The stimulation of TH2 cells results in increased IL-5 and IL-13 production via the H4 receptor.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. OBJECTIVE: In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. METHODS: Total CD4+ T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. RESULTS: H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. CONCLUSION: We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.

The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells.

INTRODUCTION: Granzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine. METHODS: Naïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker. RESULTS: By RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells. CONCLUSION: Our findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.

oxLDL inhibits differentiation of mesenchymal stem cells into osteoblasts via the CD36 mediated suppression of Wnt signaling pathway.

Bone abnormalities as a consequence of osteoblast deregulation are associated with several diseases such as diabetes and chronic kidney disease. Important role for oxidized low density lipoproteins (oxLDL) in the pathophysiology of bone disorders has been reported. However, little is known about the effects and mechanisms of oxLDL on the process of osteoblastogenesis in human mesenchymal stem cells (MSCs). We show that oxLDL concentrations of ~ 10-25 µg protein (0.43-1.0 µM MDA/mg protein) inhibited the differentiation of MSCs to osteoblasts. We demonstrate that the underlying mechanism entails the suppression of the Wnt signaling through the down-regulation of ß-catenin. Further, we show the association of scavenger receptor CD36 with the receptors LRP5/6 and Frizzled in mediating the oxLDL effects on the differentiation of MSCs to pre-osteoblasts. Inhibiting CD36 restored osteoblasts differentiation in the presence of oxLDL. Our findings suggest that oxLDL interferes with the canonical Wnt signaling pathway in a CD36 dependent manner leading to an inhibition of osteoblastogenesis.

Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling.

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-ß-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

oxLDL inhibits differentiation and functional activity of osteoclasts via scavenger receptor-A mediated autophagy and cathepsin K secretion.

Resorptive activity of osteoclasts is important for maintaining bone homeostasis. Endogenous compounds such as oxidized low density lipoprotein (oxLDL) have been shown to disturb this activity. While some studies have investigated the effects of oxLDL on the process of osteoclastogenesis, the underlying mechanism are not fully understood. We show here that oxLDL concentrations of ~10-25 µg protein (0.43-1.0 µM MDA/mg protein) completely blocked the formation of functional osteoclasts. The underlying mechanism implies an inhibition of autophagy that in turn leads to a decreased fusion of cathepsin K (CatK)-loaded lysosomal vesicles with the ruffled border membrane. As result, a lower secretion of CatK and impaired protonation of the resorption lacunae by vacuolar-ATPase (v-ATPase) is observed in the presence of oxLDL. We demonstrate that scavenger receptor A (SR-A) mediates oxLDL effects on osteoclastogenesis and repressing this receptor partially rescued oxLDL effects. Collectively, our data provides an insight into the possible mechanism of oxLDL on osteoclastogenesis suggesting that it does not perturb the packaging of CatK and v-ATPase (V-a3) in the secretory lysosome, but inhibits the fusion of these lysosomes to the ruffled border. The relevance of our findings suggests a distinct link between oxLDL, autophagy and osteoclastogenesis.