Inflammation-induced Expression Research Articles

Collagenase-Responsive Hydrogel Loaded with GSK2606414 Nanoparticles for Periodontitis Treatment through Inhibiting Inflammation-Induced Expression of PERK of Periodontal Ligament Stem Cells.

GSK2606414 is a new, effective, highly selective PERK inhibitor with adenosine-triphosphate-competitive characteristics. It can inhibit endoplasmic reticulum stress and has the possibility of treating periodontitis. However, owing to its strong hydrophobicity and side effects, highly efficient pharmaceutical formulations are urgently needed to improve the bioavailability and therapeutic efficacy of GSK2606414 in the treatment of periodontitis. Herein, a novel local GSK2606414 delivery system was developed by synthesizing GSK2606414 nanoparticles (NanoGSK) and further loading NanoGSK into a collagenase-responsive hydrogel. The drug release results showed that the drug-loaded hydrogels had outstanding enzymatic responsive drug release profiles under the local microenvironment of periodontitis. Furthermore, in vitro studies showed that the drug-loaded hydrogel exhibited good cellular uptake and did not affect the growth and proliferation of normal cells, while the drug-loaded hydrogel significantly improved the osteogenic differentiation of inflammatory cells. In the evaluations of periodontal tissue repair, the drug-loaded hydrogels showed a great effect on inflammation inhibition, as well as alveolar bone regeneration. Therefore, this work introduces a promising strategy for the clinical treatment of periodontitis.

Anti-Inflammatory Activity of a Demineralized Bone Matrix: An In Vitro Pilot Study

Demineralized bone matrix (DBM) is commonly used for the reconstruction of bone defects. Early graft consolidation involves a transient inflammatory process. It is, however, unclear whether DBM can modulate this process. To test this possibility, we prepared acid lysates of demineralized ground cortical (DGC) and moldable demineralized fibers (MDF). Murine RAW 264.7 and primary bone marrow macrophages were exposed to acid lysates of DGC and MFD prior to provoking an inflammatory response with lipopolysaccharide (LPS). Similarly, murine ST2 mesenchymal cells were exposed to DGC and MFD with and without interleukin 1β (IL1) and TNFα. We show here that acid lysates of DGC and MFD reduced the expression of IL1 and IL6 in RAW 264.7 macrophages, as determined by RT-PCR and, for IL6, by immunoassay. This response was confirmed with primary macrophages. Likewise, desalted acid lysates exert anti-inflammatory properties on RAW 264.7 cells and in ST2 cells, the forced expression of IL6, inducible nitric oxide synthase (iNOS) and chemokine ligand 5 (CCL5) was reduced. These in vitro findings suggest that DGC and MFD lower the inflammation-induced expression of inflammatory mediators in murine cell-based bioassays.

High miR-451 expression in peripheral blood mononuclear cells from subjects at risk of developing rheumatoid arthritis

Individuals carrying anti-citrullinated protein antibodies (ACPA) are considered at high risk of developing rheumatoid arthritis (RA). The altered expression of miRNAs contributes to the pathogenesis of RA. We aimed to identify differentially expressed miRNAs in the peripheral blood of ACPA-positive individuals with arthralgia at risk of RA compared to healthy controls (HC) and to determine their implications in the preclinical phase of RA. A comprehensive analysis of miRNAs revealed the dysregulation of miR-451 in peripheral blood mononuclear cells (PBMC) and plasma from RA-risk individuals. Higher miR-451 expression in PBMC from RA-risk individuals was further validated. Notably, miR-451 was previously shown to regulate CXCL16, a protein involved in RA pathogenesis. The expression of miR-451 in PBMC positively correlated with the CXCL16 mRNA, which could be secondary to the inflammation-induced expression of miR-451. Transfection of monocytes with pre-miR-451 in vitro resulted in the downregulation of CXCL16. Moreover, flow cytometry revealed a lower count of CXCL16-positive monocytes in RA-risk individuals. We propose that the constitutive or inflammation-induced upregulation of miR-451 in PBMC downregulates the expression of CXCL16, reduces the inflammatory milieu and thereby strives to delay the shift from the preclinical phase to the clinical manifestation of RA. This hypothesis warrants further investigation.

Mechanism of action and potential applications of selective inhibition of microsomal prostaglandin E synthase-1-mediated PGE2 biosynthesis by sonlicromanol\u2019s metabolite KH176m

Increased prostaglandin E2 (PGE2) levels were detected in mitochondrial disease patient cells harboring nuclear gene mutations in structural subunits of complex I, using a metabolomics screening approach. The increased levels of this principal inflammation mediator normalized following exposure of KH176m, an active redox-modulator metabolite of sonlicromanol (KH176). We next demonstrated that KH176m selectively inhibited lipopolysaccharide (LPS) or interleukin-1β (IL-1β)-induced PGE2 production in control skin fibroblasts. Comparable results were obtained in the mouse macrophage-like cell line RAW264.7. KH176m selectively inhibited mPGES-1 activity, as well as the inflammation-induced expression of mPGES-1. Finally, we showed that the effect of KH176m on mPGES-1 expression is due to the inhibition of a PGE2-driven positive feedback control-loop of mPGES-1 transcriptional regulation. Based on the results obtained we discuss potential new therapeutic applications of KH176m and its clinical stage parent drug candidate sonlicromanol in mitochondrial disease and beyond.

PXR Functionally Interacts with NF-κB and AP-1 to Downregulate the Inflammation-Induced Expression of Chemokine CXCL2 in Mice.

Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated intraperitoneally with the PXR agonist pregnenolone 16α-carbonitrile (PCN) and/or carbon tetrachloride (CCl4). Liver injury was evaluated, and hepatic mRNA levels were determined via quantitative reverse transcription polymerase chain reaction. Reporter assays with wild-type and mutated mouse Cxcl2 promoter-containing reporter plasmids were conducted in 293T cells. Results showed that the hepatic expression of inflammation-related genes was upregulated in CCl4-treated mice, and PCN treatment repressed the induced expression of chemokine-encoding Ccl2 and Cxcl2 among the genes investigated. Consistently, PCN treatment suppressed the increased plasma transaminase activity and neutrophil infiltration in the liver. In reporter assays, tumor necrosis factor-α-induced Cxcl2 expression was suppressed by PXR. Although an NF-κB inhibitor or the mutation of an NF-κB-binding motif partly reduced PXR-dependent suppression, the mutation of both NF-κB and activator protein 1 (AP-1) sites abolished it. Consistently, AP-1-dependent gene transcription was suppressed by PXR with a construct containing AP-1 binding motifs. In conclusion, the present results suggest that PXR exerts anti-inflammatory effects by suppressing both NF-κB- and AP-1-dependent chemokine expression in mouse liver.

IL-6 Induced by Periodontal Inflammation Causes Neuroinflammation and Disrupts the Blood-Brain Barrier.

Background: Periodontal disease (PD) is a risk factor for systemic diseases, including neurodegenerative diseases. The role of the local and systemic inflammation induced by PD in neuroinflammation currently remains unclear. The present study investigated the involvement of periodontal inflammation in neuroinflammation and blood–brain barrier (BBB) disruption. Methods: To induce PD in mice (c57/BL6), a ligature was placed around the second maxillary molar. Periodontal, systemic, and neuroinflammation were assessed based on the inflammatory cytokine mRNA or protein levels using qPCR and ELISA. The BBB permeability was evaluated by the mRNA levels and protein levels of tight junction-related proteins in the hippocampus using qPCR and immunofluorescence. Dextran tracing in the hippocampus was also conducted to examine the role of periodontal inflammation in BBB disruption. Results: The TNF-α, IL-1β, and IL-6 levels markedly increased in gingival tissue 1 week after ligation. The IL-6 serum levels were also increased by ligature-induced PD. In the hippocampus, the IL-1β mRNA expression levels were significantly increased by ligature-induced PD through serum IL-6. The ligature-induced PD decreased the claudin 5 expression levels in the hippocampus, and the neutralization of IL-6 restored its levels. The extravascular 3-kDa dextran levels were increased by ligature-induced PD. Conclusions: These results suggest that the periodontal inflammation-induced expression of IL-6 is related to neuroinflammation and BBB disruption in the hippocampus, ultimately leading to cognitive impairment. Periodontal therapy may protect against neurodegenerative diseases.

Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

BackgroundDiseases and disorders with a chronic neuroinflammatory component are often linked with changes in brain metabolism. Among neurodegenerative disorders, people living with human immunodeficiency virus (HIV) and Alzheimer’s disease (AD) are particularly vulnerable to metabolic disturbances, but the mechanistic connections of inflammation, neurodegeneration and bioenergetic deficits in the central nervous system (CNS) are poorly defined. The particularly interesting new cysteine histidine-rich-protein (PINCH) is nearly undetectable in healthy mature neurons, but is robustly expressed in tauopathy-associated neurodegenerative diseases including HIV infection and AD. Although robust PINCH expression has been reported in neurons in the brains of patients with HIV and AD, the molecular mechanisms and cellular consequences of increased PINCH expression in CNS disease remain largely unknown.MethodsWe investigated the regulatory mechanisms responsible for PINCH protein-mediated changes in bioenergetics, mitochondrial subcellular localization and bioenergetic deficits in neurons exposed to physiological levels of TNFα or the HIV protein Tat. Changes in the PINCH-ILK-Parvin (PIP) complex association with cofilin and TESK1 were assessed to identify factors responsible for actin depolymerization and mitochondrial mislocalization. Lentiviral and pharmacological inhibition experiments were conducted to confirm PINCH specificity and to reinstate proper protein-protein complex communication.ResultsWe identified MEF2A as the PINCH transcription factor in neuroinflammation and determined the biological consequences of increased PINCH in neurons. TNFα-mediated activation of MEF2A via increased cellular calcium induced PINCH, leading to disruption of the PIP ternary complex, cofilin activation by TESK1 inactivation, and actin depolymerization. The disruption of actin led to perinuclear mislocalization of mitochondria by destabilizing the kinesin-dependent mitochondrial transport machinery, resulting in impaired neuronal metabolism. Blocking TNFα-induced PINCH expression preserved mitochondrial localization and maintained metabolic functioning.ConclusionsThis study reported for the first time the mechanistic and biological consequences of PINCH expression in CNS neurons in diseases with a chronic neuroinflammation component. Our findings point to the maintenance of PINCH at normal physiological levels as a potential new therapeutic target for neurodegenerative diseases with impaired metabolisms.

Anti-inflammatory effects of gallic acid in human gestational tissues in vitro.

Spontaneous preterm birth is the leading cause of neonatal mortality and morbidity globally. Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions and the rupture of the foetal membranes. Anti-inflammatory agents may be a novel therapeutic approach to prevent inflammation-induced myometrial contractions and premature rupture of foetal membranes. The polyphenol gallic acid has been previously shown to exert potent anti-inflammatory effects. Thus, this study aimed to determine the effect of gallic acid on proinflammatory and pro-labour mediators in cytokine-stimulated gestational tissues in vitro. In primary human cells isolated from myometrium and foetal membranes (decidua, and amnion mesenchymal and epithelial cells), gallic acid treatment suppressed inflammation-induced expression of proinflammatory cytokines and chemokines and extracellular matrix-degrading and matrix-remodelling enzymes. Gallic acid also significantly inhibited inflammation-induced myometrial activation as evidenced by decreased expression of contraction-associated proteins, the uterotonic PGF2α and collagen cell contractility. Using a global proteomic approach, gallic acid may differentially regulate proteins associated with collagen synthesis, cell contractility and protein synthesis in primary myometrial and decidual cells. In summary, gallic acid inhibited inflammation-induced mediators involved in active labour in primary cells isolated from myometrium and foetal membranes. These in vitro studies suggest that the polyphenol gallic acid may be able to suppress the production of proinflammatory and pro-labour mediators involved in myometrial contractions and rupture of foetal membranes. Future preclinical studies may elucidate the efficacy of gallic acid in preventing inflammation-driven preterm birth.

The short-chain fatty acids butyrate and propionate protect against inflammation-induced activation of mediators involved in active labor: implications for preterm birth.

Spontaneous preterm birth is a global health issue affecting up to 20% of pregnancies and leaves a legacy of neurodevelopmental complications. Inflammation has been implicated in a significant proportion of preterm births, where pro-inflammatory insults trigger production of additional pro-inflammatory and pro-labor mediators. Thus, novel therapeutics that can target inflammation may be a novel avenue for preventing preterm birth and improving adverse fetal outcomes. Short-chain fatty acids (SCFAs), such as butyrate and propionate, are dietary metabolites produced by bacterial fermentation of fiber in the gut. SCFAs are known to possess anti-inflammatory properties and have been found to function through G-coupled-receptors and histone deacetylases. Therefore, this study aimed to investigate the effect of SCFAs on pro-inflammatory and pro-labor mediators in an in vitro model of preterm birth. Primary human cells isolated from myometrium and fetal membranes (decidua, amnion mesenchymal and amnion epithelial cells) were stimulated with the pro-inflammatory cytokines tumor necrosis factor alpha (TNF) or interleukin 1B (IL1B). The SCFAs butyrate and propionate suppressed inflammation-induced expression of pro-inflammatory cytokines and chemokines, adhesion molecules, the uterotonic prostaglandin PGF2alpha and enzymes involved in remodeling of myometrium and degradation of the fetal membranes. Notably, propionate and butyrate also suppressed inflammation-induced prostaglandin signaling and myometrial cell contraction. These effects appear to be mediated through suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) activation. These results suggest that the SCFAs may be able to prevent myometrial contractions and rupture of membranes. Further in vivo studies are warranted to identify the efficacy of SCFAs as a novel anti-inflammatory therapeutic to prevent inflammation-induced spontaneous preterm birth.

Taurocholic acid lowers the inflammatory response of gingival fibroblasts, epithelial cells, and macrophages.

Taurocholic acid (TCA), a conjugation of cholic acid with taurine, is one of the main bile acids that is elevated in liver disease. Considering the epidemiologic linkage of periodontal disease to liver disease, the question arises about the possible effect of elevated TCA levels on periodontal cells. To answer this question, gingival fibroblasts and human oral squamous cell carcinoma cell line (HSC-2) were pretreated with interleukine1β (IL1β) and tumor necrosis factorα (TNFα) in the presence and absence of TCA. Also, mouse macrophages (RAW 264.7) were incubated with sterile-filtered human saliva with and without TCA. Inflammatory cytokines were measured by real time polynucleotide chain reaction (RT-PCR) and an immunoassay. The nuclear translocation of the p65 subunit was visualized by immunostaining. In pretreated gingival fibroblasts and HSC-2 cells, TCA considerably reduced the expression of IL1β, IL6, and IL8. In support of these observations, TCA lowered the saliva-induced expression of IL1α, IL1β and IL6 in RAW 264.7 cells. An immunoassay confirmed the capacity of TCA to diminish inflammation-induced expression of IL6 in gingival fibroblasts, HSC-2 and RAW 264.7 cells. Consistently, TCA blocked the nuclear translocation of p65 in fibroblasts. These findings suggest that TCA has anti-inflammatory activity in gingival fibroblasts, human oral squamous cell carcinoma cells and macrophages in vitro.

Abstract A42: Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4

Abstract Pancreatic ductal adenomcarcinoma (PDAC) is one of the most lethal forms of cancer with a 5-year survival rate of 8%. To determine the composition of different cell types in PDAC, we performed single-cell RNA-sequencing (scRNA-seq) analysis of two resected patient tumors. Nine distinct cell types were identified: (1) ductal carcinoma cells expressing mutant KRAS, (2) normal pancreatic islet cells, (3) B-lymphocytes, (4) macrophages, (5) mast cells, (6) nerve cells, (7-8) two types of cancer-associated fibroblasts (CAFs), and (9) a previously unidentified epithelial type with distinctly high expression of OLFM4 and CRISP3. Histologic analysis of human PDAC and chronic pancreatitis (CP) tissue sections confirmed the presence of this distinct cell type and furthermore demonstrated that it corresponded to inflamed/injured ducts and early pancreatic intraepithelial neoplasms (PanINs) but that actual PDACs did not express OLFM4. CRISP3 was not always expressed in this new cell type, but the expression of OLFM4 was always detected in these histologic structures. This distinct pancreatic epithelial cell type was also observed in mouse models for chronic pancreatitis and in mutant-KRAS induced PanINs. We hypothesize that inflammation-induced OLFM4 expression helps drive PanIN formation, and that its expression could be exploited for early PDAC detection. We are currently performing functional studies using organoids to address the role that OLFM4 might play in generation of this distinct cell type. Citation Format: Manisha Rao, Alice Nemajerova, Jinyu Li, Mei Gao, Ki Oh, Richard Moffit, Joseph Kim, George Georgakis, Aaron Sasson, Agnieszka Bialkowkska, Scott Powers. Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A42.

Targeting bromodomain-containing proteins to prevent spontaneous preterm birth.

Preterm birth is a global healthcare challenge. Spontaneous preterm birth (sPTB) is commonly caused by inflammation, yet there are currently no effective therapies available. The Bromodomain and Extra-Terminal motif (BET) proteins, Bromodomain-containing protein (Brd) 2 (Brd2), Brd3 and Brd4 regulate inflammation in non-gestational tissues. The roles of Brd2-4 in human pregnancy are unknown. Using human and mouse models, the present study has identified the Brd proteins part of the process by which inflammation induces parturition. Using human clinical samples, we demonstrate that labor and infection increase the expression of Brds in the uterus and fetal membranes. In primary human myometrial, amnion and decidual cells, we found that global Brd protein inhibition, as well as selective inhibition of Brds, suppressed inflammation-induced expression of mediators involved in myometrial contractions and rupture of fetal membranes. Importantly, studies in the mouse model demonstrate that the pan-Brd inhibitor JQ1 reduced intrauterine inflammation induced by bacterial endotoxin LPS as well as decreasing the effectiveness of LPS to induce parturition. These results implicate BET proteins as novel therapeutic targets for reducing inflammation associated with spontaneous preterm labor.

Investigation of the preventive effect of calcium on inflammation-mediated choroidal neovascularization

AimsAge-related macular degeneration (AMD) is a leading cause of irreversible blindness in elderly people. The pathogenesis of neovascular AMD is known but is closely related to inflammation and choroidal neovascularization (CNV). The aim of this study was to investigate the anti-inflammatory and anti-angiogenic effects of calcium on neovascular AMD. Main methodsHuman retinal pigment epithelial cells (ARPE-19) were used to identify protein markers of inflammation induced by differentiated macrophages. Choroidal neovascularization (CNV) mouse model was established by rupturing the Bruch's membrane using laser photocoagulation in C57BL/6 mice. Mice were divided into the following groups: untreated control and calcium supplemented. The expression levels of toll-like receptor isotype (TLR) 4, nuclear factor kappa B (NF-κB), hypoxia-inducible factor-1α (Hif-1α), and vascular endothelial growth factor (VEGF) were investigated to check whether calcium supplementation results in suppression of inflammation and has an anti-angiogenic effect. CNV was evaluated by immunofluorescence staining on choroidal flat mounts. Key findingThe inflammation-induced expression of TLR4, NF-κB, and Hif-1α was decreased in ARPE-19 cells after calcium supplementation. Inhibition of the transcriptional activation of ARPE-19 cells by Hif-1α suppression resulted in decreased VEGF expression. In the laser-induced CNV mouse model, calcium supplementation inhibited inflammatory mediators and neovascularization in the retinal tissue. SignificanceSupplementation with calcium seems to constrain inveterate symptoms of neovascular AMD by inhibiting inflammation and angiogenesis in the laser-induced CNV mouse model.

Inflammation-Induced Expression of α7 nAChR and Phosphorylated GSK-3β in Periodontal Ligament Stem Cells Contributes to the Synergistic Effect of Nicotine and Inflammation in Periodontitis

Backgroud: Periodontitis is the leading cause of adult tooth loss, and those who smoke are at an increased risk of developing periodontitis. α7 nicotinic acetylcholine receptor (α7 nAChR) is proposed to mediate the potential synergistic effect of nicotine and inflammation in smoking-related periodontitis. However, this has not been experimentally demonstrated. Methods: We isolated and cultured human periodontal ligament stem cells (PDLSCs) from healthy and inflamed tissues. PDLSCs were treated with either inflammatory factors and/or nicotine. We measured expression of genes that are associated with osteogenic differentiation and osteoclast formation using RT-qPCR and western blot analyses. Besides, Immunohistochemical staining, micro-CT analysis, shRNA lentivirus infection, and tartaric acid phosphatase staining were used to measure α7 nAChR expression and function. Findings: Inflammation up-regulated α7 nAChR expression in both periodontal ligament tissues and PDLSCs. The up-regulated α7 nAChR contributed to the synergistic effect of nicotine and inflammation, leading to a decreased capability of osteogenic differentiation and increased capability of osteoclast formation-induction of PDLSCs. Moreover, the inflammation-induced up-regulation of α7 nAChR expression was partially dependent on the level of phosphorylated GSK-3β. Interpretation: This study provides experimental evidence for the pathological development of smoking-related periodontitis, and sheds new light on developing inflammation and α7 nAChR targeted therapeutics to treat and prevent the disease. Funding Statement: This study was supported by the National Natural Science Foundation of China (Grant No. 81670988, 81800924, 81601620 and 81800979) and the in-hospital scientific research program (Grant No. 2018LQ04) of Northwest Women’s and Children’s Hospital, Xi’an, Shaanxi Province, China. Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Ethical approval was obtained from the ethics committee of the School of Stomatology, Air Force Military Medical University (Committee’s reference number: IRB-REV-2016093). All participants and guardians of the patients were informed on the objectives of this study. Written consents were obtained from all participants prior to conducting the study.

Abstract 1290: Development of anti inflammatory agents targeting TonEBP for treatment of chronic inflammatory diseases

Abstract Tonicity-responsive enhancer binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), belongs to the Rel family of transcriptional factors which include NF-κB and NFAT. TonEBP is a key regulator in chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, diabetic nephropathy and hepatocellular carcinoma. TonEBP is a rate-limiting component of “pro-inflammatory enhanceosome” in which TonEBP provides a physical tether between activated transcription factors NF-κB and AP-1 to p300 and RNA polymerase II. Assembled in response to inflammatory signals, the pro-inflammatory enhanceosome drives the transcription of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. We discovered a class of compounds (CXs) targeting TonEBP. CXs blocked the assembly of the pro-inflammatory enhancesome and potently suppressed the inflammation-induced expression of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. Orally administered CXs exhibited remarkable therapeutic activities in mouse models of inflammatory arthritis. Using an alkynyl derivative of a CX and click reaction, we found that CX was covalently attached to TonEBP by alkylation but not to other components of the enhanceosome. Cells harboring a site-directed mutant TonEBP molecule whose amino acid residues for the alkylation were removed exhibited a markedly reduced sensitivity to CXs. These data demonstrate that CXs are a new class of anti-inflammatory agents with a novel mode of action. Citation Format: Byeongjin YE, Hyug Moo Kwon, Soo Youn Choi, Jun Ho Lee, Hyun Je Kang, Cheol-Min Park. Development of anti inflammatory agents targeting TonEBP for treatment of chronic inflammatory diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1290.

The commensal bacterium Bacteroides fragilis down-regulates ferroportin expression and alters iron homeostasis in macrophages.

The intestinal microbiota has several effects on host physiology. Previous work from our laboratory demonstrated that the microbiota influences systemic iron homeostasis in mouse colitis models by altering inflammation-induced expression of the iron-regulating hormone hepcidin. In the present study, we examined the impact of the gut commensal bacterium Bacteroides fragilis on the expression of the iron exporter ferroportin, the target of hepcidin action, in macrophages, the cell type that plays a pivotal role in iron recycling. Mouse bone marrow-derived macrophages were exposed to B. fragilis and were analyzed by quantitative real-time polymerase chain reaction and Western blotting. We found that B. fragilis down-regulated ferroportin transcription independently of bacterial viability. Medium conditioned by the bacteria also reduced ferroportin expression, indicating the involvement of soluble factors, possibly Toll-like receptor ligands. Consistent with this idea, several of these ligands were able to down-regulate ferroportin. The B. fragilis-induced decrease in ferroportin was functionally important since it produced a significant increase in intracellular iron concentrations that prevented the effects of the iron chelator deferoxamine on Salmonella-induced IL-6 and IL-1β production. Our results thus reveal that B. fragilis can influence macrophage iron handling and inflammatory responses by modulating ferroportin expression.

Association of serum deiodinase type 2 level with chronic obstructive pulmonary disease in the Polish population.

Deiodinase type 2 (DIO2) is a selenoenzyme involved in the synthesis of thyroid hormones. Chemerin is a newly investigated adipokine known also as novel chemokine. Both molecules have been recently expected and found to play an important role in inflammation and immunity. DIO2, for example, is upregulated during acute and chronic inflammation. In addition, inflammation-induced expression of DIO2 in macrophages has been confirmed, while chemerin modulates the activation and chemotaxis of immune cells. It is widely known that chronic obstructive pulmonary disease (COPD) - the most common lung disease in the world - is accompanied by an inflammatory process and immune activation. There are no studies demonstrating an association between DIO2, chemerin and COPD. The aim of this study was to estimate DIO2 and chemerin concentration in serum collected from patients suffering from COPD and to compare it with healthy subjects, as well as to correlate with basic and clinical characteristics. The study group included 50 patients with COPD and 30 healthy subjects. DIO2 and chemerin serum levels as well as c-reactive protein levels were determined in all the subjects using commercial enzyme-linked immunosorbent assay kits. The association between serum DIO2 and chemerin with sociodemographic and clinical variables was assessed. DIO2 serum levels were significantly higher in the patients with COPD as compared to the control group (50.3±23.2 U/L vs. 13.3±13.1; p<0.00001). No differences were observed in serum chemerin levels between the patients and controls (107.559±86.695.6 vs. 100.701±53.805; p=0.54). Furthermore, there was no association between DIO2 and chemerin levels and other variables, and no correlation between both molecules. This study demonstrated that DIO2 levels were higher in the patients with COPD than in the control subjects. The examined molecules should be further investigated if they are intended to be considered markers of processes involved in COPD mechanisms.

GIT2 deficiency attenuates inflammation-induced expression of pro-labor mediators in human amnion and myometrial cells†.

Untimely activation of the inflammatory response by sterile or infective insults in uterine tissues can result in preterm birth. Pro-inflammatory cytokines and pathogenic activation of toll-like receptors (TLRs) initiate a biochemical cascade of events leading to myometrial activation and contractility, cervical dilatation, and rupture of the chorioamniotic membranes. GIT2 is a signaling protein known to play a role in innate and adaptive immunity; however, its role in the inflammatory pathways of human labor is not known. In this article, we report that GIT2 expression is lower in human myometrium and fetal membranes with term labor, and in preterm amnion with histological chorioamnionitis. GIT2 knockdown by siRNA in primary myometrial and amnion cells exhibited reduced expression of pro-inflammatory cytokines and chemokines in response to inflammatory challenge by cytokines or TLR ligands. In addition, the pro-inflammatory cytokines IL1B and TNF could not induce the expression of extracellular matrix degrading enzymes in GIT2-deficient amnion cells. Myometrial activation in response to pro-inflammatory cytokines was also significantly suppressed in GIT2-deficient cells as evidenced by decreased prostaglandin release and expression of contraction-associated proteins. Further to this, collagen gel assays demonstrated that TNF had a reduced ability to induce myometrial contractility in situ in GIT2-deficient myometrial cells compared to control-transfected cells. In summary, the loss of GIT2 diminishes the effects inflammatory mediators have in promoting myometrial contraction and fetal membrane rupture in vitro, suggesting that GIT2 could be a possible target for preterm birth therapies.

NOD-like receptor pyrin domain-containing-3 (NLRP3) regulates inflammation-induced pro-labor mediators in human myometrial cells.

Inflammation plays a major role in preterm birth. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) plays a role in inflammatory diseases. The aims of this study were to determine the effect of term labor on the expression of NLRP3 in human myometrium and the effect of NLRP3 silencing on pro-labor mediators in myometrial cells. NLRP3 expression was assessed in myometrium from non-laboring and laboring women by qRT-PCR and Western blotting. Human primary myometrial cells were transfected with NLRP3 siRNA (siNLRP3), treated with pro-inflammatory cytokines and toll-like receptor (TLR) ligands, and assayed for pro-inflammatory mediators' expression. NLRP3 expression was higher in myometrium after term spontaneous labor and by TNF, IL1B, fsl-1, and flagellin. In siNLRP3-transfected cells, there was a significant decrease in the expression of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), and adhesion molecules (ICAM1 and VCAM1) stimulated with IL1B, TNF, or TLR ligands; decrease in IL1B-stimulated PTGS2 and PTGFR mRNA expression and PGF2α release; and increase in TNF-stimulated myometrial gel shrinkage as assessed by an in vitro cell contraction assay. NLRP3 is increased with labor in myometrial, and knockdown of NLRP3 is associated with an attenuation of inflammation-induced expression of pro-inflammatory and pro-labor mediators in human myometrium.

Interferonregulatoryfactor-8(IRF-8) regulates the expression of matrix metalloproteinase-13 (MMP-13) in chondrocytes.

Low levels of inflammation-induced expression of matrix metalloproteinase (MMP) play a crucial role in articular cartilage matrix destruction in osteoarthritis (OA) patients. Interferon regulatory factor-8 (IRF-8), an important member in the IRF family, plays a key role in regulating the inflammation-related signaling pathway. The aim of this study is to investigate the physiological roles of IRF-8 in the pathological progression of OA. We found that IRF-8 was expressed in human primary chondrocytes. Interestingly, the expression of IRF-8 was upregulated in OA chondrocytes. In addition, IRF-8 was increased in response to interleukin-1β (IL-1β) treatment, mediated by the Janus kinase 2 (JAK2) pathway. Overexpression of IRF-8 in human chondrocytes by transduction with lentiviral-IRF-8 exacerbated IL-1β-induced expression of matrix metalloproteinase-13 (MMP-13) in human chondrocytes. In contrast, knockdown of IRF-8 inhibited IL-1β-induced expression of MMP-13. Importantly, IRF-8 could bind to the promoter of MMP-13 and stimulate its activity. Additionally, overexpression of IRF-8 exacerbated IL-1β-induced degradation of type II collagen. However, silencing IRF-8 abrogated the degradation of type II collagen. Taken together, our findings identified a novel function of IRF-8 in regulating articular cartilage matrix destruction by promoting the expression of MMP-13.