were seeded at a density of 5×10⁴cells/well on 24-well plates. Cells were stimulated with LPS (100 ng/mL) in the absence or presence of GLA (5 µM) added 1 h before stimulation. At 30 min after adding the LPS, the sub-cellular location of the NF-κB p65 subunit was determined by immunofluorescence assay. B: Cells were treated with the indicated dose of GLA 30 min before LPS (100 ng/mL) treatment. Total nuclear protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using anti-NF-κB p65. Densitometry analysis of NF-κB p65 is shown in the lower panel. Results are expressed as a ratio of NF-κB p65 to nucleolin. Data are mean ± S.E.M. (n = 3) for three independent experiments. ^# P<0.001 compared with control group; *P<0.05, **P<0.01, and ***P<0.001 compared with LPS-treated group by One-Way analysis of variance (ANOVA) followed by <i>Bonferroni’s</i> multiple comparison test.</p

Inhibitory effects of glaucocalyxin-A (GLA) on phosphorylation of p38 mitogen activated protein kinase (MAPK) and expression of lipocalin-2 (LCN-2) in lipopolysaccharide (LPS)-stimulated microglia cells.

<p>Cells were pre-treated with indicated doses of GLA for 1 h before LPS treatment (100 ng/mL) for 30 min. A: Primary Microglia, B: BV-2 microglial cells. Total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using anti-p38 MAPKs. Representative densitometric analysis of the p38 bands were shown in the lower panel. Results are expressed as a ratio of p-p38 to total p38. C: Cells were pre-treated with the indicated doses of GLA for 1 h before LPS (100 ng/mL) treatment. LCN-2 mRNA levels were determined by RT-PCR. GAPDH was used as an internal control for the RT-PCR analysis. Representative quantification data was shown in the lower panel. Results are expressed as a ratio of LCN-2 to GAPDH. Data are mean ± S.E.M. (n = 3) for three independent experiments. ^#P<0.001 compared with control group; *P<0.05, **P<0.01, and ***P<0.001 compared with LPS-treated group by One-Way analysis of variance (ANOVA) followed by <i>Bonferroni’s</i> multiple comparison test.</p