Neuroinflammation is required for tissue clearance and repair after infections or insults. To prevent excessive damage, it is crucial to limit the extent of neuroinflammation and thereby the activation of its principal effector cell, microglia. The two main major innate immune cell types in the CNS are astrocytes and microglia. Histone deacetylases (HDACs) have been implicated in regulating the innate inflammatory response, and here we addressed their role in pure astrocyte and microglia cultures. Endogenous HDAC expression levels were determined in microglia and astrocytes and after treatment with lipopolysaccharide (LPS) or LPS and interferon γ (IFNγ). The relative expression level of HDACs was reduced in LPS- or LPS/IFNγ (with the exception of HDAC1 and -7)-stimulated astrocytes and increased in microglia after LPS treatment both in primary cultures and in microglia acutely isolated from LPS-treated mice, so we focused on the inflammatory response in microglia. Primary microglia cultures were treated with LPS in the presence or absence of HDAC inhibitors (HDACi). Expression and release of inflammatory cytokines was determined by quantitative RT-PCR, flow cytometry, and ELISA. HDACi strongly suppressed LPS-induced cytokine expression and release by microglia. Furthermore, expression of M1- and M2-associated activation markers was suppressed, and the migratory behavior of microglia was attenuated. Our findings strongly suggest that HDACi suppress innate immune activation in microglia.
New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.
Background and Purpose Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor‐like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine‐like receptor proteins D6 and DARC as well as CCX‐CKR. Here, we provide evidence for an additional biological function of human (h)CCX‐CKR. Experimental Approach We used transfection strategies in HEK293 and human T cells. Key Results Co‐expression of hCCX‐CKR completely inhibits hCXCR3‐induced chemotaxis. We found that hCCX‐CKR forms complexes with hCXCR3, suggesting a relationship between CCX‐CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX‐CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX‐CKR‐induced inhibition of chemotaxis. Conclusions and Implications These findings suggest that hCCX‐CKR prevents hCXCR3‐induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration.
Rheumatoid Arthritis (RA) is a chronic autoimmune disease associated with inflammation and joint remodeling. Adenosine deaminase (ADA), a risk factor in RA, degrades adenosine, an anti-inflammatory molecule, resulting in an inflammatory bias. We present an integrative analysis of clinical data, cytokines, serum metabolomics in RA patients and mechanistic studies on ADA-mediated effects on in vitro cell culture models. ADA activity differentiated patients into low and high ADA sets. The levels of the cytokines TNFα, IFNγ, IL-10, TGFβ and sRANKL were elevated in RA and more pronounced in high ADA sets. Serum metabolomic analysis shows altered metabolic pathways in RA which were distinct between low and high ADA sets. Comparative analysis with previous studies shows similar pathways are modulated by DMARDs and biologics. Random forest analysis distinguished RA from control by methyl-histidine and hydroxyisocaproic acid, while hexose-phosphate and fructose-6-phosphate distinguished high ADA from low ADA. The deregulated metabolic pathways of High ADA datasets significantly overlapped with high ADA expressing PBMCs GEO transcriptomics dataset. ADA induced the death of chondrocytes, synoviocyte proliferation, both inflammation in macrophages and their differentiation into osteoclasts and impaired differentiation of mesenchymal stem cells to osteoblasts and mineralization. PBMCs expressing elevated ADA had increased expression of cytokines and P2 receptors compared to synovial macrophages which has low expression of ADA. Our data demonstrates increased cytokine levels and distinct metabolic signatures of RA based on the ADA activity, suggests an important role for ADA in the pathophysiology of RA joints and as a potential marker and therapeutic target in RA patients.
Glaucoma of which primary open angle glaucoma (POAG) constitutes 75%, is the second leading cause of blindness. Elevated intra ocular pressure and Nitric oxide synthase (NOS) dysfunction are hallmarks of POAG. We analyzed clinical data, cytokine profile, ATP level, metabolomics and GEO datasets to identify features unique to POAG. N9 microglial cells are used to gain mechanistic insights. Our POAG cohort showed elevated ATP in aqueous humor and cytokines in plasma. Metabolomic analysis showed changes in 21 metabolites including Dimethylarginine (DMAG) and activation of tryptophan metabolism in POAG. Analysis of GEO data sets and previously published proteomic data sets bins genes into signaling and metabolic pathways. Pathways from reanalyzed metabolomic data from literature significantly overlapped with those from our POAG data. DMAG modulated purinergic signaling, ATP secretion and cytokine expression were inhibited by N-Ethylmaleimide, NO donors, BAPTA and purinergic receptor inhibitors. ATP induced elevated intracellular calcium level and cytokines expression were inhibited by BAPTA. Metabolomics of cell culture supernatant from ATP treated sets showed metabolic deregulation and activation of tryptophan metabolism. DMAG and ATP induced IDO1/2 and TDO2 were inhibited by N-Ethylmaleimide, sodium nitroprusside and BAPTA. Our data obtained from clinical samples and cell culture studies reveal a strong association of elevated DMAG, ATP, cytokines and activation of tryptophan metabolism with POAG. DMAG mediated ATP signaling, inflammation and metabolic remodeling in microglia might have implications in management of POAG.
P2 receptors are nucleotide-activated receptors involved in inflammation, cell proliferation osteoblastogenesis, osteoclastogenesis and their function. They can be potential role players in the pathophysiology of rheumatoid arthritis (RA). Our analysis of gene expression datasets of synovial tissue biopsy from the GEO database shows changes in the expression levels of P2 receptors. HIG-82, a synovial fibroblast cell line and RAW 264.7, a macrophage cell line are good in vitro models to study RA. Nucleotide addition experiments showed UDP Glucose significantly increased the proliferation of synovial fibroblasts (HIG-82). Similarly, nucleotides such as Adenosine tri-phosphate (ATP), Adenosine di-phosphate (ADP), Uridine tri-phosphate (UTP), Uridine di-phosphate (UDP) and Uridine diphosphoglucose (UDPG) induced elevated reactive oxygen species (ROS) and tartrate Resistant Acid Phosphatase (TRAP) activity in RAW264.7 cells. The ADP-induced TRAP could be inhibited by clopidogrel a P2Y 12 inhibitor. ATP, ADP, UTP, UDP and UDPG also induced osteoclastogenesis as evident from fused multinucleate cells and expression of osteoclast markers (TRAP, Cathepsin K [CTSK]) as determined by Q-PCR. Apyrase (APY) a nucleotidase and an enzyme that is used to modulate extracellular nucleotide concentration is sufficient to induce osteoclastogenesis. Taken together our results show that nucleotides modulate synoviocyte proliferation and macrophage differentiation into osteoclast and play an important role in RA. Nucleotide receptors might be potential therapeutic targets in RA.
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