Chronic TNF-α Research Articles

Age Related Senescence, Apoptosis, and Inflammation Profiles in Periodontal Ligament Cells from Canine Teeth.

The periapical tissues, including periodontal ligament cells (PDLCs) play an important role in repairing the surrounding tissue of the teeth. A decrease in the regenerative potentiality of resident stem cells (PDLCs) has been suggested to be attributed to the decline of pulp regeneration. Therefore, examining the functional changes in periodontal tissue and cells that occur during the aging process is necessary. The changes in the cementum extract (CE) and PDLCs isolated from young and aged dog teeth were evaluated. PDLCs growth rate, senescence markers, p16 and p21, and proinflammatory cytokines, IL-6, IL-1β, and TNF-α, were analyzed by RT-PCR. Bax, an apoptosis marker, Bcl-2, a marker for cell survival, and IL-6 were examined by Western blot analyses to detect their variance expression in the CE. Our results demonstrated that aged PDLCs exhibit a low growth rate and an increased expression of p16; however, no change has been demonstrated in the expression of p21. The chronic inflammatory molecules, IL-6 and TNF-α, were significantly upregulated compared to young PDLCs. Western blot analyses showed decreased expression of Bcl-2 in the CE of the aged tooth (p < 0.001). Taken together, aging influences the functional changes of PDLCs and CE and increases senescence, chronic inflammation, and apoptosis markers. As a result, donor age is a key factor influencing the utilization of PDLCs for tooth regeneration.

Reduction in Endothelial Adhesion of Red Blood Cells Under Crizanlizumab: Assessment of Standardized Microfluidic Platform in Sickle Cell Disease Population

Introduction: Monoclonal P-selectin blocking antibody (Crizanlizumab, ADAKVEO®, Novartis, Switzerland) targets reduction of vaso-occlusive crises (VOCs) in adult and pediatric patients (≥16 years) with sickle cell disease (SCD). Patients with SCD have been reported with endothelial p-selectin to be initiatory for endothelial adhesion of sickle red blood cells (RBCs). Herein, we report the novel endothelium-on-a-chip (microfluidic platform - MFP) that enables assessment of Crizanlizumab on the adhesion of RBCs to perfusion-cultured human endothelial cells (ECs) in acute and chronic activation setup. Methods: Whole blood samples collected from SCD subjects (n=14), HbSS and in EDTA and sodium citrate. RBCs were isolated via centrifugation from whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were cultured within the microfluidic platform channels at 15 dyne/cm2 for at least 48 hours prior to experiments. To mimic the acute EC activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml Crizanlizumab and injected through the microfluidic channels for 15 minutes. Instead to mimic chronic pre-activation in SCD HUVECs were pretreated for 4 hours with TNFα (20 ng/ml) or 50% plasma of SCD patients in basal media before, +/- 100 µg/ml Crizanlizumab followed by injection of blood samples through the microfluidic channels. Non-adherent RBCs were removed via washing solution with or without Crizanlizumab and the remaining RBCs were imaged using an inverted microscope and the manual quantification was completed based on characteristics previous published. Paired t-test to evaluate the effects of the Crizanlizumab on RBCs adherent to activated EC has been used. Results: We observed a statistically significant reduction of RBC adhesion due to Crizanlizumab treatment in 15-minutes acutely heme activated ECs (135±40 vs 1513±617, p≤0.05). The effect of Crizanlizumab on decreasing RBC adhesion was also statistically significant when ECs were chronically pre-activated by TNF-α followed by a 15-minute heme activation (4404±1393 vs 2016±609, p≤0.05). To further elaborate on the effects of Crizanlizumab in the atherogenic SCD population, we activated the EC with subjects' autologous plasma for 4 hours followed by a 15-minute heme activation, (5876±2579 vs 2397±1381p≤0.05). Discussion and Conclusions: Per label use of Crizanlizumab to reduce VOC, application of novel MFP associated with Crizanlizumab use displayed a reduced RBC adhesion to acutely heme-activated EC. Effects were preserved in presence or absence of chronic TNFα or autologous plasma pre-activation. Endothelium-on-a-chip MFP has been shown as reliable in monitoring patient response to anti-adhesive therapies in SCD population. Fig 1. Endothelium-on-a-chip MFP set up (A). The phase-contrast image of HUVECs demonstrate a confluent layer of cells aligned with flow. (C) RBCs adhesion to activated HUVECs. Effects of Crizanlizumab on RBC adhesion levels to HUVECs activated for 15-minutes with heme (D); 4-hours with TNF-α and 15-minutes with heme (E); 4-hours with plasma and 15-minutes with heme(F). Microscope images at 10X. Scale bare 100um. Error bars represent the standard error of the mean (SEM). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P-030: CRIZANLIZUMAB REDUCES ENDOTHELIAL ADHESION OF RED BLOOD CELLS FROM SCD PATIENTS IN STANDARDIZED MICROFLUIDIC PLATFORM: IN VITRO TECHNOLOGY ASSESSMENT

Purpose: Crizanlizumab (ADAKVEO®, Novartis, Switzerland) a monoclonal P-selectin blocking antibody designed to disrupt the blood cells and endothelium interactions in sickle cell disease (SCD) patients, which leads to reduction of vaso-occlusive crises, the major manifestation of the disease. We report the novel microfluidic platform (endothelium-on-a-chip), to test the effects of Crizanlizumab on the adhesion of red blood cells (RBCs) to perfusion-cultured, acutely and chronically activated human endothelial cells (ECs). Materials and methods: Whole blood samples collected from SCD subjects (n=21), 20 HbSS and 1 HbSC in EDTA and sodium citrate. RBCs were isolated via centrifugation from whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were cultured within the microfluidic platform channels at 15 dyne/cm2 for at least 48 hours prior to experiments. To mimic chronic pre-activation in SCD HUVECs were pretreated for 4 hours with heme (40 µM), TNFα (20 ng/ml) or 50% plasma of SCD patients in basal media, +/- 100 µg/ml Crizanlizumab followed by injection of blood samples through the microfluidic channels. For acute EC activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml Crizanlizumab and injected through the microfluidic channels for 15 minutes. Thereafter, non-adherent RBCs were rinsed via washing solution with or without Crizanlizumab and the remaining RBCs were quantified based on previous published methods. Paired t-test was used to calculate statistical significance. Results: The inhibitory effects of Crizanlizumab on blood cell adhesion to ECs was linked to the type and duration of EC activation (Fig. 1). Crizanlizumab slightly reduced RBC adhesion to 4-hours heme activated ECs (1170±413vs1671±522 p>0.05). Reduction of RBC adhesion due to Crizanlizumab treatment was significant in 15-minutes heme activated ECs (135±40 vs 1513±617, p≤0.05). The effect of Crizanlizumab on decreasing RBC adhesion was significant when ECs were pre-activated by TNF-α (4404±1393 vs 2016±609, p≤0.05) or subjects’ autologous plasma for 4 hours followed by a 15-minute heme activation (5876±2579 vs 2397±1381p≤0.05). Conclusion: In accordance with the label of Crizanlizumab to reduce VOC, application of microfluidic platform associated with Crizanlizumab use displayed a reduced RBC adhesion to acutely heme-activated EC. Effects were preserved in presence or absence of chronic TNFα or autologous plasma pre-activation. Endothelium-on-a-chip microfluidic platform has been shown as reliable in monitoring patient response to anti-adhesive therapies in SCD.Effects of Crizanlizumab on RBC adhesion levels to HUVECs activated for (A) 4-hours with heme (B) 15-minutes with heme (C) 4-hours with TNF-? and 15-minutes with heme, (D) 4-hours with plasma and 15-minutes with heme. Error bars represent the standard err J. ZAK declares a conflict of interest: Research support/Scientific studies: I am the Administrative Officer on several federal grants that support the development of the BioChip Labs assay platform: NIH phase I & II grant NSF phase I grant State of Ohio TVSF grant Stock shareholder: I am the founder of BioChip Labs Inc. and own a majority stake in the company Other: I hold the administrative title of CEO at BioChip Labs Inc. U. GURKAN declares a conflict of interest: Consultancy, Expert: Hemex Health Inc, BioChip Labs Inc Patent or product inventor: Hemex Health Inc, BioChip Labs Inc, Xatek Inc, DxNow Inc Research support/Scientific studies: Hemex Health Inc, BioChip Labs Inc, Stock shareholder: Hemex Health Inc, BioChip Labs Inc, Xatek Inc, DxNow Inc

Late phase endothelial cell inflammation is characterized by interferon response genes and driven by JAK/STAT, not NFκB

Chronic vascular inflammation underlies many diseases, including atherosclerosis, autoimmune vasculitides and transplant rejection. The resolution of inflammation is critical for proper healing and restoration of homeostasis, but the timing and signaling mechanisms involved in the return to a non-inflamed state are not well understood. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells was measured in primary human aortic endothelium under chronic TNFα stimulation, and after short-term TNFα priming followed by withdrawal. The effects of NFκB, MAPK and JAK1/2 inhibitors on TNFα-induced gene expression were tested. The majority of inducible TNFα effectors, such as E-selectin, VCAM-1 and most chemokines, required continuous exposure for reinforcement of the altered phenotype, and were NFκB dependent. However, 3 h priming with TNFα induced late phase STAT activation and interferon response genes after 18 h, as well as enhanced ICAM-1, BST2 and CXCR3 ligand expression. Chronic activation was autonomous of continuous TNFα, and could be blocked by the JAK1/2 inhibitor ruxolitinib. The results demonstrate that NFκB is not a significant driver of the later phase of endothelial cell activation by TNFα, but that sustained inflammation is JAK1/2-dependent and characterized by adaptive chemokines.

TNF-α blockade improves immunotherapy efficacy by altering the tumor microenvironment and enhancing tumor-specific T cell function in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a particularly lethal malignancy with a 5-year survival rate of 9%. A recent phase 1 clinical trial suggests CD40 agonist has antitumor activity in some patients. We developed an orthotopic PDA mouse model to track tumor specific CD8 T cells, identify critical antitumor mechanisms, and determine pathways of immunotherapy resistance. Here, we exploit this model to uncover a novel combination immunotherapy that includes CD40 agonist, PD-L1 blockade and TNF-α neutralization (e.g., 4PT). Interfering with TNF-α significantly improves overall mouse survival and cure rate compared to CD40+PDL1 only (4P). Critically, 4PT enhanced the generation of tumor-specific long-lived effector and central memory T cells. TNF-α neutralization significantly reduced T cell exhaustion, as indicated by reduced Lag-3 and increased IFN-γ production by intratumoral tetramer+ CD8 T cells. Additionally, 4PT increased CD4+Foxp3− T cell frequency and decreased CD4+Foxp3+ T cells as compared to 4P-treated mice, consistent with enhanced antitumor CD4 T cell reactivity in the absence of chronic TNF-α signaling. Lastly, abrogating Tnfr1 significantly reduced splenic and intratumoral Ly6G+ granulocytes following 4P. Thus, disrupting TNF-α via genetic deletion or monoclonal antibodies alters the tumor microenvironment to promote highly functional tumor-specific CD8 T cells. We conclude that perturbation of TNF-α-mediated chronic inflammation is an appealing approach to enhance immunotherapy efficacy for pancreatic cancer patient treatment. Supported by NIH 1R01CA249393-01A1

Presence of Tumor Necrosis Factor-Alpha in Urine Samples of Patients With Chronic Low Back Pain Undergoing Chiropractic Care: Preliminary Findings From a Prospective Cohort Study.

Background and aimsLow back pain is the leading cause of years lived with disability worldwide. Chiropractors employ different interventions to treat low back pain, including spinal manipulative therapy, although the mechanisms through which chiropractic care improves low back pain are still unclear. Clinical research and animal models suggest that spinal manipulation might modulate plasma levels of inflammatory cytokines, which have been involved in different stages of low back pain. More specifically, serum levels of Tumor Necrosis Factor-alpha (TNF-α) have been found to be elevated in patients with chronic low back pain. We aimed to investigate whether urine from chronic low back pain patients could be an appropriate medium to measure concentrations of TNF-α and to examine possible changes in its levels associated to chiropractic care.MethodsUrine samples were collected from 24 patients with chronic low back pain and TNF-α levels were analyzed by ELISA before and after 4–6 weeks of care compared to a reference value obtained from 5 healthy control subjects, by means of a Welch’s t-test. Simultaneously, pain intensity and disability were also evaluated before and after care. Paired t-tests were used to compare mean pre and post urinary concentrations of TNF-α and clinical outcomes.ResultsSignificantly higher baseline levels of urinary TNF-α were observed in chronic low back pain patients when compared to our reference value (p < 0.001), which were significantly lower after the period of chiropractic treatment (p = 0.03). Moreover, these changes were accompanied by a significant reduction in pain and disability (both p < 0.001). However, levels of urinary TNF-α were not correlated with pain intensity nor disability.ConclusionThese results suggest that urine could be a good milieu to assess TNF-α changes, with potential clinical implications for the management of chronic low back pain.

Potential contribution of early endothelial progenitor cell (eEPC)-to-macrophage switching in the development of pulmonary plexogenic lesion

BackgroundPlexiform lesions, which have a dynamic appearance in structure and cellular composition, are the histological hallmark of severe pulmonary arterial hypertension in humans. The pathogenesis of the lesion development remains largely unknown, although it may be related to local inflammation and dysfunction in early progenitor endothelial cells (eEPCs). We tested the hypothesis that eEPCs contribute to the development of plexiform lesions by differentiating into macrophages in the setting of chronic inflammation.MethodsThe eEPC markers CD133 and VEGFR-2, macrophage lineage marker mannose receptor C-type 1 (MRC1), TNFα and nuclear factor erythroid 2-related factor 2 (Nrf2) in plexiform lesions in a broiler model were determined by immunohistochemistry. eEPCs derived from peripheral blood mononuclear cells were exposed to TNFα, and macrophage differentiation and angiogenic capacity of the cells were evaluated by phagocytotic and Matrigel plug assays, respectively. The role of Nrf2 in eEPC-to-macrophage transition as well as in MRC1 expression was also evaluated. Intratracheal installation of TNFα was conducted to determine the effect of local inflammation on the formation of plexiform lesions.ResultsCells composed of the early lesions have a typical eEPC phenotype whereas those in more mature lesions display molecular and morphological characteristics of macrophages. Increased TNFα production in plexiform lesions was observed with lesion progression. In vitro studies showed that chronic TNFα challenge directed eEPCs to macrophage differentiation accompanied by hyperactivation of Nrf2, a stress-responsive transcription factor. Nrf2 activation (Keap1 knockdown) caused a marked downregulation in CD133 but upregulation in MRC1 mRNA. Dual luciferase reporter assay demonstrated that Nrf2 binds to the promoter of MRC1 to trigger its expression. In good agreement with the in vitro observation, TNFα exposure induced macrophage differentiation of eEPCs in Matrigel plugs, resulting in reduced neovascularization of the plugs. Intratracheal installation of TNFα resulted in a significant increase in plexiform lesion density.ConclusionsThis work provides evidence suggesting that macrophage differentiation of eEPCs resulting from chronic inflammatory stimulation contributes to the development of plexiform lesions. Given the key role of Nrf2 in the phenotypic switching of eEPCs to macrophages, targeting this molecular might be beneficial for intervention of plexiform lesions.

622 TNFα in impaired diabetic wound healing: A role for GM3

Type 2 diabetes (T2D) is associated with chronic exposure to increases in tumor necrosis factor alpha (TNFα), which contributes to insulin resistance and the development of foot ulcers in 25% of affected individuals. The mechanism by which TNFα leads to insulin resistance and poor wound healing is unclear. We hypothesized that glycosphingolipid GM3 is a critical mediator of TNFα-induced pathology in diabetic skin. In undifferentiated, cultured normal human epidermal keratinocytes (NHEKs), chronic TNFα treatment (100 pM x 4 days) increased the expression of both GM3 (8-fold increase, P<0.001) and its synthesizing enzyme GM3 synthase (GM3S) (4-fold increase, P<0.01), as shown by lipidomics, flow-cytometry and RT-qPCR.

Regulators of TNF\u03b1 mediated insulin resistance elucidated by quantitative proteomics

Obesity is a growing epidemic worldwide and is a major risk factor for several chronic diseases, including diabetes, kidney disease, heart disease, and cancer. Obesity often leads to type 2 diabetes mellitus, via the increased production of proinflammatory cytokines such as tumor necrosis factor-α (TNFα). Our study combines different proteomic techniques to investigate the changes in the global proteome, secretome and phosphoproteome of adipocytes under chronic inflammation condition, as well as fundamental cross-talks between different cellular pathways regulated by chronic TNFα exposure. Our results show that many key regulator proteins of the canonical and non-canonical NF-κB pathways, such as Nfkb2, and its downstream effectors, including Csf-1 and Lgals3bp, directly involved in leukocyte migration and invasion, were significantly upregulated at the intra and extracellular proteomes suggesting the progression of inflammation. Our data provides evidence of several key proteins that play a role in the development of insulin resistance.

Modulation of Epidermal γδ T Cell Functionality by Obesity due to Acute versus Chronic TNF-α Reception

Abstract Epidermal γδ T cells exhibit functional plasticity with roles varying from wound healing to tumor cell lysis. Activation of epidermal γδ T cells occurs via the γδ TCR along with costimulation, suggesting that costimulatory ligand expression regulates functional outcome. Data from our lab indicates that the cytokine milieu adds an additional pressure on epidermal γδ T cells to mediate or inhibit a particular functionality. Previous work from our lab shows that obesity impairs epidermal γδ T cell wound repair function in a TNF-α dependent manner. Here we identify TNF-α as a modulator of epidermal γδ T cell function in wound repair or cellular lysis depending on reception of acute or chronic signals. TNF-α signals through two receptors, tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), thus the signaling dynamics are complex and induce varied responses including apoptosis, survival and inflammation. We show that epidermal γδ T cells exhibit low TNFR1 and TNFR2 expression prior to TCR activation. Even with low TNFR expression epidermal γδ T cells produce cytokines and chemokines involved in inflammation and macrophage recruitment in response to acute TNF-α signals without TCR activation. During TCR activation or IL-2 stimulation epidermal γδ T cells to upregulate TNFR2 indicating a reliance on TNFR2 signaling during activation. Alternatively, chronic TNF-α stimulation, similar to levels in obese patients, induces a reduction in epidermal γδ T cell cytotoxicity, proliferation and viability. Normal function is restored with IL-2 addition suggesting the inhibition is not permanent, but instead can be modulated. These studies provide potential targets for regulating epidermal γδ T cell functions such as wound repair and tumor lysis.

Interactions among glomerulus infiltrating macrophages and intrinsic cells via cytokines in chronic lupus glomerulonephritis

Inflammation plays a key role in the pathogenesis of lupus nephritis (LN) and inflammatory cytokines within the glomeruli are critical in this process. However, little information is available for the identities of the cell types that are primarily responsible for the production and function of the various cytokines. We have devised a novel method to visualize cytokine signals in the kidney by confocal microscopy and found that cytokine production within the glomerulus is cell type-specific and under translational control. In the lupus-prone NZM2328 mice with chronic glomerulonephritis, IL-6, IL-1β, and TNF-α in the glomerulus were produced predominantly by mesangial cells, podocytes, and glomerulus-infiltrating blood-derived macrophages, respectively. Microarray and RNASeq analyses showed that these cells expressed the receptors for these cytokines. Together the 3 cell types form a cytokine circuit in amplifying cytokine responses in LN. The intrinsic cells and infiltrating macrophages also produced other cytokines including M-CSF, SCF, and IL-34 that constituted within the enclosed glomerular space the soluble effector milieu which may mediate cellular damage and proliferation, and cytokine transcriptional and translation regulation. IL-10 and IL-1β were translationally regulated in the glomeruli in the intact kidney in a cell type-specific manner. The production of these 2 cytokines by infiltrating macrophages was undetectable in a visualization system for in situ protein accumulation despite high mRNA expression levels. However, these macrophages in isolated glomeruli which are released from Bowman's capsules produced large amounts of IL-10 and IL-1β. These data reveal the complexity of cytokine regulation, production, and function in the glomerulus and provide a model in which cytokine blocking may be beneficial in LN treatment.

Tumor Necrosis Factor-Alpha Alters Electrophysiological Properties of Rabbit Hippocampal Neurons.

Previous studies have shown tumor necrosis factor-alpha (TNF-α) may impact neurodegeneration in Alzheimer's disease (AD) by regulating amyloid-β and tau pathogenesis. However, it is unclear whether TNF-α has a role in a cholesterol-fed rabbit model of AD or TNF-α affects the electrophysiological properties of rabbit hippocampus. This study was designed to investigate whether long-term feeding of cholesterol diet known to induce AD pathology regulates TNF-α expression in the hippocampus and whether TNF-α would modulate electrophysiological properties of rabbit hippocampal CA1 neurons. TNF-α ELISA showed dietary cholesterol increased hippocampal TNF-α expression in a dose-dependent manner. Whole-cell recordings revealed TNF-α altered the membrane properties of rabbit hippocampal CA1 neurons, which was characterized by a decrease in after-hyperpolarization amplitudes; Field potential recordings showed TNF-α inhibited long-term potentiation but did not influence presynaptic function. Interestingly, TNF-α did not significantly affect the after-hyperpolarization amplitudes of hippocampal CA1 neurons from cholesterol fed rabbits compared to normal chow fed rabbits. In conclusion, dietary cholesterol generated an in vivo model of chronic TNF-α elevation and TNF-α may underlie the learning and memory changes previously seen in the rabbit model of AD by acting as a bridge between dietary cholesterol and brain function and directly modulating the electrophysiological properties of hippocampal CA1 neurons.

Chronic TNFα-driven injury delays cell migration to villi in the intestinal epithelium.

The intestinal epithelium is a single layer of cells which provides the first line of defence of the intestinal mucosa to bacterial infection. Cohesion of this physical barrier is supported by renewal of epithelial stem cells, residing in invaginations called crypts, and by crypt cell migration onto protrusions called villi; dysregulation of such mechanisms may render the gut susceptible to chronic inflammation. The impact that excessive or misplaced epithelial cell death may have on villus cell migration is currently unknown. We integrated cell-tracking methods with computational models to determine how epithelial homeostasis is affected by acute and chronic TNFα-driven epithelial cell death. Parameter inference reveals that acute inflammatory cell death has a transient effect on epithelial cell dynamics, whereas cell death caused by chronic elevated TNFα causes a delay in the accumulation of labelled cells onto the villus compared to the control. Such a delay may be reproduced by using a cell-based model to simulate the dynamics of each cell in a crypt-villus geometry, showing that a prolonged increase in cell death slows the migration of cells from the crypt to the villus. This investigation highlights which injuries (acute or chronic) may be regenerated and which cause disruption of healthy epithelial homeostasis.

Association of tumor necrosis factor alpha gene polymorphisms with Helicobacter pylori infection in dyspeptic patients in Sri Lanka.

Single nucleotide polymorphisms present on the promoter sequence of the TNF-α gene may affect production of TNF-α, a pro-inflammatory cytokine, during immune responses. The presence of TNF-α polymorphisms is also reportedly associated with more severe manifestations of Helicobacter pylori infection. However, the frequency of TNF-α polymorphisms and the associated disease severity vary between different patient groups. In this study, gastric biopsies and blood specimens were collected from 138 patients with dyspepsia undergoing routine upper gastrointestinal endoscopy. Our institution's Ethics Review Committee approved the study and written informed consent was obtained from all participants. The presence of H. pylori was confirmed histologically in all patients. The frequency of TNF-α polymorphisms in the study cohort was investigated using PCR-restriction fragment length polymorphism and expression of serum TNF-α quantitated using a commercial ELISA assay. The proportions of selected TNF-α polymorphisms (TNF-α -238, -308 and -863) were similar in H. pylori-positive and -negative patients. Homozygous mutations of TNF-α polymorphisms were rarely detected in the study group. There was a significant difference in TNF-α concentrations between patients with mild chronic gastritis and TNF-α -308 GG genotype and patients with moderate to severe chronic gastritis (P = 0.008). It was not possible to identify an association between these genotypes and disease severity because of the low frequency of heterozygous and homozygous mutated genes in Sri Lankan patients with dyspepsia.

An inflammatory environment containing TNFα favors Tet2-mutant clonal hematopoiesis

Clonal hematopoiesis of aging and indeterminate potential (ARCH or CHIP), driven mainly by mutations in DNMT3A and TET2, is an emerging public health issue, affecting at least 10-15% of adults older than 65 years. CHIP is associated with increased risks of de novo and therapy-related hematological neoplasms and serves as a reservoir for leukemic relapse. CHIP is also associated with increased all-cause mortality and risk of cardio-metabolic disease. The latter association may be explained, at least in part, by the effects of inactivating mutations in TET2 on progeny macrophages. We and others have shown recently that TET2-deficient macrophages are hyperinflammatory and this may exacerbate processes such as atherosclerosis. We postulated an inflammatory state associated with TET2 inactivation and/or unhealthy aging may also favor TET2-mutant hematopoietic stem and progenitor cell (HSPC) expansion. Herein, we demonstrate a clonogenic advantage for Tet2-knockout murine and TET2-mutant human HSPCs in an in vitro environment that contains the proinflammatory cytokine tumor necrosis factor-alpha (TNFα). This phenotype emerges on chronic TNFα exposure and is associated with myeloid skewing and resistance to apoptosis. To our knowledge, this is the first evidence to suggest that TET2 mutations promote clonal dominance with aging by conferring TNFα resistance to sensitive bone marrow progenitors while also propagating such an inflammatory environment. Normalizing the immune environment may present a novel strategy to control or eradicate mutant CHIP clones.

Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling

Inflammation influences cancer development, progression, and the efficacy of cancer treatments, yet the mechanisms by which immune signaling drives alterations in the cancer cell transcriptome remain unclear. Using ChIP-seq, RNA-seq, and GRO-seq, here we demonstrate a global overlap in the binding of tumor-promoting p53 mutants and the master proinflammatory regulator NFκB that drives alterations in enhancer and gene activation in response to chronic TNF-α signaling. We show that p53 mutants interact directly with NFκB and that both factors impact the other’s binding at diverse sets of active enhancers. In turn, the simultaneous and cooperative binding of these factors is required to regulate RNAPII recruitment, the synthesis of enhancer RNAs, and the activation of tumor-promoting genes. Collectively, these findings establish a mechanism by which chronic TNF-α signaling orchestrates a functional interplay between mutant p53 and NFκB that underlies altered patterns of cancer-promoting gene expression.

Association of TNF-α Gene Polymorphisms with Production of Protein and Susceptibility to Chronic Hepatitis B Infection in the South East Iranian Population.

BackgroundThe host genetic background regulates the natural history of chronic hepatitis B virus (HBV) infection.ObjectivesThe aim of this study was to investigate the association between TNF-α gene polymorphism in the promoter region and susceptibility to chronic hepatitis B virus infection.MethodsFour polymorphisms of TNF-α gene including -238 A/G, -308 A/G, -857 C/T, and -863 A/C were analyzed by PCR-RFLP in 100 chronic HBV infected patients (HBV group), 40 spontaneously recovered HBV subjects (SR group), and 100 healthy controls (C group). Also, serum levels of protein were monitored.ResultsThe study showed that the existence of -308 G, -857 C, and -863 A alleles significantly increased susceptibility to chronic HBV infection. In addition, GGCA haplotype had a higher frequency in HBV patients than C and SR groups that might be related to the natural history of the infection. Chronic HBV patients with -308 GG, -857 CC, and -863 AA genotypes had lower serum levels of TNF-α compared to those with other genotypes.ConclusionsThe results indicated that there was a positive association between susceptibility to chronic HBV infection and TNF-α polymorphism. In addition, HBV patients carrying -308 GG, -857 CC, and -863 AA genotypes with lower serum levels of TNF-α had an increased risk of infection.

Pharmacological inhibition of protein kinase C (PKC)ζ downregulates the expression of cytokines involved in the pathogenesis of chronic obstructive pulmonary disease (COPD)

The protein kinase PKCζ is involved in the fine regulation of the NF-κB transcriptional activity and, therefore, represents a potential pharmacological target in inflammatory diseases. We previously developed a selective, allosteric inhibitor (MA130) of PKCζ. Now, we investigated which of the NF-κB–regulated gene expressions are suppressed by MA130 after TNFα-stimulation of the macrophage model cell line U937. The analysis of gene expressions using a qPCR array revealed that many cytokines contributing to the pathogenesis and systemic inflammation in chronic obstructive pulmonary disease (COPD), including CCL2, CCL20, CSF2, CXCL1, CXCL10, IL1B and TNFα, were down-regulated by MA130 but not by a PKCζ–inactive control compound. Thus, we provided the first evidence that PKCζ is a potential target for the treatment of COPD by selective small molecules. MA130 inhibited only a subset of NF-κB–dependent gene expressions, suggesting that targeting PKCζ will be more tolerable than total inhibition of NF-κB activation.

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction.

Sclerostin, an inhibitor of the Wnt/β-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities.

Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels.

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.