TNF mRNA Research Articles

Diesel exhaust impairs TREM2 to dysregulate neuroinflammation

BackgroundAir pollution has been linked to neurodegenerative diseases, including Alzheimer’s disease (AD), and the underlying neuroimmune mechanisms remain poorly understood. TREM2 is a myeloid cell membrane receptor that is a key regulator of disease-associated microglia (DAM) cells, where loss-of-function TREM2 mutations are associated with an increased risk of AD. At present, the basic function of TREM2 in neuroinflammation is a point of controversy. Further, the impact of air pollution on TREM2 and the DAM phenotype is largely unknown. Using diesel exhaust (DE) as a model of urban air pollution exposure, we sought to address its impact on TREM2 expression, the DAM phenotype, the association of microglia with the neurovasculature, and the role of TREM2 in DE-induced neuroinflammation.MethodsWYK rats were exposed for 4 weeks to DE (0, 50, 150, 500 μg/m3) by inhalation. DE particles (DEP) were administered intratracheally once (600 μg/mouse) or 8 times (100 μg/mouse) across 28 days to male mice (Trem2+/+, Trem2−/−, PHOX+/+, and PHOX−/−).ResultsRats exposed to DE exhibited inverted-U patterns of Trem2 mRNA expression in the hippocampus and frontal cortex, while TREM2 protein was globally diminished, indicating impaired TREM2 expression. Analysis of DAM markers Cx3Cr1, Lyz2, and Lpl in the frontal cortex and hippocampus showed inverted-U patterns of expression as well, supporting dysregulation of the DAM phenotype. Further, microglial-vessel association decreased with DE inhalation in a dose-dependent manner. Mechanistically, intratracheal administration of DEP increased Tnf (TNFα), Ncf1 (p47PHOX), and Ncf2 (p67PHOX) mRNA expression in only Trem2+/+ mice, where Il1b (IL-1β) expression was elevated in only Trem2−/− mice, emphasizing an important role for TREM2 in DEP-induced neuroinflammation.ConclusionsCollectively, these findings reveal a novel role for TREM2 in how air pollution regulates neuroinflammation and provides much needed insight into the potential mechanisms linking urban air pollution to AD.

HPA axis regulation and epigenetic programming of immune-related genes in chronically stressed and non-stressed mid-life women

Hypothalamic-pituitary-adrenal (HPA) axis dysregulation has been associated with altered immune function, but the underlying molecular mechanisms are unclear. Epigenetic processes, including DNA methylation, respond to the glucocorticoid end-products of the HPA axis (cortisol in humans) and could be involved in this neuroendocrine-immune crosstalk. Here we examined the extent to which variations in HPA axis regulation are associated with peripheral blood DNA (CpG) methylation changes in 57 chronically stressed caregivers and 67 control women. DNA methylation was determined with the Illumina 450k array for a panel of genes involved in HPA axis and immune function. HPA axis feedback was assessed with the low-dose dexamethasone suppression test (DST), measuring the extent to which cortisol secretion is suppressed by the synthetic glucocorticoid dexamethasone. After multiple testing correction in the entire cohort, higher post-DST cortisol, reflecting blunted HPA axis negative feedback, but not baseline waking cortisol, was associated with lower DNA methylation at eight TNF and two FKBP5 CpG sites. Caregiver group status was associated with lower methylation at two IL6 CpG sites. Since associations were most robust with TNF methylation (32% of the 450k-covered sites), we further examined functionality of this epigenetic signature in cultured peripheral blood mononuclear cells in 33 participants; intriguingly, lower TNF methylation resulted in higher ex vivo TNF mRNA following immune stimulation. Taken together, our findings link chronic stress and HPA axis regulation with epigenetic signatures at immune-related genes, thereby providing novel insights into how aberrant HPA axis function may contribute to heightened inflammation and disease risk.

Indomethacin augments lipopolysaccharide-induced expression of inflammatory molecules in the mouse brain.

Indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and fever including during infections. However, some studies suggest that NSAIDs protect against neuroinflammation, while some find no effects or worsening of neuroinflammation. We evaluated the effect of indomethacin alone on in combination with minocycline, a drug that inhibits neuroinflammation, on the expression of transcripts of neuroinflammatory molecules-induced by lipopolysaccharide (LPS) in the brain of mice. Inoculation of male BALB/c mice with LPS induced the expression of the microglia marker ionized calcium binding adaptor molecule protein, mRNA expression of the genes for cytokines interleukin-1beta (Il1b) and tumor necrosis factor-alpha (Tnf) and inducible nitric oxide synthase gene (Nos2), but not Il10, in the brain. Treatment with indomethacin had no significant effect on the cytokines or Nos2 mRNA expression in naïve animals. However, pretreatment with indomethacin increased LPS-induced Nos2 mRNA and inducible nitric oxide (iNOS) protein expression, but had no significant effect on LPS-induced mRNA expression of the cytokines. Minocycline reduced LPS-induced Il1b and Tnf, but not Nos2, mRNA expression. Treatment with indomethacin plus minocycline had no effect on LPS-induced Il1b, Tnf and Nos2 mRNA expression. In conclusion these results show that indomethacin significantly augments LPS-induced Nos2 mRNA and iNOS protein expression in the brain. In the presence of indomethacin, minocycline could not inhibit LPS-induced pro-inflammatory cytokine expression. Thus, indomethacin could exacerbate neuroinflammation by increasing the expression of iNOS and also block the anti-inflammatory effects of minocycline.

Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in Human T Cells.

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

Severe Impairment of TNF Post-transcriptional Regulation Leads to Embryonic Death.

SummaryPost-transcriptional regulation mechanisms control mRNA stability or translational efficiency via ribosomes, and recent evidence indicates that it is a major determinant of the accurate levels of cytokine mRNAs. Transcriptional regulation of Tnf has been well studied and found to be important for the rapid induction of Tnf mRNA and regulation of the acute phase of inflammation, whereas study of its post-transcriptional regulation has been largely limited to the role of the AU-rich element (ARE), and to a lesser extent, to that of the constitutive decay element (CDE). We have identified another regulatory element (NRE) in the 3′ UTR of Tnf and demonstrate that ARE, CDE, and NRE cooperate in vivo to efficiently downregulate Tnf expression and prevent autoimmune inflammatory diseases. We also show that excessive TNF may lead to embryonic death.

Inhibition of sphingosine‐1‐phosphate receptor 2 attenuated ligature‐induced periodontitis in mice

ObjectivesPeriodontitis is an inflammatory bone loss disease initiated by oral bacterial inflammation. Herein, we determined whether inhibition of sphingosine‐1‐phosphate receptor 2 (S1PR2, a G protein‐coupled receptor) by its specific antagonist, JTE013, could alleviate ligature‐induced periodontitis in mice.Materials and MethodsC57BL/6 mice were placed with silk ligatures at the left maxillary second molar to induce experimental periodontitis. Mice were treated with JTE013 or control vehicle (dimethyl sulfoxide, DMSO) oral topically on the ligatures once daily. After 15 days of treatment, RNA was extracted from the lingual mucosal tissues to quantify IL‐1β, IL‐6, and TNF mRNA levels in the tissues. Alveolar bone loss was determined by micro‐computed tomography. Sagittal periodontal tissue sections were cut and stained by hematoxylin and eosin (H&E) for general histology, or stained by tartrate‐resistant acid phosphatase (TRAP) for osteoclasts.ResultsTreatment with JTE013 attenuated ligature‐induced alveolar bone loss compared with DMSO treatment. Treatment with JTE013 reduced IL‐1β, IL‐6, and TNF mRNA levels in murine gingival mucosal tissues, inhibited leukocyte infiltration in the periodontal tissues, and decreased the number of osteoclasts in the periodontal tissues compared with controls.ConclusionOral topical administration of JTE013 alleviated periodontal inflammatory bone loss induced by ligature placement in mice.

Activated microglia-derived macrophage-like cells exacerbate brain edema after ischemic stroke correlate with astrocytic expression of aquaporin-4 and interleukin-1 alpha release

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3–7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema.

Tristetraprolin Overexpression in Non-hematopoietic Cells Protects Against Acute Lung Injury in Mice.

Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3′ untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.

Abstract 358: Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer

Abstract Tumor necrosis factor alpha (TNF) is a potent cytokine reported to have several functions, including stimulation of inflammation, directing cell death and cell proliferation. The cellular origin of TNF has generally believed to be the inflammatory cells especially because macrophages are prone to induction of TNF. Using in situ hybridization (ISH) based on TNF mRNA probes (RNAscope) we recently reported that TNF mRNA is also seen in cytokeratin positive cancer cells (CC) and is associated with morphologic appearance interpreted as CC branching (Møller et al, IJMS 2019, doi: 10.3390/ijms20081907). To determine the fraction of CC-derived TNF mRNA signal at different areas in colon cancers we developed a cell and color segmentation-based image analysis tool to process fluorescence signals in digital slides. The approach allowed quantification of the TNF mRNA-positive CC and TNF mRNA-positive stromal cells in the digital slides previously reported, that were also stained for cytokeratin, microRNA-21 (miR-21) and DAPI nuclear stain. In the two cases with most abundant TNF mRNA ISH signal, both showing moderate-to-high budding index, the fraction of TNF-mRNA positive CC in the outer periphery (approximately 500 µm) was 8% and 20%, respectively. The frequency of the TNF-positive CC increased towards the invasive front from 7% (33/500 CC per 0.25 mm2) to 35% (48/137 CC), and from 8% (127/1536 CC per 0.50 mm2) to 28% (108/389 CC), respectively. The upregulation is in agreement with initial qualitative evaluation. In the same areas, the fraction of TNF mRNA positive stromal cells among all TNF mRNA-positive cells was only 8% in both cases, indicating that the majority of the TNF mRNA originates from CC. The fraction of the TNF mRNA-positive CC that were also miR-21 positive was 85% and 43%, respectively, which was higher than the prevalence of the overall miR-21 positive CC, 55% and 30%, suggesting frequent co-expression with miR-21. In conclusion, we have developed a method to obtain quantitative estimates from digital multiplex-stained slides with RNAscope and LNA fluorescence ISH and immunofluorescence. More samples will need to be processed to better understand the importance of TNF expression in cancer cell invasion. Citation Format: Boye Schnack Nielsen, Kim Holmstrom. Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 358.

Monocytes from infliximab-resistant patients with Crohn\u2019s disease exhibit a disordered cytokine profile

Crohn's disease (CD) is a chronic inflammatory disorder characterized by immune response dysregulation. Tumor necrosis factor-α (TNFα) is a key cytokine in the pathogenesis of CD, as indicated by the efficacy of anti-TNF-α therapy with infliximab (IFX). However, approximately 30–40% of CD patients fail to respond to IFX with still unclear underlying mechanisms. This study compares the inflammatory phenotype of monocytes from CD patients, who respond or non-respond to IFX. Under basal conditions, the mRNA for the cytokines TNFα, IL-23, IL-1β and the chemokines CXCL8/IL-8, CCL5/RANTES and CCL2/MCP-1 was up-regulated in monocytes from non-responders than responders. The expression of the same cytokines and CCL2/MCP-1 was higher in non-responders also upon LPS treatment. Moreover, higher secretion of TNFα, IL-1β, IFNγ and IL-2 proteins occurred in the supernatants of LPS-treated non-responders cells. Resistance to IFX in CD may result from a transcriptional dysregulation of circulating monocytes, leading to hyperactivation of pro-inflammatory pathways. Monocytes’ cytokine profile may thus represent a predictive marker of response to IFX. Monocytes were isolated from blood samples of 19 CD patients (11 responders, 8 non-responders) and incubated with or without LPS. Cytokine profiles were assessed by RT-qPCR and, in the supernatants, by ELISA assay.

Glaucoma after Ocular Surgery or Trauma: The Role of Infiltrating Monocytes and Their Response to Cytokine Inhibitors

Glaucoma is a frequent and devastating long-term complication following ocular trauma, including corneal surgery, open globe injury, chemical burn, and infection. Postevent inflammation and neuroglial remodeling play a key role in subsequent ganglion cell apoptosis and glaucoma. To this end, this study was designed to investigate the amplifying role of monocyte infiltration into the retina. By using three different ocular injury mouse models (corneal suture, penetrating keratoplasty, and globe injury) and monocyte fate mapping techniques, we show that ocular trauma or surgery can cause robust infiltration of bone marrow-derived monocytes into the retina and subsequent neuroinflammation by up-regulation of Tnf, Il1b, and Il6 mRNA within 24 hours. This is accompanied by ganglion cell apoptosis and neurodegeneration. Prompt inhibition of tumor necrosis factor-α or IL-1β markedly suppresses monocyte infiltration and ganglion cell loss. Thus, acute ocular injury (surgical or trauma) can lead to rapid neuroretinal inflammation and subsequent ganglion cell loss, the hallmark of glaucoma. Infiltrating monocytes play a central role in this process, likely amplifying the inflammatory cascade, aiding in the activation of retinal microglia. Prompt administration of cytokine inhibitors after ocular injury prevents this infiltration and ameliorates the damage to the retina-suggesting that it may be used prophylactically for neuroprotection against post-traumatic glaucoma.

Combined Effects Of Unloading And Radiation On Skeletal Muscle In Mice

In preparation for upcoming space missions to the Moon and Mars, there is a need to understand how space stressors (e.g. microgravity, radiation) affect different physiological systems. As skeletal muscle is a critical organ, not only for locomotion but also for overall body homeostasis, defining the molecular impact of microgravity and radiation on this tissue will help developing new, or fine-tuning current countermeasures to maintain health and physiological function of space travellers. PURPOSE: To investigate the effects of combined radiation and unloading on anabolic/catabolic and immune/inflammatory processes on skeletal muscle in mice. METHODS: Ten C57/BL6J mice were subjected to 14-d hind-limb unloading by tail suspension with an acute radiation session (dose=25 mGy, X-ray) on day 7 of unloading (HLUR). Ten mice were used as control (CTRL; similar cages, sham radiation). Mice were sacrified and soleus muscle was immediately dissected, weighed and frozen. Then, RNA was extracted and converted to cDNA. Gene expression of anabolic/catabolic (i.e. myostatin, MuRF-1, Atrogin-1, PGC-1α) and immune/inflammatory markers (i.e. CD4, CD8, IFNγ, CD11b, MHCII, TNF, IL-6) was assessed by RT-PCR. Independent t-tests were use to compare HLUR vs. CTRL. RESULTS: Soleus muscle weight was ~30% lower in HLUR vs. CTRL (P<0.001). Myostatin expression was greater in HLUR vs. CTRL (1.8-fold, P=0.014). MHCII expression was higher in HLUR vs. CTRL (2.4-fold, P<0.001). There was a trend for group differences (P<0.08) in CD11b and TNF mRNA content with HLUR showing greater values than CTRL. Gene expression of CD4, CD8 and IFNγ was barely detected in either group. CONCLUSIONS: The combination of unloading and radiation has a major impact on skeletal muscle. Apart from inducing muscle atrophy, as indicated by the decreased muscle weight and increased myostatin levels, these two space stressors altered the immune profile within the muscle. The increased gene expression of MHCII and CD11b indicates that the myeloid component of the immune system is activated upon unloading and radiation in skeletal muscle. In contrast, the almost undetected mRNA levels of CD4, CD8 and IFNγ may imply that unloading and acute radiation have little impact on the lymphoid component. These findings should be followed up with immunohistochemical analysis.

TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity

Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1β. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.

Deletion of H-ferritin in macrophages alleviates obesity and diabetes induced by high-fat diet in mice.

Iron accumulation affects obesity and diabetes, both of which are ameliorated by iron reduction. Ferritin, an iron-storage protein, plays a crucial role in iron metabolism. H-ferritin exerts its cytoprotective action by reducing toxicity via its ferroxidase activity. We investigated the role of macrophage H-ferritin in obesity and diabetes. Conditional macrophage-specific H-ferritin (Fth, also known as Fth1) knockout (LysM-Cre Fth KO) mice were used and divided into four groups: wild-type (WT) and LysM-Cre Fth KO mice with normal diet (ND), and WT and LysM-Cre Fth KO mice with high-fat diet (HFD). These mice were analysed for characteristics of obesity and diabetes, tissue iron content, inflammation, oxidative stress, insulin sensitivity and metabolic measurements. RAW264.7 macrophage cells were used for in vitro experiments. Iron concentration reduced, and mRNA expression of ferroportin increased, in macrophages from LysM-Cre Fth KO mice. HFD-induced obesity was lower in LysM-Cre Fth KO mice than in WT mice at 12weeks (body weight: KO 34.6 ± 5.6g vs WT 40.1 ± 5.2g). mRNA expression of inflammatory cytokines and infiltrated macrophages and oxidative stress increased in the adipose tissue of HFD-fed WT mice, but was not elevated in HFD-fed LysM-Cre Fth KO mice. However, WT mice fed an HFD had elevated iron concentration in adipose tissue and spleen, which was not observed in LysM-Cre Fth KO mice fed an HFD (adipose tissue [μmol Fe/g protein]: KO 1496 ± 479 vs WT 2316 ± 866; spleen [μmol Fe/g protein]: KO 218 ± 54 vs WT 334 ± 83). Moreover, HFD administration impaired both glucose tolerance and insulin sensitivity in WT mice, which was ameliorated in LysM-Cre Fth KO mice. In addition, energy expenditure, mRNA expression of thermogenic genes, and body temperature were higher in KO mice with HFD than WT mice with HFD. In vitro experiments showed that iron content was reduced, and lipopolysaccharide-induced Tnf-α (also known as Tnf) mRNA upregulation was inhibited in a macrophage cell line transfected with Fth siRNA. Deletion of macrophage H-ferritin suppresses the inflammatory response by reducing intracellular iron levels, resulting in the prevention of HFD-induced obesity and diabetes. The findings from this study highlight macrophage iron levels as a potential therapeutic target for obesity and diabetes.

Novel Combinatorial Regimen of Garcinol and Curcuminoids for Non-alcoholic Steatohepatitis (NASH) in Mice

Non-alcoholic steatohepatitis (NASH) is a progressive form of Non-alcoholic fatty liver disease (NAFLD), a chronic liver disease with a significant unmet clinical need. In this study, we examined the protective effects of Garcinia indica extract standardized to contain 20% w/w of Garcinol (GIE) and 95% Curcuminoids w/w from Curcuma longa (Curcuminoids) in a Stelic animal model (STAM) of NASH. The STAM mice developed steatosis, hepatocyte ballooning, and inflammation, which were significantly reduced by the combination of GIE and Curcuminoids, resulting in a lower NAFLD activity score. The treatment reduced fibrosis as observed by Sirius red staining, liver hydroxyproline content and mRNA levels of TGF- β and collagen in the liver. Immunostaining with alpha-smooth muscle actin (α SMA) revealed a significant reduction in hepatic stellate cells. Intriguingly, the combination regimen markedly decreased the mRNA levels of MCP1 and CRP and both mRNA and protein levels of TNF-α. NF-kB, reduced the hepatic and circulating FGF21 levels and altered the nonenzymatic (glutathione) and enzymatic antioxidant markers (Glutathione peroxidase, and superoxide dismutase). Our results suggest that the combination of GIE and Curcuminoids can reduce the severity of NASH by reducing steatosis, fibrosis, oxidative stress, and inflammation. The results suggest that the combinatorial regimen could be an effective supplement to prevent the progression of liver steatosis to inflammation and fibrosis in NASH.

Epigenetics in non-classical monocytes support their pro-inflammatory gene expression.

Non-classical human monocytes are characterized by high-level expression of cytokines like TNF, but the mechanisms involved are elusive. We have identified miRNAs and CpG-methylation sites that are unique to non-classical monocytes, defined via CD14 and CD16 expression levels. For down-regulated miRNAs that are linked to up-regulated mRNAs the dominant gene ontology term was intracellular signal transduction. This included down-regulated miRNA-20a-5p and miRNA-106b-5p, which both are linked to increased mRNA for the TRIM8 signaling molecule. Methylation analysis revealed 16 hypo-methylated CpG sites upstream of 14 differentially increased mRNAs including 2 sites upstream of TRIM8. Consistent with a positive role in signal transduction, high TRIM8 levels went along with high basal TNF mRNA levels in non-classical monocytes. Since cytokine expression levels in monocytes strongly increase after stimulation with toll-like-receptor ligands, we have analyzed non-classical monocytes (defined via slan expression) after stimulation with lipopolysaccharide (LPS). LPS-stimulated cells continued to have low miRNA-20a and miRNA-106b and high TRIM8 mRNA levels and they showed a 10-fold increase in TNF mRNA. These data suggest that decreased miRNAs and CpG hypo-methylation is linked to enhanced expression of TRIM8 and that this can contribute to the increased TNF levels in non-classical human monocytes.

Anticancer and antimetastatic activity of Hypomyces chrysospermus, a cosmopolitan parasite in different human cancer cells.

The importance of microbial natural compounds in drug research is increasing every year and they are used to prevent or treat a variety of diseases. Hypomyces chrysospermus is a cosmopolitan parasite of many Boletaceae members. Since not much work has been conducted to date, this study is undertaken to explore the anticancer effect, including the antiproliferative and antimetastatic activity of Hypomyces chrysospermus. The aim of this study is to determine the antiproliferative and antimetastatic activity of Hypomyces chrysospermus ethyl acetate extract, having antioxidant activity, against A549, Caco2, MCF-7 human cancer and CCD-19 Lu and CCD 841 CoN healthy human cell lines. Firstly, cytotoxic activity was determined by the WST-1 assay. After cell proliferations and anti-metastatic effects were investigated by a real-time cell analysis system (RTCA-DP) and IC50 concentrations were calculated for each cell line. In addition, the expression levels of Apaf-1, TNF and NF-kB mRNA in cancer cells were investigated with RealTime-PCR. The ethyl acetate extract of Hypomyces chrysospermus presented anticancer activities including antiproliferative and antimetastatic effects. Hypomyces chrysospermus as a source of biologically active metabolites can be used as an important resource in the development of new anticancer effective agents.

Sleep- and time of day-linked RNA transcript expression in wild-type and IL1 receptor accessory protein-null mice.

Sleep regulation involves interleukin-1β (IL1) family members, TNF, and circadian clock genes. Previously, we characterized spontaneous sleep and sleep after 8 h of sleep deprivation (SD) ending at zeitgeber time (ZT)4 and ZT16 in wild-type (WT) and IL1 receptor accessory protein (AcP)- and brain-specific AcP (AcPb)-knockout (KO) mice. Here, we applied quantitative reverse transcriptase polymerase chain reaction and Spearman gene pair expression correlation methods to characterize IL1, IL1 receptor 1 (IL1R1), AcP, AcPb, Period 1 (Per1), Clock, adenosine deaminase (Ada), peptidoglycan recognition protein 1 (Pglyrp1), and TNF mRNA expressions under conditions with distinct sleep phenotypes. In WT mice, IL1, IL1R1, AcP, Ada, and Clock mRNAs were higher at ZT4 (mid-sleep period) than at ZT16. mRNA expressions differed substantially in AcP and AcPb KO mice at those times. After SD ending at ZT4, only WT mice had a non-rapid eye movement sleep (NREMS) rebound, and AcPb and IL1R1 mRNA increases were unique to WT mice. In AcPb KO mice, which have spontaneous high EEG slow wave power, AcP and Pglyrp1 mRNAs were elevated relative to WT mice at ZT4. At ZT4, the AcPb KO - WT Spearman correlation difference networks showed high positive correlations between IL1R1 and IL1, Per1, and Clock and high negative correlations between TNF and Pglyrp1 and Ada. At ZT16, the WT mice gene pair expression network was mostly negative, whereas in AcP KO mice, which have substantially more rapid eye movement sleep than WT mice, it was all positive. We conclude that gene pair expression correlations depend on the presence of AcP and AcPb.NEW & NOTEWORTHY Spearman gene pair expression correlations depend upon the presence or absence of interleukin-1 receptor accessory protein and upon sleep phenotype.

Inflammation due to Chronic Alcohol Exposure Promotes Cardiac Atrophy and Dysfunction Resulting from Impaired Fibroblast‐Myocyte Crosstalk

Alcoholic cardiomyopathy (ACM) is a cardiac disease caused by chronic heavy alcohol abuse. ACM is most notably a type of dilated cardiomyopathy (DCM), accounting for approximately 21–50% of all idiopathic non‐ischemic DCM cases in Western countries. ACM is typically characterized by left ventricular dilation and LV muscle wasting (atrophy), with diastolic and systolic dysfunction (ejection fraction &lt; 40%) occurring in later stages. Previous studies in our lab have shown that chronic alcohol exposure prevents compensatory hypertrophy in both volume‐ and pressure‐overload models of heart failure. However, the mechanisms of alcohol‐induced cardiac atrophy and dysfunction are not clear. The purpose of this study was to investigate mechanisms of chronic alcohol‐induced cardiac remodeling and dysfunction. We also sought to determine the role of cardiac fibroblasts, which play a dynamic role in cardiac remodeling, in mediating these effects.Adult male Wistar rats were exposed to ethanol (EtOH) vapor inhalation for 16 weeks. Echocardiography was performed to assess terminal cardiac structure and function. Cardiac fibroblasts were isolated from the left ventricle (LV) for both ex vivo and in vitro analysis. Gene expression was determined by real‐time PCR and flow cytometry. Protein expression was determined via Western blot analysis. EtOH led to cardiac wall thinning and impaired systolic function, as evident by decreased LV wall thickness at diastole (LVPWd) and fractional shortening, respectively, Additionally, EtOH decreased mRNA levels of various contractile proteins including Acta1, Actc1, and Myh7. EtOH also increased mRNA and protein levels of LV inflammatory markers, TNF‐α and NF‐κB. Furthermore, EtOH induced increased activation of JNK and decreased mTOR activation, suggestive of decreased hypertrophic signaling and atrophy. EtOH led to myofibroblast activation as assessed by flow cytometry and led to ECM remodeling (increased collagen III content). EtOH increased Tnf and Il6 mRNA in cardiac fibroblasts ex vivo, and increased mRNA levels of several pro‐inflammatory cytokines and chemokines in vitro. Administration of conditioned media from EtOH‐treated fibroblasts decreased contractile protein mRNA levels and impaired Akt and mTOR signaling in differentiated H9C2 cardiomyocytes.Our results indicate that EtOH‐induced cardiac atrophy and dysfunction is associated with activation of inflammatory pathways. Furthermore, EtOH may induce a pro‐inflammatory cardiac fibroblast phenotype, leading to aberrant fibroblast‐myocyte cross‐talk.Support or Funding InformationFunding provided by the NIH/NIAAA 1R21AA022690 (JDG) and the Saving tiny Hearts Society (JDG).

Western diet promotes renal injury, inflammation, and fibrosis in a murine model of Alström syndrome

Alström syndrome is a rare recessive genetic disease caused by mutations in ALMS1, which encodes a protein that is related to cilia function and intracellular endosome trafficking. The syndrome has been linked to impaired glucose metabolism and chronic kidney disease (CKD). Polymorphisms in ALMS1 have likewise been linked to CKD, but little is known about the modification of the phenotype by environmental factors. To gain further insights, the fat aussie (foz) mouse strain, a genetic murine model of Alström syndrome, was exposed to a normal chow (NC) or to a Western diet (WD, 20% fat, 34% sucrose by weight and 0.2% cholesterol). Body weight was 50% higher in foz vs WT mice on NC, and WD enhanced the difference to 64% (Pinteraction=0.0002, two‐way ANOVA). Kidney weight to body weight ratio was not different between groups. Plasma creatinine was lower in foz vs WT mice independent of diet, potentially indicating glomerular hyperfiltration in foz mice. Renal mRNA expression of injury marker Kim1 was not affected by WD in WT and was not different between foz and WT mice on NC (P=0.9995), but it was 26‐fold higher in foz vs WT mice on WD (P&lt;0.0001). Likewise, other renal injury markers (Ngal mRNA; histology assessment) and renal markers of inflammation (Tnf, Il6, Il10 and Ccl2 mRNA; CD68‐positive cells) and fibrosis (Col1A1, Timp1 and Tgf‐beta mRNA; Masson’s trichrome staining) were similar in both genotypes on NC, but significantly greater in foz vs WT mice fed a WD. Albuminuria was higher in foz vs WT mice on both diets but WD significantly increased the difference (Pinteraction=0.0019, two‐way ANOVA). A glucose tolerance test performed in foz and WT mice on a WD revealed similar basal blood glucose levels and subsequent blood glucose profiles. These results indicate that a Western diet sensitizes a murine model of Alström syndrome to kidney injury, inflammation and fibrosis, an effect that may not be solely due to effects on glucose metabolism. Polymorphisms in ALMS1 may induce CKD in part by modulating the deleterious effects of high dietary fat and sucrose on the kidney.Support or Funding InformationThe authors were supported by NIH grants R01DK112042, R01DK106102 (VV), the UAB/UCSD O’Brien Center of Acute Kidney Injury NIH‐P30DK079337 (VV), the Department of Veterans Affairs (VV), and by an investigator‐initiated research project by Janssen (DB, VV). DD was supported by NIH/CTRI 1KL2TR001444 and the American Liver Foundation (ALF) “Liver Scholar” grants.