Transcription Of Inflammatory Cytokines Research Articles

Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice

Inflammatory reactions activated by pattern recognition receptors (PRRs) on the membrane of innate immune cells play an important role in atherosclerosis. Whether the PRRs of the C-type lectin receptor (CLR) family including Dectin-2 may be involved in the pathogenesis of atherosclerosis remains largely unknown. Recently, the CLR-adaptor molecule caspase recruitment domain family member 9 (CARD9) has been suggested to play a role in cardiovascular pathologies as it provides the link between CLR activation and transcription of inflammatory cytokines as well as immune cell recruitment. We therefore evaluated whether hematopoietic deletion of Dectin-2 or CARD9 reduces inflammation and atherosclerosis development. Low-density lipoprotein receptor (Ldlr)-knockout mice were transplanted with bone marrow from wild-type, Dectin-2- or Card9-knockout mice and fed a Western-type diet containing 0.1% (w/w) cholesterol. After 10 weeks, lipid and inflammatory parameters were measured and atherosclerosis development was determined. Deletion of hematopoietic Dectin-2 or CARD9 did not influence plasma triglyceride and cholesterol levels. Deletion of hematopoietic Dectin-2 did not affect atherosclerotic lesion area, immune cell composition, ex vivo cytokine secretion by peritoneal cells or bone marrow derived macrophages. Unexpectedly, deletion of hematopoietic CARD9 increased atherosclerotic lesion formation and lesion severity. Deletion of hematopoietic CARD9 did also not influence circulating immune cell composition and peripheral cytokine secretion. Besides a tendency to a reduced macrophage content within these lesions, plasma MCP-1 levels decreased upon WTD feeding. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development in hyperlipidemic mice. The absence of CARD9 unexpectedly increased atherosclerotic lesion size and severity, suggesting that the presence of CARD9 may protect against initiation of atherosclerosis development.

Inhibitory effect of polysaccharide of Sargassum weizhouense on PCV2 induced inflammation in mice by suppressing histone acetylation

Seaweeds are excellent source of bioactive compounds and seaweed-derived polysaccharides have demonstrated an array of biological effects. Here, we investigated the effect of polysaccharide of Sargassum weizhouense (PSW) on the inflammatory response in porcine circovirus type 2 (PCV2) infected mice and the underlying mechanism was studied according to the histone acetylation. After PCV2 infection, the levels of TNF-α, IL-1β, IL-6, IL-8, IL-10, MCP-1, COX-1, COX-2 and HAT in both serum and spleen were significantly increased (P <0.05). The mRNA expression of TNF-α, IL-6, IL-10 and NF-κB p65 were elevated in PCV2 infected mice (P <0.05). The HDAC content in both serum and spleen as well the mRNA expression of HDAC1 were greatly decreased (P <0.05). PSW treatment dramatically inhibited the secretions of inflammatory cytokines and HATs, reduced mRNA expression of TNF-α, IL-6, IL-10 and NF-κB p65, but promoted HDAC secretion and mRNA expression of HDAC1 in PCV2-infected mice. The acetylation of both H3 and H4 was significantly up-regulated in PCV2-infected mice, and strongly inhibited by PSW treatment (P <0.01). These results suggested that PCV2 mediate the equilibrium between HATs and HDACs, alternate the histone acetylation and thus DNA packaging, and then activate the transcription of inflammatory cytokines. PSW could inhibit the histone acetylation and the production of inflammatory cytokines, showing excellent potentials in improving the resistance of host against PCV2 infection.

Curcumin inhibits inflammatory cytokine transcription via the apoptosis pathway in THP-1 cells

Curcumin inhibits inflammatory cytokine transcription via the apoptosis pathway in THP-1 cells

MyD88 hypermethylation mediated by DNMT1 is associated with LTA-induced inflammatory response in human odontoblast-like cells.

Dental caries is a chronic, infectious, and destructive disease that allows bacteria to break into the dental pulp tissue. As caries-related bacteria invade the human dentinal tubules, odontoblasts are the first line of dental pulp that trigger the initial inflammatory and immune responses. DNA methylation is a key epigenetic modification that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, whether DNA methylation regulates the inflammatory response of human odontoblasts is still unknown. In the present study, we investigated the expression of DNA methyltransferase (DNMT)-1 in lipoteichoic acid (LTA)-stimulated human odontoblast-like cells (hOBs) and found that DNMT1 expression showed a decline that is contrary to the transcription of inflammatory cytokines. Knockdown of the DNMT1 gene increased the expression of several cytokines, including IL-6 and IL-8, in the LTA-induced inflammatory response. DNMT1 knockdown increased the phosphorylation of IKKα/β, IκBα, and p65 in the NF-κB pathway and the phosphorylation of p38 and ERK in the MAPK pathway; however, only the NF-κB pathway inhibitor PDTC suppressed both IL-6 and IL-8 expression, whereas inhibitors of the MAPK pathway (U0126, SB2035580, and SP600125) did not. Furthermore, DNMT1 knockdown upregulated the expression of MyD88 and TRAF6 but only attenuated the MyD88 gene promoter methylation in LTA-treated hOBs. Taken together, these results demonstrated that DNMT1 depletion caused hypomethylation and upregulation of MyD88, which resulted in activation of the NF-κB pathway and the subsequent release of LTA-induced inflammatory cytokines in hOBs. This study emphasizes the critical role of DNA methylation in the immune defense of odontoblasts when dental pulp reacted to caries.

Retinoic Acid Receptor Alpha Represses a Th9 Transcriptional and Epigenomic Program to Reduce Allergic Pathology

CD4+ T helper (Th) differentiation is regulated by diverse inputs, including the vitamin A metabolite retinoic acid (RA). RA acts through its receptor RARα to repress transcription of inflammatory cytokines, but is also essential for Th-mediated immunity, indicating complex effects of RA on Th specification and the outcome of the immune response. We examined the impact of RA on the genome-wide transcriptional response during Th differentiation to multiple subsets. RA effects were subset-selective and were most significant in Th9 cells. RA globally antagonized Th9-promoting transcription factors and inhibited Th9 differentiation. RA directly targeted the extended Il9 locus and broadly modified the Th9 epigenome through RARα. RA-RARα activity limited murine Th9-associated pulmonary inflammation, and human allergic inflammation was associated with reduced expression of RA target genes. Thus, repression of the Th9 program is a major function of RA-RARα signaling in Th differentiation, arguing for a role for RA in interleukin 9 (IL-9) related diseases.

Notch4 activation aggravates NF-κB-mediated inflammation in HIV-1-associated nephropathy.

ABSTRACTNotch pathway activation plays a central role in the pathogenesis of many glomerular diseases. We have previously shown that Notch4 expression was upregulated in various renal cells in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) patients and rodent models of HIVAN. In this study, we examined whether the Notch pathway can be distinctly activated by HIV-1 gene products and whether Notch4, in particular, can influence disease progression. Using luciferase reporter assays, we did not observe activation of the NOTCH4 promoter with the HIV protein Nef in podocytes. Further, we observed upregulated expression of a gamma secretase complex protein, presenilin 1, but not Notch4, in podocytes infected with an HIV-1 expression construct. To assess the effects of Notch4 on HIVAN disease progression, we engineered Tg26 mice with global deletion of the Notch4 intracellular domain (Notch4dl), which is required for signaling function. These mice (Notch4d1/Tg26+) showed a significant improvement in renal function and a significant decrease in mortality compared to Tg26 mice. Histological examination of kidneys showed that Notch4d1/Tg26+ mice had overall glomerular, tubulointerstitial injury and a marked decrease in interstitial inflammation. A significant decrease in the proliferating cells was observed in the tubulointerstitial compartments of Notch4d1/Tg26+ mice. Consistent with the diminished inflammation, kidneys from Notch4d1/Tg26+ mice also showed a significant decrease in expression of the inflammatory cytokine transcripts Il-6 and Ccl2, as well as the master inflammatory transcription factor NF-κB (Nfkb1 transcripts and p65 protein). These data identify Notch4 as an important mediator of tubulointerstitial injury and inflammation in HIVAN and a potential therapeutic target.

(+)-JQ1 attenuated LPS-induced microglial inflammation via MAPK/NF\u03baB signaling

BackgroundMicroglia activation is a crucial event in neurodegenerative disease. The depression of microglial inflammatory response is considered a promising therapeutic strategy. NFκB signaling, including IKK/IκB phosphotylation, p65 nucelus relocalization and NFκB-related genes transcription are prevalent accepted to play important role in microglial activation. (+)-JQ1, a BRD4 inhibitor firstly discovered as an anti-tumor agent, was later confirmed to be an anti-inflammatory compound. However, its anti-inflammatory effect in microglia and central neural system remains unclear.ResultsIn the current work, microglial BV2 cells were applied and treatment with lipopolysaccharide (LPS) to induce inflammation and later administered with (+)-JQ1. In parallel, LPS and (+)-JQ1 was intracerebroventricular injected in IL-1β-luc transgenic mice, followed by fluorescence evaluation and brain tissue collection. Results showed that (+)-JQ1 treatment could significantly reduce LPS induced transcription of inflammatory cytokines both in vitro and in vivo. (+)-JQ1 could inhibit LPS induced MAPK but not PI3K signaling phosphorylation, NFκB relocalization and transcription activity. In animal experiments, (+)-JQ1 postponed LPS induced microglial and astrocytes activation, which was also dependent on MAPK/NFκB signaling.ConclusionsThus, our data demonstrated that (+)-JQ1 could inhibit LPS induced microglia associated neuroinflammation, via the attenuation of MAPK/NFκB signaling.

MicroRNA-182-5p attenuates cerebral ischemia-reperfusion injury by targeting Toll-like receptor 4

Cerebral ischemia-reperfusion-induced microglial activation causes neuronal death through the release of inflammatory cytokines. Increasing evidence suggests that microRNAs (miRNAs) exert a neuroprotective effect by modulating the inflammatory process in cerebral ischemia-reperfusion injury. Furthermore, Toll-like receptor 4 (TLR4) is increasingly being considered to have a significant role in the regulation of inflammation. However, whether miRNAs mediate their neuroprotective effects by regulating TLR4-mediated inflammatory responses remains unknown. To explore this gap in the literature, we conducted both in vitro and in vivo experiments. In vitro: BV2 cells were activated by oxygen-glucose deprivation (OGD). TLR4 and inflammatory cytokine (TNF-a, IL-6, and IL-1β) transcription and translation expression levels were assessed using RT-PCR, ELISA, and western blot. BV2 cells were transfected with miR-182-5p mimics, inhibitors, siTLR4, or negative control (NC) using lipofectamine 2000 reagent. To confirm whether TLR4 is a direct target of miR-182-5p, we performed a luciferase reporter assay. In BV2 cells, we observed that OGD upregulated TLR4 expression, but downregulated miR-182-5p expression. We determined that miR-182-5p inhibited TLR4 by directly binding to its 3′-UTR. Furthermore, miR-182-5p suppressed the release of TNF-a, IL-6, and IL-1β. In vivo: A middle cerebral artery occlusion (MCAO) rat model was used to mimic cerebral ischemia-reperfusion. Iba1 and TLR4 double staining was used to demonstrate that the target of miR-182-5p in microglial cells, and the mediator of the anti-inflammatory effect, is TLR4. TTC staining was performed to evaluate the infarct volume. Compared to the animals treated with miR-182-5p NC and normal saline, rats treated with miR-182-5p mimics demonstrated significantly enhanced neurological functions. TTC staining results were consistent with neurological function test findings. In summary, our data suggested that miR-182-5p exhibits potential neuroprotective effects in the cerebral ischemia-reperfusion injury via the regulation of the TLR4-mediated inflammatory response.

Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate.

Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1, cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.

Transcriptional Factor FOXP3 in Renal Allograft Biopsies Predicts Tolerance in Non-Human Primates

NIH NHP Transplantation Tolerance Cooperative Study Group. Identification of reliable biomarkers for tolerance is critically important in transplantation. We reasoned that intragraft transcripts may be more revealing than those in peripheral blood leukocytes if tolerance is mediated in part by events in the graft. Using the NanoString nCounter platform, we retrospectively studied 256 kidney allograft formalin-fixed paraffin-embedded (FFPE) serial samples taken from non-human primate recipients after combined kidney and bone marrow transplantation (CKBMT) to measure 49 genes, which were previously reported significantly associated with tolerance or rejection. We found dynamic time-dependent kinetics of intragraft biomarkers, which indicated the timing of analysis is critical to interpret the results (Figure 1).. To identify biomarkers that can predict the transplant outcome at the early post-transplant period, we focused on analyzing mRNAs in biopsies taken between 30 and 120 days after CKBMT when no rejection was yet observed. The recipients that subsequently achieved tolerance (TOL, n=11, allograft survival>1995 ± 557 days) showed significantly higher mRNA expression of FOXP3, BCL2, GATA3 and RPS6KB1 compared with biopsies from recipients that later developed chronic antibody-mediated rejection (cAMR, n=11, 850 ± 134 days) or T cell mediated rejection (TCMR, n=9, 109 ± 22 days). In contrast, transcripts of inflammatory cytokines or adaptive immunity (IFNG, CXCL11, FCGR3A, GNLY, GZMB, IL4 and CD4) were higher in TCMR, while endothelium associated transcripts, such as PECAM1 and TEK, were higher in cAMR (Figure 2A and 2B). The ROC curve analyses revealed that intragraft FOXP3 mRNA alone reliably differentiate TOL from both cAMR and TCMR, and that ratios of FOXP3 to several inflammatory cytokine signatures showed better AUC to differentiate TOL from TCMR The significance of intragraft FOXP3 for tolerance was validated by the prospective study with 11 CKBMT recipients. We conclude that intragraft mRNA transcripts, especially FOXP3, measured early post transplantation can serve as biomarkers to reliably predict subsequent tolerance induction via the mixed chimerism approach. Grant 5U19AI102405.

Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-\u03baB activation

Apoptotic cells modulate the function of macrophages to control and resolve inflammation. Here, we show that neutrophils induce a rapid and sustained suppression of NF-κB signalling in the macrophage through a unique regulatory relationship which is independent of apoptosis. The reduction of macrophage NF-κB activation occurs through a blockade in transforming growth factor β-activated kinase 1 (TAK1) and IKKβ activation. As a consequence, NF-κB (p65) phosphorylation is reduced, its translocation to the nucleus is inhibited and NF-κB-mediated inflammatory cytokine transcription is suppressed. Gene Set Enrichment Analysis reveals that this suppression of NF-κB activation is not restricted to post-translational modifications of the canonical NF-κB pathway, but is also imprinted at the transcriptional level. Thus neutrophils exert a sustained anti-inflammatory phenotypic reprogramming of the macrophage, which is reflected by the sustained reduction in the release of pro- but not anti- inflammatory cytokines from the macrophage. Together, our findings identify a novel apoptosis-independent mechanism by which neutrophils regulate the mediator profile and reprogramming of monocytes/macrophages, representing an important nodal point for inflammatory control.

998 EMT-like phenotype in normal keratinocytes driven by TLR3 activation

Toll-Like-Receptor 3 (TLR3), a pattern recognition receptor that recognizes dsRNA, activation results in NFkB or interferon-regulatory factor 3 (IRF3) mediated gene transcription of inflammatory cytokines (e.g. IL-6) and type I and III interferons (e.g. IFN-λ). TLR3 also contributes to induced-pluripotent stem cell (IPS) programming, wound healing and skin regeneration through Wnt, SHH, and epithelial-mesenchymal transition (EMT) pathways. Our hypothesis is that TLR3 signaling within normal keratinocytes contributes to non-melanoma skin cancer (NMSC) through activation of EMT. In vitro, normal neonatal human embryonic keratinocytes (NHEKs) treated with a TLR3 agonist, polyinosinic:polycytidylic acid (poly IC), exhibit sustained EMT morphology changes that correlate with elevated levels of the intermediate filament Vimentin (∼2 fold), and transcription factors, ZEB1 (∼3-fold), SNAI1 (∼2-fold), and TWIST1 (∼2-fold), all key EMT markers. Poly IC treatment significantly increased levels of IL-6 (∼8-fold) and IFN-λ1 (∼200-fold) in normal keratinocytes. However, treatment of NHEKs with recombinant IL-6 or IFN-λ1 proteins do not induce an EMT-like morphology or increase in EMT gene expression. UVB radiation, the major player in NMSC development, also activates TLR3 signaling in keratinocytes. In vitro, NHEKs exposed to UVB (10mJ/cm2) exhibit similar EMT-like morphology and gene expression changes as poly IC treated cells. Chemical inhibition of TLR3 (CU CPT 4a) prior to poly IC or UVB exposure prevents EMT gene expression. We performed RNA-sequencing to investigate common pathways activated by both Poly IC and UVB to uncover additional downstream players responsible for driving the TLR3 induced EMT-phenotype. Taken together, TLR3 activation and the resulting EMT-like phenotype is a result of multiple players, and not likely driven by a single downstream mediator of TLR3 signaling.

Baicalin modulates the Treg/Teff balance to alleviate uveitis by activating the aryl hydrocarbon receptor

Autoimmune uveitis is a sight-threatening ocular inflammatory disorder. Immunological inflammation is regarded as the key to pathogenesis in autoimmune uveitis. Baicalin, the major bioactive component of Scutellaria baicalensis, possesses immunomodulatory properties. However, the role of baicalin in uveitis and its underlying mechanisms remain unclear. In the current study, we found that baicalin treatment obviously inhibited the intraocular inflammatory process in mice with experimental autoimmune uveitis, along with clear declines in infiltrated inflammatory cells and inflammatory cytokine transcription in the retina and draining lymph nodes. Furthermore, baicalin treatment increased the frequency and number of regulatory T cells and decreased the frequency and number of effector T cells (Th1 and Th17 cells) in the draining lymph nodes of mice with experimental autoimmune uveitis. In vitro, baicalin treatment suppressed interphotoreceptor retinoid binding protein-specific CD4+ T cell proliferation and converted CD4+ T cell differentiation. Furthermore, the expression of aryl hydrocarbon receptor was activated by baicalin treatment. Baicalin-mediated modulation of CD4+ T cell differentiation was partially abrogated by the suppression of aryl hydrocarbon receptor. These findings suggest that baicalin modulates the Treg/Teff balance and CD4+ T cell proliferation to ameliorate experimental autoimmune uveitis by activating the aryl hydrocarbon receptor.

PKR inhibition mediates endotoxin tolerance in macrophages through inactivation of PI3K/AKT signaling.

Following long‑term exposure to endotoxins, macrophages enter an immunosuppressive state that renders them unable respond to subsequent exposures to endotoxin, a phenomenon that is termed 'endotoxin tolerance'. Endotoxin tolerance increases the risks of secondary infection and mortality in patients with sepsis. In endotoxin‑tolerant macrophages, the mixed variation of gene transcription is referred to as macrophage reprogramming. The mechanisms underlying macrophage reprogramming remain unclear at present. Interferon‑induced double‑stranded RNA‑dependent protein kinase (PKR) is a widely expressed serine/threonine protein kinase. In addition to antiviral effects, PKR regulates the transcription of inflammatory cytokines by affecting transcription factors. However, the role of PKR in macrophage reprogramming remains to be elucidated. In the present study, the expression of inflammatory cytokines differed in lipopolysaccharide (LPS)‑tolerant RAW264.7 macrophages compared with LPS‑activated macrophages. Specifically, reverse transcription‑quantitative polymerase chain reaction results demonstrated that the mRNA levels of tumor necrosis factor‑α, interleukin‑1β (IL‑1β), C‑X‑C motif chemokine ligand 11, C‑C motif chemokine ligand (CCL17), CCL22 and suppressor of cytokine signaling 3 were decreased, and mRNAs levels of arginase‑1 (Arg1) and nitric oxide synthase 2 (iNOS) were increased, in LPS‑tolerant macrophages compared with LPS‑activated macrophages. Furthermore, western blot analysis demonstrated that the protein levels of phosphorylated (p)‑PKR were significantly decreased in the LPS‑tolerant cells. PKR activation with rotenone (10µM) abrogated endotoxin tolerance by increasing the levels of the IL‑1β, CCL17 and CCL22 mRNAs and decreasing the levels of the Arg1 and iNOS mRNAs. Furthermore, western blotting demonstrated that AKT was markedly inactivated in endotoxin‑tolerant cells, as indicated by reduced p‑AKT levels. However, levels of p‑AKT were markedly increased following rotenone‑induced PKR activation in endotoxin‑tolerant cells. Ly294002 (10µM), a phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K)/AKT signaling inhibitor, partially reversed the rotenone‑induced alleviation of endotoxin tolerance. These results demonstrated that PKR inhibition mediated endotoxin tolerance in macrophages, and these effects were partially mediated by PI3K/AKT signaling. PKR may be a potential target for the treatment of endotoxin tolerance in patients with sepsis.

Lysophosphatidic Acid Regulates the Vitamin D Receptor and its Metabolism‐Related Genes in Human Gingival Fibroblasts

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA 18:1 controls immediate‐early GF gene transcription of inflammatory cytokines, and of their receptors and regulators. Vitamin D3 is a key hormone in decreasing inflammation, and has been shown by Nastri et al. (2018) to decrease pro‐inflammatory and increase anti‐inflammatory cytokine production by P. gingivalis‐ and S. pyogenes‐infected human GF. Therefore, we hypothesized that LPA would also modulate transcription of its receptor, VDR, since chronic periodontal disease (PD) is an inflammatory condition in which we have reported that LPA is elevated in human gingival crevicular fluid and saliva (Bathena et al., 2011). After GF treatment with 1 × 10−5 M LPA 18:1 for 2h or 8h, mRNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analyses [n=3; each n= pooled mRNA derived from 3 healthy young donors; (9 total donors; the study conformed to the Declaration of Helsinki guidelines, was approved by the Creighton University IRB, and informed written consent was obtained from all donors)] revealed that LPA consistently and significantly down‐regulated the VDR vs. control (≤ −2.0 ± 0.5‐fold at 2h, and −3.1 ± 1.1 fold at 8h, respectively). Of great interest, the receptor cubilin (CUBN), which has been shown in other systems to bind vitamin D binding protein (DBP), was also down‐regulated in groups 1 (−2.0) and 2 (−4.1) at 8h; it may be involved in the secretion of vitamin D by GF, as postulated by Liu et al., (2012). LPA treatment also significantly affected transcription of the cytochromes (CYP) CYP27A1 [−3.0 ± 0.6 (2h); −3.2 ± 1.4 (8h)] and CYP27B1 (induction or repression varied by group) which is involved in the production of metabolically active metabolites and which catalyzes the hydroxylation at the C‐1α position of 25(OH)D3 and 24R, 25(OH)2D3, respectively. These results for the VDR (minimum level of significance, p &lt; 1×10−7) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by regulating it, along with other key receptors and enzymes in the vitamin D pathway.Support or Funding InformationHealth Future Foundation (D.R.C).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Valsartan attenuates bleomycin-induced pulmonary fibrosis by inhibition of NF-κB expression and regulation of Th1/Th2 cytokines

Objective: Pulmonary fibrosis (PF) is a chronic respiratory system disease. The role of inflammation and angiotensin in the development and progression of PF has previously been demonstrated. Alternation in antifibrotic/profibrotic mediators and NF-κB activation have important roles in PF development. NF-κB, a nuclear factor, induces the transcription of inflammatory and pro-inflammatory cytokines. The aim of this study was to evaluate the effect of valsartan as an angiotensin receptor blocker on IL-4, INF-γ, and NF-κB expression in the treatment of PF.Materials and methods: Rats were divided into five groups: groups I (bleomycin) and II (control) received a single injection of bleomycin (7.5 IU/kg) or vehicle, respectively. Groups III–V received valsartan (20, 40, and 80 mg/kg, respectively) orally a week before and for 3 weeks after the bleomycin injection. Serum levels of IL-4 and INF- γ were then measured. Relative NF-κB expression was investigated by real-time PCR.Results: Histopathological examination showed the anti-inflammation effect of valsartan. Bleomycin significantly increased IL-4 serum level and decreased that of INF-γ in the serum. Valsartan could restore their levels to normal. Valsartan raised the decreased ratio of INF-γ/IL-4. Exposure to bleomycin elevated NF-κB expression; and valsartan decreased the increased gene expression.Discussion: Valsartan as an angiotensin receptor antagonist presumably by blocking angiotensin receptor causes to ameliorated PF, which was at least partly due to antifibrotic/profibrotic cytokine regulation and reduced NF-κB expression.Conclusions: Valsartan showed a significant protective effect against bleomycin-induced PF.

Ultrasound microbubble-mediated transfection of NF-κB decoy oligodeoxynucleotide into gingival tissues inhibits periodontitis in rats in vivo.

Periodontitis is a chronic infectious disease for which the fundamental treatment is to reduce the load of subgingival pathogenic bacteria by debridement. However, previous investigators attempted to implement a nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) as a suppressor of periodontitis progression. Although we recently reported the effectiveness of the ultrasound-microbubble method as a tool for transfecting the NF-κB decoy ODN into healthy rodent gingival tissue, this technique has not yet been applied to the pathological gingiva of periodontitis animal models. Therefore, the aim of this study was to investigate the effectiveness of the technique in transfecting the NF-κB decoy ODN into rats with ligature-induced periodontitis. Micro computed tomography (micro-CT) analysis demonstrated a significant reduction in alveolar bone loss following treatment with the NF-κB decoy ODN in the experimental group. RT-PCR showed that NF-κB decoy ODN treatment resulted in significantly reduced expression of inflammatory cytokine transcripts within rat gingival tissues. Thus, we established a transcutaneous transfection model of NF-κB decoy ODN treatment of periodontal tissues using the ultrasound-microbubble technique. Our findings suggest that the NF-κB decoy ODN could be used as a significant suppressor of gingival inflammation and periodontal disease progression.

Interplay of inflammatory gene expression in pericytes following Japanese encephalitis virus infection

Neuroinflammation is a pathological hallmark and has been implicated in the pathogenesis of Japanese encephalitis. Although brain pericytes show regulatory effects on neuroinflammation, their involvement in Japanese encephalitis-associated neuroinflammation is not understood. Here, we demonstrated that brain microvascular pericytes could be an alternative cellular source for the induction and/or amplification of neuroinflammation caused by Japanese encephalitis virus (JEV) infection. Infection of cultured pericytes with JEV caused profound production of IL-6, RANTES, and prostaglandin E2 (PGE2). Mechanistic studies revealed that JEV infection elicited an elevation of the toll-like receptor 7 (TLR7)/MyD88 signaling axis, leading to the activation of NF-κB through IKK signaling and p65 phosphorylation as well as cAMP response element-binding protein (CREB) via phosphorylation. We further demonstrated that extracellular signal-regulated kinase (ERK) could be an alternative regulator in transducing signals to NF-κB, CREB, and cytosolic phospholipase A2 (cPLA2) through the phosphorylation mechanism. Released IL-6 and RANTES played an active role in the disruption of endothelial barrier integrity and leukocyte chemotaxis, respectively. cPLA2/PGE2 had a role in activating NF-κB and CREB DNA-binding activities and inflammatory cytokine transcription via the EP2/cAMP/PKA mechanism in an autocrine loop. These inflammatory responses and biochemical events were also detected in the brain of JEV-infected mice. The current findings suggest that pericytes might have pathological relevance in Japanese encephalitis-associated neuroinflammation through a TLR7-related mechanism. The consequences of pericyte activation are their ability to initiate and/or amplify inflammatory cytokine expression by which cellular function of endothelial cells and leukocytes are regulated in favor of CNS infiltration by leukocytes.

Involvement of receptor-interacting protein 140 in palmitate-stimulated macrophage infiltration of pancreatic beta cells.

Receptor-interacting protein 140 (RIP140) in macrophages stimulates the nuclear factor-κB subunit RelA to activate tumor necrosis factor (TNF)-α and interleukin (IL)-6 transcription. However, under lipotoxic conditions, the involvement of RIP140 in the infiltration of beta cells by macrophages remains unknown. In the present study, murine RAW264.7 macrophages were transfected with a RIP140 overexpression plasmid or siRNA prior to macrophage activation with 500 µM palmitate. Palmitate-free conditioned media was then collected and added to murine insulinoma MIN6 cells. Significant decreases were observed in cell viability (P<0.01), glucose-stimulated insulin secretion (P<0.01) and levels of peroxisome proliferator-activated receptor-γ coactivator-1α (P<0.05), phosphoenolpyruvate carboxykinase and proliferating cell nuclear antigen mRNA (P<0.01) in MIN6 cells. In addition, conditioned media from palmitate-treated and RIP140-upregulated macrophages significantly increased the levels of uncoupling protein-2 (P<0.01), inducible nitric oxide synthase 1 (P<0.01) and pancreatic and duodenal homeobox 1 (P<0.05) mRNA and levels of activated Jun N-terminal kinase (JNK) (P<0.01) and extracellular signal-regulated kinase (ERK) 1/2 (P<0.01). In turn, the conditioned media was found to be significantly enriched in TNF-α and IL-6 (both P<0.05). These results were the opposite of those obtained from MIN6 cells treated with conditioned media from palmitate-treated and RIP140-knockdown macrophages. MIN6 cells were transfected with RIP140 overexpression plasmid or siRNA prior to treatment with 500 µM palmitate and supernatant was collected for use in macrophage chemotaxis assays. In the palmitate-activated and RIP140-overexpressing MIN6 cells, TNF-α and IL-6 secretion increased significantly (both P<0.05) and macrophage chemotaxis towards MIN6 cells was enhanced. By contrast, downregulating RIP140 in MIN6 cells had the opposite effect. These data suggest that RIP140 in macrophages mediates the transcription of inflammatory cytokines when concentration of palmitate is high. Macrophage RIP140 may also impair beta cell function by activating the JNK and ERK1/2 signaling pathways and promoting specific gene transcription. Furthermore, expression of RIP140 in pancreatic beta cells may stimulate macrophage chemotaxis, thus triggering local low-grade inflammation.

Damage-associated molecular patterns in the pathogenesis of osteoarthritis: potentially novel therapeutic targets.

Osteoarthritis (OA) is a chronic disease that degrades the joints and is often associated with increasing age and obesity. The two most common sites of OA in adults are the knee and hip joints. Increased mechanical stress on the joint from obesity can cause the articular cartilage to degrade and release damage-associated molecular patterns (DAMPs). These DAMPs are involved in various molecular pathways that interact with nuclear factor-kappa B and result in the transcription of inflammatory cytokines and activation of matrix metalloproteinases that progressively destroy cartilage. This review focuses on the interactions and contribution to the pathogenesis and progression of OA through the DAMPs: high-mobility group box 1 (HMGB-1), the receptor for advanced glycation end-products (RAGE), the alarmin proteins S100A8 and S100A9, and heparan sulfate. HMGB-1 is released from damaged or necrotic cells and interacts with toll-like receptors (TLRs) and RAGE to induce inflammatory signals, as well as behave as an inflammatory cytokine to activate innate immune cells. RAGE interacts with HMGB-1, advanced glycation end-products, and innate immune cells to increase local inflammation. The alarmin proteins are released following cell damage and interact through TLRs to increase local inflammation and cartilage degradation. Heparan sulfate has been shown to facilitate the binding of HMGB-1 to RAGE and could play a role in the progression of OA. Targeting these DAMPs may be the potential therapeutic strategies for the treatment of OA.