Elevated Expression Of Inflammatory Cytokines Research Articles

CTNNAL1 enhances glucocorticoid sensitivity in HDM-induced asthma mouse model through deactivating hsp90 signaling pathway

AimsAdhesion molecules play vital roles in the induction of airway hyperresponsiveness (AHR) or airway inflammation. The down-regulation of catenin alpha-like 1 (CTNNAL1) in the bronchial epithelial cells of asthma patients and mice models has been noted in our previous study. In this work, we further explore the underlying mechanism of CTNNAL1 in asthma. Main methodsWe constructed a house dust mite (HDM)-induced asthma animal model on control mice and applied CTNNAL1-siRNA transfection to create CTNNAL1-deficient mice. Key findingsWe documented much more severe airway inflammation and increased leukocyte infiltration in the lungs of the CTNNAL1-deficient mice comparing to control mice, along with elevated expression of inflammatory cytokines. Dexamethasone (DEX) treatment led to less reduced inflammation in CTNNAL1-deficient mice compared with control mice. Immunoprecipitation confirmed the interaction between heat shock protein90 (hsp90) and CTNNAL1. The expression of hsp90 was upregulated after CTNNAL1 silencing. Meanwhile, the use of hsp90 inhibitor geldanamycin significantly decreased the expression of NR3C1, ICAM-1 and the ratio of p-p65/p65 in CTNNAL1-silenced 16HBE14o- cells. Both geldanamycin and DEX could function to suppress the expression of ICAM-1 and the phosphorylation level of p65. Nevertheless, the anti-inflammatory effect of DEX proved less potent than geldanamycin in the CTNNAL1-silenced group. The combined therapy of geldanamycin and DEX significantly decreased the inflammatory responses in CTNNAL1-deficient HBE cells than DEX monotherapy. SignificanceOur study corroborates that CTNNAL1 deficiency induced aggravated airway inflammation and rendered insensitivity to glucocorticoids via triggering hsp90 signaling pathway.

Pyruvate: Ferredoxin oxidoreductase is involved in IgA-related microbiota dysbiosis and intestinal inflammation.

Inflammatory bowel diseases (IBDs) are associated with both immune abnormalities and dysbiosis, characterized by a loss of Faecalibacterium prausnitzii (F. prausnitzii). However, the reason for F. prausnitzii deficiency remains unclear. 16S rDNA seque-ncing and IgA enzyme-linked immunosorbent assay (ELISA) were applied to identify bacterial community and IgA changes in ulcerative colitis (UC) patients. Forced immunization with F. prausnitzii in rabbits was conducted. To screen for potential IgA-reactive proteins in F. prausnitzii lysates, we performed western blotting and mass spectrometry analyses. Pyruvate: ferredoxin oxidoreductase (PFOR) was cloned and purified, then the immunoreactivity of PFOR was verified in peripheral blood mononuclear cells (PBMCs) through PCR, ELISpot assay and single-cell sequencing (scRNA-seq). Finally, the UC fecal dysbiosis was re-analyzed in the context of the phylogenetic tree of PFOR. F. prausnitzii was underrepresented in UC patients with elevated F. prausnitzii-reactive IgA in the fecal supernatant. Forced immunization with F. prausnitzii in rabbits led to high interferon-γ (IFN-γ) transcription in the colon, along with beta diversity disturbance and intestinal inflammation. PFOR was identified as an IgA-binding antigen of F. prausnitzii and the immunoreactivity was validated in PBMCs, which showed elevated expression of inflammatory cytokines. The scRNA-seq revealed enhanced signals in both T regulatory cells (Tregs) and monocytes after PFOR incubation. Furthermore, phylogenetic analysis revealed that PFOR was a common but conserved protein among the gut bacteria. Our results collectively suggest that PFOR is a bioactive protein in the immune system and may contribute to host-microbial crosstalk. Conserved but bioactive microbial proteins, such as PFOR, warrant more attention in future host-microbial interaction studies.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

Inhibition of Gas6 promotes crystalline silica-induced inflammatory response of macrophages via blocking autophagy flux.

Inhalation of crystalline silica (CS) can cause silicosis, which is one of the most serious interstitial lung diseases worldwide. Autophagy dysfunction is an essential step in silicosis progression. In this study, we aim to identify the effect of growth arrest-specific protein 6 (Gas6) during autophagy induction and macrophage inflammatory response caused by CS. After RAW 264.7 macrophages exposed to CS, the levels of Gas6 and autophagy markers (p62, Beclin1, and LC3-II/LC3-I) were increased, accompanied with enhanced inflammatory cytokines secretion. Using autophagy activator (rapamycin) repressed, whereas autophagy inhibitor (3-methyladenine) promoted inflammatory cytokines release. Besides, inhibition of Gas6 aggravated CS-induced inflammatory response, and autophagy inhibition facilitated the promoted effect of Gas6 silencing, resulting in elevated expression of inflammatory cytokines. These findings reveal the protective effects of Gas6 and autophagy in macrophages in response to CS exposure, and highlight the autophagy regulated by Gas6 may be a potential prevention target for CS-induced lung inflammatory response.

Inflammation as a Driver of Prostate Cancer Metastasis and Therapeutic Resistance.

Simple SummaryProstate cancer is the most common malignancy in men, with a high mortality rate when disease progresses to metastasis and therapeutic resistance. Evidence implicates inflammation as a driver of prostate cancer risk and has a significant impact on processes in the tumor microenvironment that facilitate progression to advanced therapeutically resistant disease. In this review, we discuss the sources of inflammation in the prostate, the functional contribution of the critical inflammatory effectors to prostate cancer initiation and metastatic progression, and the therapeutic challenges that they impose on treatment of advanced disease and overcoming therapeutic resistance. Full understanding of the role of inflammation in prostate cancer progression to advanced metastatic disease and tumor relapse will aid in the development of personalized predictive biomarkers and therapy to reduce the burden and mortality in prostate cancer patients.Prostate cancer is the most common malignancy among men, and progression to metastasis and the emergence of therapeutically resistant disease confers a high mortality rate. Growing evidence implicates inflammation as a driver of prostate cancer development and progression, resulting in increased cancer risk for prostate cancer. Population-based studies revealed that the use of antinflammatory drugs led to a 23% risk reduction prostate cancer occurrence, a negative association that was stronger in men who specifically used COX-2 inhibitors. Furthermore, patients that were taking aspirin had a 21% reduction in prostate cancer risk, and further, long-term users of daily low dose aspirin had a 29% prostate cancer risk reduction as compared to the controls. Environmental exposure to bacterial and viral infections, exposure to mutagenic agents, and genetic variations predispose the prostate gland to inflammation, with a coordinated elevated expression of inflammatory cytokines (IL-6, TGF-β). It is the dynamics within the tumor microenvironment that empower these cytokines to promote survival and growth of the primary tumor and facilitate disease progression by navigating the immunoregulatory network, phenotypic epithelial-mesenchymal transition (EMT), angiogenesis, anoikis resistance, and metastasis. In this review, we discuss the sources of inflammation in the prostate, the functional contribution of the critical inflammatory effectors to prostate cancer initiation and metastatic progression, and the therapeutic challenges that they impose on treatment of advanced disease and overcoming therapeutic resistance. Growing mechanistic evidence supports the significance of inflammation in localized prostate cancer, and the systemic impact of the process within the tumor microenvironment on disease progression to advanced therapeutically-resistant prostate cancer. Rigorous exploitation of the role of inflammation in prostate cancer progression to metastasis and therapeutic resistance will empower the development of precise biomarker signatures and effective targeted therapeutics to reduce the clinical burden and lethal disease in the future.

Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

Immune responses in Mayaro infected mice and its relation with viral replication and muscle damage

Abstract Mayaro virus (MAYV) is a tropical arbovirus first described in forest regions of the South America, and recently associated with urban circulation. Mayaro infections leads to a disease with high rate of persistent arthralgia and myalgia in humans. Despite this, pathogenesis and host immune response of MAYV infections are still limited. The aim of this work was to evaluate the immune response and cell profile during MAYV infections. We developed a disease model infecting SV129 wild-type (WT), SVA129 Type I Interferon receptor deficient (IFNAR−/−), C57BL/6 WT and C57BL/6 recombination activation gene-1 deficient (RAG−/−) mice, and accessing replication, tissue damage, and inflammation through time. While adult WT mice are resistant to infection, IFNAR−/− mice exhibited increasing viral load up to 6 days after infection, footpad swelling, lethargy and weight loss. Histological analysis showed extensive necrosis sites in infected IFNAR−/− mice and also presence of mononucleated cell infiltrate. We detected elevated expression of inflammatory cytokines like TNF, IL-6, KC, IL-1, MCP-1 and RANTES, and decreased TGF-beta in infected muscle. In RAG−/− mice, MAYV infection lead to a persistent replication being detected at blood and tissues up 40 days post infection. However, infected RAG−/− mice did not develop any signs of muscle damage or infiltrate during early and late infection and neither the expression inflammatory cytokines. So far, our data demonstrates that innate and adaptive immunity are critical to restrict MAYV replication. Adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute in underlying MAYV pathogenesis and further experiments must to be made to elucidate the mechanisms involved.

Dietary Menthol Attenuates Inflammation and Cardiac Remodeling After Myocardial Infarction via the Transient Receptor Potential Melastatin 8.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cold-sensing cation channel, mainly localized in the sensory neurons, which can be activated by menthol, a compound with a naturally cold sensation in mint. However, the effect of TRPM8 activation in inflammation and cardiac remodeling after myocardial infarction (MI) is not well defined. TRPM8 knockout (KO) mice (TRPM8-/-) and their wild-type littermates, aged 8 weeks, were randomly divided into sham and MI groups and were fed with chow or chow plus menthol. Dietary menthol significantly attenuated MI injury, evidenced by decreased survival rates and plasma cardiac troponion I levels, reduced infarct size and cardiomyocytes, declined collagen deposition, and rescued cardiac function and hemodynamics. However, these effects of menthol disappeared when mice were lacking TRPM8. Furthermore, feeding of menthol ameliorated elevated expression of inflammatory cytokines and chemokines, and aggravated inflammation infiltration in the MI mice but not in TRPM8-/- mice. In addition, menthol treatment increased the release of calcitonin gene-related peptide (CGRP), which were absent in TRPM8-/- mice. In conclusion, our results suggest that dietary menthol can protect against inflammation and cardiac remodeling after MI through activation of TRPM8.

Inflammation in PTSD is a consequence of dysregulated WNT signaling orchestrated by long non-coding RNA LINC00926 and lysine methyltransferase MLL1

Abstract Posttraumatic Stress Disorder patients experience chronic inflammation. It is not known whether immune dysregulation causes PTSD or the psychiatric imbalance occurs first. Moreover, little is known about the molecular mechanisms involved in the immune dysregulation. Our past work indicates the involvement of epigenetic mechanisms such as histone methylation in the regulation of inflammation in PTSD. By employing RNA-Seq, we identified upregulated expression of WNT10B and LINC00926 in the PBMCs of PTSD. Thus, we hypothesized that LINC00926 overexpression in PTSD results in increased H3K4me3 (gene activating histone methylation) around the promoter of WNT10B leading to its upregulation which in turn, regulated the elevated expression of inflammatory cytokines like IFNg and IL17. Thus, we first confirmed the interaction between LINC00926 and the histone H3K4 methyltransferase, MLL1. Then, by in vitro knockdown of LINC00926 we confirmed lowered expression of WNT10B implying that the interaction between LINC00926 and MLL1 possibly leads to increased H3K4me3 on WNT10B promoter and thereby its upregulation. Following this, we confirmed increased H3K4me3 around WNT10B promotor of PTSD patients by ChIP-Seq. Finally, we confirmed that upregulated WNT10B indeed contributed to the elevated expression of IFNg and IL17. In conclusion, our data implies that upregulation of LINC00926 in PTSD leads to inflammatory state by a mechanism that involves interaction with MLL1 resulting in increased H3K4me3 around WNT10B promotor and, eventually leads to elevated expression of proinflammatory cytokines. Our report is the first to show that inflammation in PTSD is a result of altered epigenetic mechanism involving LINC00926 and WNT10B.

RETRACTED: Angelica polysaccharide protects PC-12 cells from lipopolysaccharide-induced injury via down-regulating microRNA-223.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concerns were initially raised regarding the Western Blots from Figures 1C, 1G, 2B, 2D, 4D, 4F, 5 (left panel), 6C and 6E. Given also the comments of Dr Elisabeth Bik regarding this article “… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated”, the journal requested the authors to provide the raw data. The authors have not provided raw data of sufficient quality and detail to support the authors’™ claim and therefore the Editor-in-Chief decided to retract the article.

Elevated inflammatory cytokine expression in CSF from patients with symptomatic thoracic disc herniation correlates with increased pain scores

BACKGROUNDThe pathophysiology of pain in patients with symptomatic thoracic disc herniation (TDH) remains poorly understood. Mere mechanical compression of the spinal cord and/or the exiting nerve root by a prolapsed disc cannot explain the pathogenesis of pain in all cases. Previous studies report a direct correlation between the levels of proinflammatory cytokines in disc biopsies and the severity of leg pain in patients with lumbar disc herniation. A similar correlation in patients with TDH has not been investigated. PURPOSETo correlate the cerebrospinal fluid (CSF) expression of cytokines and pain-related amino acids with preoperative pain scores in patients with symptomatic TDH. STUDY DESIGNA prospective human study of CSF samples and clinical outcome scores. METHODSUsing enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), we determined inflammatory cytokine levels (TNF-α, IL-1β, and IL-10) and amino acid levels (glutamate, aspartate, gamma-aminobutyric acid, glycine, and arginine) in CSF samples from 10 patients with TDH and 10 control subjects who did not suffer an inflammatory disease nor pain related to spinal cord compression and subsequently correlated these levels with preoperative pain scores. Differences between both groups were evaluated by a Mann-Whitney U test. In order to estimate the correlation between cytokine or amino acid expression and pain scores, data were analyzed using a linear regression analysis. RESULTSNo inflammatory cytokines were found in CSF samples from control subjects, whereas TNF-α, IL-1β, and IL-10 were detectable by ELISA in all CSF samples from patients with TDH. TNF-α and IL-10 but not IL-1β levels moderately correlated with preoperative pain scores. Elevated TNF-αlevels positively correlated with high pain scores; elevated IL-10 levels negatively correlated with high pain scores. Amino acids were detectable in all samples from both groups. There were no significant differences between the groups in any of the amino acids measured with HPLC. CONCLUSIONIncreased proinflammatory cytokine expression is associated with elevated pain scores in patients with symptomatic TDH. On the other hand, there is no conclusive correlation between the intensity of pain and the local or systemic presence of amino acids associated with pain transmission.

Luteolin alleviates LPS-induced bronchopneumonia injury in vitro and in vivo by down-regulating microRNA-132 expression

Bronchopneumonia is a common multiple infection disease under 2 years old. Luteolin is a natural flavonoid widely distributed in plants with anti-inflammatory effect. This study aimed to explore the effects of luteolin on lipopolysaccharide (LPS)-induced bronchopneumonia injury in vitro and in vivo. Firstly, the viability and apoptosis of human bronchial epithelial BEAS-2B cells after luteolin treatment were assessed. Then, cells were treated with 10 μM LPS to simulate inflammatory injury. The potential protective effects of luteolin on LPS-induced BEAS-2B cell inflammatory injury were detected. Moreover, after LPS and/or luteolin treatment, the expression of microRNA-132 (miR-132) in BEAS-2B cells was measured. The roles of miR-132 in protective activity of luteolin were investigated. Finally, the LPS-induced bronchopneumonia murine model was established and the anti-inflammatory effects of luteolin in vivo were analyzed. The results showed that LPS decreased BEAS-2B cell viability, increased cell apoptosis and enhanced inflammatory cytokines expression. Luteolin alleviated the LPS-induced viability loss, apoptosis and elevated expression of inflammatory cytokines in a dose-dependent manner. Moreover, luteolin alleviated the LPS-induced miR-132 expression increase in BEAS-2B cells. Overexpression of miR-132 reversed the protective effects of luteolin on LPS-induced inflammatory injury. Mechanistically, luteolin mitigated LPS-induced activation of NF-κB signaling pathway by down-regulation of miR-132. Furthermore, we also found that luteolin alleviated LPS-induced bronchopneumonia model in vivo. In conclusion, this study revealed that luteolin alleviated LPS-induced bronchopneumonia injury in vitro and in vivo through down-regulating miR-132. These findings provide theoretical basis for deeply exploring the treatment of bronchopneumonia in children by using luteolin.

A Novel Catecholopyrimidine Based Small Molecule PDE4B Inhibitor Suppresses Inflammatory Cytokines in Atopic Mice.

Degradation of cyclic adenosine mono phosphate (cAMP) by phosphodiesterase-4B (PDE-4B) in the inflammatory cells leads to elevated expression of inflammatory cytokines in inflammatory cells. Suppression of cytokines has proved to be beneficial in the treatment of atopic dermatitis (AD). Henceforth, application of PDE4B specific inhibitor to minimize the degradation of cAMP can yield better results in the treatment of AD. PDE4B specific inhibitor with a limited side effect is highly warranted. Herein, we synthesized a novel PDE4 inhibitor, compound 2 comprising catecholopyrimidine core functionalized with trifluoromethyl (-CF3) group. PDE4B inhibitory potential and specificity of novel compounds were evaluated by PDE inhibitor assay. In vivo efficacy of the compounds was analyzed using DNCB-induced NC/Nga mice. IgE, CD4+ T-helper cell infiltration, and cytokine profiles were analyzed by ELISA and immunohistochemistry techniques. Toluidine blue staining was performed for mast cell count. PDE4 inhibitor assay confirmed that compound 2 specifically inhibits PDE4B. In vivo analysis with DNCB-induced NC/Nga mice confirmed that compound 2 suppressed the levels of pro-inflammatory cytokines such as TNF-α, IL-4, IL-5, and IL-17. Furthermore, compound 2 significantly reduced the infiltrative CD4+ T-helper cells, mast cells and IgE levels in atopic tissue. The in vitro and in vivo data suggested that compound 2 specifically inhibit the PDE4B and the symptoms of the AD in atopic mice. Compound 2 might constitute a good candidate molecule for the treatment of AD.

Loss of IRF2BP2 in Microglia Increases Inflammation and Functional Deficits after Focal Ischemic Brain Injury.

Ischemic stroke causes neuronal cell death and triggers a cascade of inflammatory signals that contribute to secondary brain damage. Microglia, the brain-resident macrophages that remove dead neurons, play a critical role in the brain’s response to ischemic injury. Our previous studies showed that IRF2 binding protein 2 (IRF2BP2) regulates peripheral macrophage polarization, limits their inflammatory response and reduces susceptibility to atherosclerosis. Here, we show that loss of IRF2BP2 in microglia leads to increased inflammatory cytokine expression in response to lipopolysaccharide challenge and impaired activation of anti-inflammatory markers in response to interleukin-4 (IL4) stimulation. Focal ischemic brain injury of the sensorimotor cortex induced by photothrombosis caused more severe functional deficits in mice with IRF2BP2 ablated in macrophages/microglia, associated with elevated expression of inflammatory cytokines in the brain. These mutant mice had larger infarctions 4 days after stroke associated with fewer anti-inflammatory M2 microglia/macrophages recruited to the peri-infarct area, suggesting an impaired clearance of injured tissues. Since IRF2BP2 modulates interferon signaling, and interferon beta (IFNβ) has been reported to be anti-inflammatory and reduce ischemic brain injury, we asked whether loss of IRF2BP2 in macrophages/microglia would affect the response to IFNβ in our stroke model. IFNβ suppressed inflammatory cytokine production of macrophages and reduced infarct volumes at 4 days after photothrombosis in wild type mice. The anti-inflammatory effect of IFNβ was lost in IRF2BP2-deficient macrophages and IFNβ failed to protect mice lacking IRF2BP2 in macrophages/microglia from ischemic injury. In summary, IRF2BP2 expression in macrophages/microglia is important to limit inflammation and stroke injury, in part by mediating the beneficial effect of IFNβ.

Metformin activation of AMPK suppresses AGE-induced inflammatory response in hNSCs

A growing body of evidence suggests type 2 diabetes mellitus (T2DM) is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Although the precise mechanisms remain unclear, T2DM may exacerbate neurodegenerative processes. AMP-activated protein kinase (AMPK) signaling is an evolutionary preserved pathway that is important during homeostatic energy biogenesis responses at both the cellular and whole-body levels. Metformin, a ubiquitously prescribed anti-diabetic drug, exerts its effects by AMPK activation. However, while the roles of AMPK as a metabolic mediator are generally well understood, its performance in neuroprotection and neurodegeneration are not yet well defined. Given hyperglycemia is accompanied by an accelerated rate of advanced glycosylation end product (AGE) formation, which is associated with the pathogenesis of diabetic neuronal impairment and, inflammatory response, clarification of the role of AMPK signaling in these processes is needed. Therefore, we tested the hypothesis that metformin, an AMPK activator, protects against diabetic AGE induced neuronal impairment in human neural stem cells (hNSCs). In the present study, hNSCs exposed to AGE had significantly reduced cell viability, which correlated with elevated inflammatory cytokine expression, such as IL-1α, IL-1β, IL-2, IL-6, IL-12 and TNF-α. Co-treatment with metformin significantly abrogated the AGE-mediated effects in hNSCs. In addition, metformin rescued the transcript and protein expression levels of acetyl-CoA carboxylase (ACC) and inhibitory kappa B kinase (IKK) in AGE-treated hNSCs. NF-κB is a transcription factor with a key role in the expression of a variety of genes involved in inflammatory responses, and metformin did prevent the AGE-mediated increase in NF-κB mRNA and protein levels in the hNSCs exposed to AGE. Indeed, co-treatment with metformin significantly restored inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) levels in AGE-treated hNSCs. These findings extend our understanding of the central role of AMPK in AGE induced inflammatory responses, which increase the risk of neurodegeneration in diabetic patients.

Spleen Tyrosine Kinase (Syk) Mediates IL-1β Induction by Primary Human Monocytes during Antibody-enhanced Dengue Virus Infection

Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1β secretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti-DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue.

Norepinephrine preferentially modulates memory CD8 T cell function inducing inflammatory cytokine production and reducing proliferation in response to activation.

Norepinephrine (NE) is one of the primary catecholamines of the sympathetic nervous system released during a stress response and plays an important role in modulating immune function. NE binds to the adrenergic receptors on immune cells, including T cells, resulting in either suppressed or enhanced function depending on the type of cell, activation status of the cell, duration of NE exposure and concentration of NE. Here, we aim to analyze the effects of NE on the functionality of naïve (Tn), central memory (Tcm) and effector memory (Tem) CD8 T cells. We isolated CD8 T cell subsets from healthy human adults and treated cells in vitro with NE (1×10(-6)M) for 16h; we then stimulated NE treated and untreated CD8 T cell subsets with antibodies for CD3 and CD28 for 24 and 72h. We assessed the level of beta-2 adrenergic receptor (ADRB2) expression in these cells as well as global gene expression changes in NE treated Tcm cells by microarray analysis. Altered expressed genes after NE treatment were identified and further confirmed by RT-qPCR, and by ELISA for protein changes. We further determined whether the observed NE effects on memory CD8 T cells are mediated by ADRB2 using specific adrenergic receptor agonist and antagonists. Finally, we examined the levels of mRNA and protein of the NE-induced genes in healthy adults with high serum levels of NE (>150pg/mL) compared to low levels (<150pg/mL). We found that memory (Tcm and Tem) CD8 T cells expressed a significantly higher level of ADRB2 compared to naïve cells. Consequently, memory CD8 T cells were significantly more sensitive than naïve cells to NE induced changes in gene expressions in vitro. Global gene expression analysis revealed that NE induced an elevated expression of inflammatory cytokines and chemokines in resting and activated memory CD8 T cells in addition to a reduced expression of growth-related cytokines. The effects of NE on memory CD8 T cells were primarily mediated by ADRB2 as confirmed by the adrenergic receptor agonist and antagonist assays. Finally, individuals with high serum levels of NE had similar elevated gene expressions observed in vitro compared to the low NE group. Our results demonstrate that NE preferentially modulates the functions of memory CD8 T cells by inducing inflammatory cytokine production and reducing activation-induced memory CD8 T cell expansion.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Abstract 11985: C1q / tnf-Related Protein 9 Protects Against Acute Myocardial Injury

Background: Obesity-related disorders are associated with an increased risk for cardiovascular disease. C1q/TNF-related protein (CTRP) 9 is an anti-diabetic adipokine that is downregulated in obese mice. Recently, we have reported that CTRP9 prevents adverse vascular remodeling in response to injury. Here, we investigated the effect of CTRP9 on acute cardiac injury with loss-of-function genetic manipulations. Methods and Results: CTRP9-deficient (CTRP9-KO) mice at the age of 12 weeks were indistinguishable from Wild-type (WT) mice under basal conditions. CTRP9-KO mice showed increased myocardial infarct size and elevated expression of inflammatory cytokines including TNF-α and IL-6 in ischemic heart following ischemia-reperfusion compared with WT mice. CTRP9-KO mice also had exacerbated contractile left ventricle (LV) dysfunction and increased myocardial expression of inflammatory cytokines following intraperitoneal injection of lipopolysaccharide (LPS) compared with WT mice. Conversely, systemic delivery of an adenoviral vector expressing CTRP9 (Ad-CTRP9) attenuated inflammatory responses to myocardial ischemia-reperfusion or LPS administration in WT mice. In cultured cardiac myocytes, treatment with CTRP9 protein suppressed LPS-stimulated expression of TNF-α and IL-6 with an accompanying reduction of NFkB phosphorylation. Treatment of cardiac myocytes with CTRP9 enhanced AMPK phosphorylation, and transduction with dominant-negative mutant form of AMPK reversed the suppressive effect of CTRP9 on TNF-α and IL-6 expression. Furthermore, systemic administration of Ad-CTRP9 improved LPS-induced cardiac dysfunction in WT mice but not in muscle-specific transgenic mice expressing dominant-negative mutant form of AMPK. Conclusions: These findings suggest that CTRP9 protects against acute cardiac damage in response to pathological stimuli by suppressing inflammatory reactions through AMPK-dependent mechanisms.

The injury-induced myokine insulin-like 6 is protective in experimental autoimmune myositis.

BackgroundThe idiopathic inflammatory myopathies represent a group of autoimmune diseases that are characterized by lymphocyte infiltration of muscle and muscle weakness. Insulin-like 6 (Insl6) is a poorly characterized member of the insulin-like/relaxin family of secreted proteins, whose expression is upregulated upon acute muscle injury.MethodsIn this study, we employed Insl6 gain or loss of function mice to investigate the role of Insl6 in a T cell-mediated model of experimental autoimmune myositis (EAM). EAM models in rodents have involved immunization with human myosin-binding protein C with complete Freund’s adjuvant (CFA) emulsions and pertussis toxin.ResultsInsl6-deficiency in mice led to a worsened myositis phenotype including increased infiltration of CD4 and CD8 T cells and the elevated expression of inflammatory cytokines. Insl6-deficient mice show significant motor function impairment when tested with treadmill or Rotarod devices. Conversely, muscle-specific overexpression of Insl6 protected against the development of myositis as indicated by reduced lymphocyte infiltration in muscle, diminished inflammatory cytokine expression and improved motor function. The improvement in myositis by Insl6 could also be demonstrated by acute hydrodynamic delivery of a plasmid encoding murine Insl6. In cultured cells, Insl6 inhibits Jurkat cell proliferation and activation in response to phytohemagglutinin/phorbol 12-myristate 13-acetate stimulation. Insl6 transcript expression in muscle was reduced in a cohort of dermatomyositis and polymyositis patients.ConclusionsThese data suggest that Insl6 may have utility for the treatment of myositis, a condition for which few treatment options exist.