TNFα-mediated Inflammation Research Articles

Evidence that extracellular HSPB1 contributes to inflammation in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. Serum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. Circulating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro , ethanol-stressed hepatocytes released HSPB1, which then triggered TNFα-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNFα release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. Our findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. HSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis.Ethanol consumption leads to early increases in HSPB1 in the mouse liver.Hepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNFα-mediated inflammation in macrophages.Anti-HSPB1 antibody blocks hepatocyte-triggered TNFα in a model of hepatocyte-macrophage communication during ethanol stress.

Abstract Tu016: Alleviating Aortic Valve Calcification By Blocking TNFα Receptors

Background: Tumor Necrosis Factor-alpha (TNFα) is a key inflammatory cytokine involved in calcific aortic valve disease (CAVD). Following mechanical stress, activated monocytes, macrophages induce TNFα signaling in aortic valve (AoV). The activated TNFα binds to its own receptors, TNFR1 and TNFR2, in which TNFR1 has a death domain likely to cause cell death in AoV. Subsequently, the release of extracellular vesicles (calcium and inorganic phosphates) following inflammatory process results in valve calcification and stenosis. Hypothesis: Blocking TNFα-mediated inflammation by genetic knockout of Tnfr1/Tnfr2 slowdown CAVD progression in mice. Methods and Results: Using a mouse model of CAVD with deletion of Ldlr, low-density lipoprotein receptor, we compared the disease progression in mice fed with standard and high fat diet. To get better understanding on the role of TNFα in CAVD, Ldlr -/- mice (3 months old) on the TNFR1 and TNFR2 knockout background, were also fed with high fat diet for 3 months ( Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- ). Aortic valve function was analyzed by using echocardiogram and it showed decreased AoV velocity and increased ejection fraction in Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- compared to Ldlr -/- ; Tnfr1 +/- : Tnfr2 +/- group. The pathological changes such as fibrosis, calcium and collagen deposition seen under routine and specialized histological staining methods were reduced in TNF receptors knockout group. Next, using customized designed for PCR arrays, we examined the expression of 62 candidate genes involved in inflammation, apoptosis, extracellular matrix remodeling or aortic valve stenosis pathways. The top downregulated genes in the Ldlr -/- ; Tnfr1 -/- : Tnfr2 -/- group compared to Ldlr -/- ; Tnfr1 +/- : Tnfr2 +/- group are (Col3a1, MMp7, Klk1) whereas the top upregulated gene are (Igf1r and MMp14). We are currently validating these findings by using qPCR and immunofluorescence. Conclusion: Our findings reveal blocking TNFα signaling receptors downregulate the genes related to inflammatory cytokine activation and aortic valve stenosis in the Ldlr knockout mice fed with high fat diet. Blockage of TNFα likely reduces apoptosis and inflammation while improves extracellular matrix remodeling to slowdown CAVD progression. Our findings thus suggest a new therapeutic strategy for CAVD.

TMAO enhances TNF-α mediated fibrosis and release of inflammatory mediators from renal fibroblasts

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

Identification of TNFα-mediated inflammation as potential pathological marker and therapeutic target for calcification progress of congenital bicuspid aortic valve

BackgroudCongenital bicuspid aortic valve (cBAV) develops calcification and stenotic obstruction early compared with degenerative tricuspid aortic valve (dTAV), which requires surgical intervention. Here we report a comparative study of patients with cBAV or dTAV to identify risk factors associated with the rapid development of calcified bicuspid valves. MethodsA total of 69 aortic valves (24 dTAV and 45 cBAV) were collected at the time of surgical aortic valve replacement for comparative clinical characteristics. Ten samples were randomly selected from each group for histology, pathology, and inflammatory factors expression and comparison analyses. OM-induced calcification in porcine aortic valve interstitial cell cultures were prepared for illustrating the underlying molecular mechanisms about calcification progress of cBAV and dTAV. ResultsWe found that cBAV patients have increased cases of aortic valve stenosis compared with dTAV patients. Histopathological examinations revealed increased collagens deposition, neovascularization and infiltrations by inflammatory cells, especially T-lymphocytes and macrophages. We identified that tumor necrosis factor α (TNFα) and its regulated inflammatory cytokines are upregulated in cBAV. Further in vitro study indicated that TNFα-NFκB and TNFα-GSK3β pathway accelerate aortic valve interstitial cells calcification, while inhibition of TNFα significantly delays this process. ConclusionThe finding of intensified TNFα-mediated inflammation in the pathological cBAV advocates the inhibition of TNFα as a potential treatment for patients with cBAV by alleviating the progress of inflammation-induced valve damage and calcification.

Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer.

Inflammation and metabolic reprogramming are hallmarks of cancer. How inflammation regulates cancer metabolism remains poorly understood. In this study, we found that 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), the enzyme that catalyzes the catabolism of leucine and promotes the synthesis of ketone bodies, was downregulated in lung cancer. Downregulation of HMGCL was associated with a larger tumor size and a shorter overall survival time. In a functional study, overexpression of HMGCL increased the content of β-hydroxybutyrate (β-HB) and inhibited the tumorigenicity of lung cancer cells, and deletion of HMGCL promoted de novo tumorigenesis in KP (KrasG12D;P53f/f) mice. Mechanistically, tumor necrosis factor α (TNFα) treatment decreased the HMGCL protein level, and IKKβ interacted with HMGCL and phosphorylated it at Ser258, which destabilized HMGCL. Moreover, NEDD4 was identified as the E3 ligase for HMGCL and promoted its degradation. In addition, mutation of Ser258 to alanine inhibited the ubiquitination of HMGCL by NEDD4 and thus inhibited the anchorage-independent growth of lung cancer cells more efficiently than did wild-type HMGCL. In summary, this study demonstrated a link between TNFα-mediated inflammation and cancer metabolism.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Suppression of RIP1 activity via S415 dephosphorylation ameliorates obesity-related hepatic insulin resistance.

Receptor-interacting serine/threonine-protein kinase 1 (RIP1) is a well-documented key regulator of TNFα-mediated inflammation, apoptosis, and necroptosis, which contribute to the development of obesity-related metabolic diseases such as nonalcoholic steatohepatitis. However, the mechanism regarding how RIP1 influences obesity-related insulin resistance remains elusive. Primary hepatocytes with necrostatin 1 treatment or RIP1 expression were exposed to palmitic acid (PA), prior to the examination of cellular insulin signaling. Phosphorylation sites of RIP1 were detected by liquid chromatography with tandem mass spectrometry, and RIP1 variants with mutated phosphorylation sites were overexpressed in hepatocytes to identify the specific residue that influenced the RIP1-mediated insulin resistance. Adenovirus expressing RIP1 (S415A) mutant were administered into diet-induced obese mice to assess the effects on insulin sensitivity. This study uncovered an aberrant increase in RIP1 activity during the development of obesity-induced insulin resistance. Inhibition of RIP1 activity with necrostatin 1 ameliorated PA- or high-fat diet-caused hepatic insulin resistance. With liquid chromatography with tandem mass spectrometry analysis and mutagenesis screening, S415, a novel phosphorylation site of RIP1, was identified to be responsible for RIP1-mediated insulin resistance. Loss-of-function mutation of S415 efficiently blunted RIP1-evoked insulin resistance in PA-treated hepatocytes or diet-induced obese mice. These findings highlight the diabetogenic role of RIP1 S415 and propose RIP1 as a promising therapeutic target for type 2 diabetes.

A Microfluidic 3D Endothelium-on-a-Chip Model to Study Transendothelial Migration of T Cells in Health and Disease.

The recruitment of T cells is a crucial component in the inflammatory cascade of the body. The process involves the transport of T cells through the vascular system and their stable arrest to vessel walls at the site of inflammation, followed by extravasation and subsequent infiltration into tissue. Here, we describe an assay to study 3D T cell dynamics under flow in real time using a high-throughput, artificial membrane-free microfluidic platform that allows unimpeded extravasation of T cells. We show that primary human T cells adhere to endothelial vessel walls upon perfusion of microvessels and can be stimulated to undergo transendothelial migration (TEM) by TNFα-mediated vascular inflammation and the presence of CXCL12 gradients or ECM-embedded melanoma cells. Notably, migratory behavior was found to differ depending on T cell activation states. The assay is unique in its comprehensiveness for modelling T cell trafficking, arrest, extravasation and migration, all in one system, combined with its throughput, quality of imaging and ease of use. We envision routine use of this assay to study immunological processes and expect it to spur research in the fields of immunological disorders, immuno-oncology and the development of novel immunotherapeutics.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Intestinal SIRT1 Deficiency-Related Intestinal Inflammation and Dysbiosis Aggravate TNFα-Mediated Renal Dysfunction in Cirrhotic Ascitic Mice.

In advanced cirrhosis, the TNFα-mediated intestinal inflammation and bacteria dysbiosis are involved in the development of inflammation and vasoconstriction-related renal dysfunction. In colitis and acute kidney injury models, activation of SIRT1 attenuates the TNFα-mediated intestinal and renal abnormalities. This study explores the impacts of intestinal SIRT1 deficiency and TNFα-mediated intestinal abnormalities on the development of cirrhosis-related renal dysfunction. Systemic and renal hemodynamics, intestinal dysbiosis [cirrhosis dysbiosis ratio (CDR) as marker of dysbiosis], and direct renal vasoconstrictive response (renal vascular resistance (RVR) and glomerular filtration rate (GFR)) to cumulative doses of TNFα were measured in bile duct ligated (BDL)-cirrhotic ascitic mice. In SIRT1IEC-KO-BDL-ascitic mice, the worsening of intestinal dysbiosis exacerbates intestinal inflammation/barrier dysfunction, the upregulation of the expressions of intestinal/renal TNFα-related pathogenic signals, higher TNFα-induced increase in RVR, and decrease in GFR in perfused kidney. In intestinal SIRT1 knockout groups, the positive correlations were identified between intestinal SIRT1 activity and CDR. Particularly, the negative correlations were identified between CDR and RVR, with the positive correlation between CDR and GFR. In mice with advanced cirrhosis, the expression of intestinal SIRT1 is involved in the linkage between intestinal dysbiosis and vasoconstriction/hypoperfusion-related renal dysfunction through the crosstalk between intestinal/renal TNFα-related pathogenic inflammatory signals.

New insights into TNFα/PTP1B and PPARγ pathway through RNF213- a link between inflammation, obesity, insulin resistance, and Moyamoya disease.

Diabetic patients are always at a higher risk of ischemic diseases like coronary artery diseases. One such ischemic carotid artery disease is Moyamoya disease (MMD) associated with diabetes Type I and II, but the causality was unclear. Ring Finger Protein 213 (RNF213) is the major susceptible gene for MMD. To understand the association between diabetes mellitus and MMD we chose the major players from both of the anomalies: insulin and RNF213. But before establishing the role of RNF213 in the insulin-regulating pathway we had to understand the involvement of RNF213 within different biological systems. For this, we have adopted a preliminary computational approach to find the prominent interactions of RNF213. Our first objective was to construct an interactome for RNF213. We have analyzed several curated databases and adapted a list of RNF213 interacting partners to develop its interactome. Then to understand the involvement of this interactome in biological functions we have analyzed major biological pathways, biological processes, and prominent clusters related to this interactome through a computational approach. Then to develop a pathway that might give clues for RNF213 involvement in the insulin regulatory pathway we have validated the intercluster and intracluster predictions and identified a regulatory pathway for RNF213. RNF213 interactome was observed to be involved in adaptive immunity with 4 major clusters; one of the clusters involved TNFα. The immune system involves several pathways, and therefore at this point, we have chosen an event-based strategy to obtain an explicit target. Immunity is mediated by pro-inflammatory cytokines like TNFα. TNFα-mediated inflammation, obesity, and insulin resistance are associated. Therefore we chose to explore the role of RNF213 in TNFα-mediated inflammation in macrophages and inflammation-mediated insulin-resistance in adipocytes. We have observed an enhancement of RNF213 gene expression by LPS mediated pro-inflammatory stimuli and suppression by PPARγ-mediated anti-inflammatory, insulin-sensitizing stimuli in macrophages, and also in adipocytes. Administration of the pro-inflammatory cytokine TNFα was able to impede the reduction in RNF213 expression during adipogenesis and this effect was observed to be mediated by PTP1B. Inactivation of PTP1B abolished RNF213 expression which in turn enhanced the adipogenesis process through enhanced PPARγ. Constitutive expression of RNF213 suppressed the adipocyte differentiation by the inhibition of PPARγ. We could show the regulation of RNF213 by TNFα/PTP1B pathway and PPARγ. The constitutive expression of RNF213 during adipogenesis appears to be an adipostatic measure that obese patients acquire to inhibit further adipogenesis. This is verified in silico by analyzing the gene expression data obtained from the Gene Expression Omnibus database, which showed a higher expression of RNF213 in adipose tissue samples of obese people. Overall this study gives new insights into the TNFα-mediated pathway in adipogenesis and suggests the role of RNF213 in adipogenesis via this pathway.

Immune-modulating activities of glucans extracted from Pleurotus ostreatus and Pleurotus eryngii

We compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophil effector functions (P. eryngii was more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability.

Mitophagy protects SH-SY5Y neuroblastoma cells against the TNFα-induced inflammatory injury: Involvement of microRNA-145 and Bnip3

Inflammatory response is involved in the development of facial neuritis. The aim of our study is to explore the role of mitophagy in facial nerve damage induced by tumor necrosis factor α (TNFα). Our results indicated that TNFα induced SH-SY5Y cell apoptosis in a dose-dependent manner. Besides, TNFα treatment also suppressed mitophagy by reducing the expression of BCL2 interacting protein 3 (Bnip3). Overexpression of Bnip3 under sustained SH-SY5Y cell viability in the setting of TNFα-mediated inflammation injury. At the molecular levels, Bnip3 overexpression maintained mitochondrial function via preserving mitochondrial membrane potential, reducing cytochrome-c leakage and inhibiting mitochondrial permeability transition pore opening. Functional studies have suggested that microRNA-145 (miR-145) was an upstream regulator of Bnip3-dependent mitophagy. MiR-145 inhibited Bnip3 transcription and expression, leading to mitophagy inhibition. In contrast, inhibition of miR-145 reversed mitophagy activity and subsequently promoted SH-SY5Y cell survival in the context of TNFα-mediated inflammation injury. Altogether, our data identified Bnip3-dependent mitophagy as one of the defensive mechanisms to sustain mitochondrial homeostasis and SH-SY5Y cell survival. Besides, miR-145/Bnip3/mitophagy axis may be considered as a potential target for the treatment of facial neuritis in clinical practice.

Rapid Communication: Prolactin and hydrocortisone impact TNFα-mediated mitogen-activated protein kinase signaling and inflammation of bovine mammary epithelial (MAC-T) cells.

The objective of this study was to evaluate the effects of the hormones prolactin (PRL) and hydrocortisone (HC) on bovine mammary alveolar (MAC-T) cells mitogen-activated protein kinase (MAPK) inflammatory signaling and inflammatory gene expression. MAC-T cells were cultured in the presence (+PRL +HC; Dulbecco's modified Eagle's medium [DMEM] 10% fetal bovine serum, 10 µg/mL of insulin, 100 IU/mL penicillin, 100 µg/mL streptomycin, 1 µg/mL ovine PRL, 0.5 µg/mL HC, and 10 m sodium acetate) or the absence (-PRL -HC; DMEM 10% fetal bovine serum, 10 µg/mL insulin, 100 IU/mL penicillin , and 100 µg/mL streptomycin) of PRL and HC, and MAPK (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38) phosphorylation and inflammatory gene expression were examined in response to tumor necrosis factor α (TNFα). Statistical analysis was assessed using 1-way ANOVA, and Tukey's post hoc analysis was used to assess statistical significance when ≤ 0.05. MAC-T cells cultured in +PRL +HC and -PRL -HC were co-stimulated with increasing concentrations of TNFα (0, 10, 30, 100, 300, and 1,000 p). Cell lysates were harvested 15 min after TNFα stimulation and assessed for MAPK phosphorylation using immunoblotting. c-Jun N-terminal kinase and p38 phosphorylation increased in a dose-dependent manner and was greater in cells cultured in -PRL -HC. MAC-T cells cultured in +PRL +HC and -PRL -HC were next stimulated with TNFα (300 p), and lysates were harvested over time (0, 15, 30, 60, 120, and 180 min) after TNFα stimulation. c-Jun N-terminal kinase and p38 phosphorylation was transiently increased in MAC-T cells stimulated with TNFα; however, JNK and p38 signaling was greater in MAC-T cells cultured in -PRL -HC. We next examined inflammatory gene expression in MAC-T cells cultured in +PRL +HC and -PRL -HC. Cells were co-stimulated with (300 p) or without TNFα. Ribonucleic acid was isolated 1 h after TNFα stimulation, and a PCR array was performed to examine the expression of 83 inflammatory genes. Gene expression was increased in MAC-T cells in response to TNFα. Consistent with enhanced MAPK signaling, inflammatory gene expression was increased in MAC-T cells cultured in -PRL -HC. Real-time quantitative PCR of 6 target genes was used to validate the PCR array findings. Collectively, our data demonstrate that -PRL -HC MAC-T cells are more responsive to TNFα stimuli. These findings suggest that cell culture conditions (e.g., treatment with hormones) greatly impact cellular response and should be considered prior to experimental design and hypothesis testing.

Novel Influences of IL-10 on CNS Inflammation Revealed by Integrated Analyses of Cytokine Networks and Microglial Morphology.

Coordinated interactions between cytokine signaling and morphological dynamics of microglial cells regulate neuroinflammation in CNS injury and disease. We found that pro-inflammatory cytokine gene expression in vivo showed a pronounced recovery following systemic LPS. We performed a novel multivariate analysis of microglial morphology and identified changes in specific morphological properties of microglia that matched the expression dynamics of pro-inflammatory cytokine TNFα. The adaptive recovery kinetics of TNFα expression and microglial soma size showed comparable profiles and dependence on anti-inflammatory cytokine IL-10 expression. The recovery of cytokine variations and microglial morphology responses to inflammation were negatively regulated by IL-10. Our novel morphological analysis of microglia is able to detect subtle changes and can be used widely. We implemented in silico simulations of cytokine network dynamics which showed—counter-intuitively, but in line with our experimental observations—that negative feedback from IL-10 was sufficient to impede the adaptive recovery of TNFα-mediated inflammation. Our integrative approach is a powerful tool to study changes in specific components of microglial morphology for insights into their functional states, in relation to cytokine network dynamics, during CNS injury and disease.

Epithelial EZH2 serves as an epigenetic determinant in experimental colitis by inhibiting TNFα-mediated inflammation and apoptosis

Epithelial barrier disruption is a major cause of inflammatory bowel disease (IBD); however, the mechanism through which epigenetic regulation modulates intestinal epithelial integrity remains largely undefined. Here we show that EZH2, the catalytic subunit of polycomb repressive complex (PRC2), is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. In accordance with reduced EZH2 expression in patients, the inactivation of EZH2 in IECs sensitizes mice to DSS- and TNBS-induced experimental colitis. Conversely, EZH2 overexpression in the intestinal epithelium renders mice more resistant to colitis. Mechanistically, the genes encoding TRAF2/5 are held in a finely tuned bivalent status under inflammatory conditions. EZH2 deficiency potentiates the expression of these genes to enhance TNFα-induced NF-κB signaling, thereby leading to uncontrolled inflammation. More importantly, we show that EZH2 depletion compromises the protective role of NF-κB signaling in cell survival by directly up-regulating ITCH, a well-known E3 ligase that degrades the c-FLIP protein. Thus, our findings highlight an epigenetic mechanism by which EZH2 integrates the multifaceted effects of TNFα signaling to promote the inflammatory response and apoptosis in colitis.

Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs—Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)–triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

504. Targeted Genome Engineering of Induced Pluripotent Stem Cells To Produce Auto-Regulated Inflammation Resistance for Musculoskeletal Regenerative Medicine

Chronic inflammatory diseases such as arthritis are characterized by aberrant and dysregulated responses to cytokines such as tumor necrosis factor α (TNFα). While exogenous anti-cytokine therapies have been shown to diminish this inflammatory response, they are often used at high, unregulated doses that can induce significant side effects. Here, we propose a novel regenerative medicine approach to treating such chronic inflammatory diseases by generating custom-designed cells that can execute real-time, user-defined responses to environmental cues, including pro-inflammatory cytokines. We designed and deployed gene editing nucleases based on the CRISPR/Cas9 system to create stem cells with the ability to antagonize TNFα-mediated inflammation in an auto-regulated, feedback-controlled manner for musculoskeletal regenerative medicine applications. Specifically, the cytokine-responsive Ccl2 gene was reprogrammed by nuclease-mediated integration of the gene encoding soluble TNFα receptor I (sTNFRI), a TNFα antagonist, immediately downstream of the Ccl2 promoter. Using a combination of qRT-PCR and site-specific integration of luciferase reporters, we observed increased transgene transcription in response to endogenous Ccl2 activation by treatment with TNFα. Transgene expression from engineered cells was feedback-controlled with rapid on/off dynamics (Fig 1 A). Importantly, engineered cells showed the ability to mitigate the inflammatory effects of TNFα in an auto-regulated manner within 48 hr of induction (Fig 1 B). Finally, while cartilage produced from control stem cells showed significant tissue degradation in response to TNFα, cartilage derived from engineered stem cells was protected from a 3-day treatment with 20 ng/ml TNFα, as evidenced by qRT-PCR, biochemical, and histological analyses (Fig 1C-G).This work demonstrates the utility of programmable nucleases for the development of “designer” stem cells with properties customized for regenerative medicine. Using CRISPR/Cas9, we engineered pluripotent stem cells with the user-specified trait of pro-inflammatory cytokine resistance. Our results show that genome engineering facilitated the rewiring of endogenous cell circuits in order to define novel input/output relationships between inflammatory mediators and their antagonists, achieving therapeutic benefit coupled to a rapidly responding, auto-regulated system. The customization of intrinsic cellular signaling pathways in therapeutic stem cell populations, as demonstrated in this work, can transform the landscape of regenerative medicine and open innovative possibilities for safer and more effective treatments applicable to a wide variety of diseases. View Large Image | Download PowerPoint Slide

Rebamipide suppresses TNF-α mediated inflammation in vitro and attenuates the severity of dermatitis in mice.

Rebamipide is a routine drug for the treatment of gastritis in a clinical setting. Recently, it has been shown to protect against various inflammatory diseases, and has provided a potential therapy for these diseases. However, whether rebamipide has a role in dermatitis remains to be elucidated. Here, we found that rebamipide alleviated the inflammatory reaction induced by tumor necrosis factor-α in RAW264.7, a stable macrophage cell line. Furthermore, rebamipide treatment repressed activation of nuclear factor-kappaB signaling, a well-established inflammatory signaling pathway. Moreover, an oxazolone-induced dermatitis mouse model was established to investigate the role of rebamipide in vivo. PBS control group exhibited typical skin inflammation, whereas treatment with rebamipide remarkably attenuated a dermatitis phenotype in this mouse model. The protective role of rebamipide in dermatitis in vivo was probably due to its inhibition of nuclear factor-kappaB signaling. Collectively, rebamipide may represent a promising molecular target for the prevention and treatment of inflammatory skin diseases.

Nutritional recovery with a soybean diet after weaning reduces lipogenesis but induces inflammation in the liver in adult rats exposed to protein restriction during intrauterine life and lactation.

We evaluated the effects of postweaning nutritional recovery with a soybean flour diet on de novo hepatic lipogenesis and inflammation in adult rats exposed to protein restriction during intrauterine life and lactation. Rats from mothers fed with protein (casein) in a percentage of 17% (control, C) or 6% (low, L) during pregnancy and lactation were fed with diet that contained 17% casein (CC and LC groups, resp.) or soybean (CS and LS groups, resp.) after weaning until 90 days of age. LS and CS rats had low body weight, normal basal serum triglyceride levels, increased ALT concentrations, and high HOMA-IR indices compared with LC and CC rats. The soybean diet reduced PPARγ as well as malic enzyme and citrate lyase contents and activities. The lipogenesis rate and liver fat content were lower in LS and CS rats relative to LC and CC rats. TNFα mRNA and protein levels were higher in LS and CS rats than in LC and CC rats. NF-κB mRNA levels were lower in the LC and LS groups compared with the CC and LC groups. Thus, the soybean diet prevented hepatic steatosis at least in part through reduced lipogenesis but resulted in TNFα-mediated inflammation.