TNF-α Receptor Expression Research Articles

MPFC TNFR2 modulated the impairment of cognitive flexibility induced by adolescent social stress and the relevant neuroplastic molecules

The impairment of cognitive flexibility (CF) exists to varying degrees in several psychiatric disorders and is increasingly recognized as the important etiological and pathological factors in these disorders. Our previous research has demonstrated that adolescent social stress (ASS) can lead to cognitive dysfunction in adult mice, accompanied by immune changes in the medial prefrontal cortex (mPFC), mainly manifested by reduced levels of cytokine TNFα. The present study aimed to further investigate the mechanisms of TNFα receptor and the downstream neuroplasticity-related molecules involved in cognitive dysfunction induced by ASS. Cognitive flexibility was assessed using Attentional Set Shifting Task (AST). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) methods were use to determine mRNA and protein level of TNFα receptor (TNFR) and downstream signaling molecules, including nuclear transcript factor NF-κB and CREB, pCREB and Brain-derived neurotrophic factor (BDNF). We found that ASS significantly decreased the mRNA and protein expression of TNFα receptor 2 (TNFR2), but not TNFR1 of the mPFC in adult mice. Direct up-regulation TNFR2 expression by microinjection of adeno-associated virus (AAV) encoding TNFR2 into the mPFC reversed the impairment of CF and the decrease of BDNF protein levels in stressed adult mice compared to that in controls. These findings demonstrated that mPFC TNFR2 plays critical roles in cognitive inflexibility induced by ASS, which effects may be mediated by neuroplastic molecules, and could be a promising target for treating cognitive dysfunction in psychiatric disorders.

TNF-α activates RELA expression via TNFRSF1B to upregulate OPA1 expression and inhibit chondrogenic differentiation of human adipose stem cells

BackgroundTumor necrosis factor-alpha (TNF-α), one of the pro-inflammatory cytokines mediating the local inflammatory process in joints, inhibits cartilage formation and has a detrimental effect on stem cell-based cartilage regeneration for the treatment of osteoarthritis (OA). However, the mechanisms behind this inhibitory effect are still poorly understood. Mitochondrial morphological changes mediated by mitochondrial fusion and fission are highly plastic, are quite sensitive to environmental stimuli and play a crucial role in maintaining cell structure and function. In our study, chondrogenic differentiated human adipose stem cells (hADSCs) were exposed to TNF-α and the effect of TNF-α on the ability of hADSCs to chondrogenic differentiate and on mitochondrial fusion and fission was observed and analyzed. The aim was to investigate the role and mechanisms of mitochondrial fusion and fission regulation in the chondrogenic differentiation of hADSCs under normal conditions and under exposure to TNF-α.MethodsWe used flow cytometry to identify hADSCs immunophenotypes CD29, CD44, CD34, CD45, and HLA-DR. Alcian blue staining and Sirius red staining were used to observe the formation of proteoglycans and collagen during the chondrogenic differentiation of hADSCs, respectively. The mRNA and protein expression levels of the cartilage formation marker SOX9, type II collagen (COL2A1), and Aggrecan were measured by real-time fluorescent quantitative PCR (RT-qPCR) and western blot, respectively. The fluorescent probes MitoTracker® Red CMXRos and JC-1 were used to visualize mitochondria morphology and detect mitochondrial membrane electricity (MMP). Affymetrix PrimeView™ chips were used for gene expression profiling.ResultsThe results showed that the chondrogenic differentiation of hADSCs was inhibited in the presence of TNF-α that optic atrophy 1 (OPA1) expression was significantly upregulated and mitochondria were prolonged and interconnected during this process. Gene microarray and RT-qPCR data showed that the presence of TNF-α led to increased expression of TNFα receptor 2 (TNFRSF1B) and RELA during chondrogenic differentiation of hADSCs.ConclusionsTNF-α inhibits chondrogenic differentiation of human adipose stem cells by activating RELA expression through TNFRSF1B upregulating OPA1 expression thereby increasing mitochondrial fusion.

Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

BackgroundMedulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive.MethodsBy taking advantage of Math1-Cre/Ptch1loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo.ResultsWe found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo.ConclusionsC3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.

Dexmedetomidine Activates Akt, STAT6 and IRF4 Modulating Cytoprotection and Macrophage Anti-Inflammatory Phenotype Against Acute Lung Injury in vivo and in vitro.

PurposeThis study aims to investigate the cytoprotective and anti-inflammatory effects of an α2-adrenoreceptor (α2-AR) agonist, dexmedetomidine (Dex), on lipopolysaccharides (LPS)-induced acute lung injury and underlying mechanisms with focus on alveolar macrophage polarization modulation.MethodsC57BL/6 mice were intraperitoneally injected LPS (10 mg/kg) with or without Dex (25 µg/kg) and/or α2-AR antagonist atipamezole (Atip, 500 µg/kg). Lung tissues were then analysed to determine injuries. In vitro, human pulmonary epithelial cells (A549) and mice alveolar macrophages (MH-S) were exposed to LPS (10 ng/mL) with or without different concentrations of Dex (0.1–100 nM). Alveolar macrophage polarization, NLRP3 inflammasome activation and inflammatory responses were determined. PTEN/Akt signaling and its downstream transcriptional factors as targets for macrophage polarization were assessed.ResultsDex treatment significantly reduced pro-inflammatory M1 macrophage polarization and NLRP3 inflammasome activation in the lungs relative to the mice treated with LPS. The similar pattern reduction of NLRP3 inflammasome activation by Dex was also found in A549 cells. Atip partly reversed the anti-inflammatory effects of Dex. In cultured alveolar macrophages, Dex reduced LPS-mediated expression of IL-1, −6 and TNF-α receptors while promoting alveolar macrophages differentiation towards a M2 anti-inflammatory phenotype. Additionally, LPS increased Akt signaling activation in a time-dependent manner, which was further activated by Dex via inhibiting phosphatase and tensin homolog (PTEN). The action of Dex on Akt signaling shifted alveolar macrophages from M1 to M2 phenotype through increasing STAT6 and IRF4 transcriptional factors.ConclusionDex protected against LPS-induced lung injury and suppressed LPS-induced pulmonary inflammatory responses by attenuating the NLRP3 inflammasome activation and promoting anti-inflammatory M2 macrophage polarization.

Effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha receptor 1 expression, granulosa cell apoptosis, and folliculogenesis repair in endometriosis mouse models.

Background and Aim:Endometriosis affects the ovaries and causes a decrease in the oocyte quality during endometrial receptivity. During the development of ovarian follicles, paracrine communication occurs between granulosa cells and oocytes. This study was conducted to determine the effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha (TNF-α) receptor 1 (TNFR1) expression, granulosa cell apoptosis, and folliculogenesis in endometriosis mouse models.Materials and Methods:This study involved 42 female mice, which were divided into three groups: Healthy mice (T0), endometriosis mice without transplantation (T1), and endometriosis mice with bone marrow mesenchymal stem cell transplantation (T2). The mice were injected intraperitoneally with endometrial fragments (200 μL) to become endometriosis models. On day 15, the endometriosis models received mesenchymal stem cells. Sample collection was performed on day 29. Granulosa cell apoptosis and TNFR1 expression were examined using immunohistochemical staining, and folliculogenesis was assessed using hematoxylin and eosin staining of ovary samples. The data obtained from both examinations were statistically analyzed using Statistical Package for the Social Sciences.Results:The results showed that TNFR1 expression is significantly decreased in T2 (p<0.004). The apoptosis of granulosa cells was lower in T2 (p<0.000). The primary, secondary, and graafian follicle counts in T2 were significantly increased.Conclusion:Bone marrow mesenchymal stem cell transplantation in endometriosis mouse models can reduce TNFR1 expression and granulosa cell apoptosis and improve folliculogenesis.

ERα-dependent estrogen-TNFα signaling crosstalk increases cisplatin tolerance and migration of lung adenocarcinoma cells

Lung adenocarcinoma is the most common type of lung cancer in women. Our previous studies demonstrated that 17β-estradiol (E2) promoted lung adenocarcinoma cell proliferation and tumor growth through estrogen receptor ERα. Transcriptomic analysis suggested that E2 potentiated TNFα-NFκB signaling in ERα-expressing lung adenocarcinoma cells. This study further demonstrated that E2 increased TNFα receptor expression and TNFα-triggered NFκB activity in ERα-expressing cells. E2-activated ERα had no physical association with NFκB p65/p50 heterodimer but facilitated TNFα-initiated IκBα degradation, NFκB nuclear translocation, and S468/S536 phosphorylation of p65 essential for NFκB activity. While knockdown of ERα prevented E2 from boosting NFκB activity, antiestrogen ICI 182,780 stimulated NFκB activity like E2. Inhibition of GSK3β hampered E2:ERα-promoted NFκB activity and abolished S468 phosphorylation of p65, suggesting that GSK3β played a role in the E2-TNFα signaling crosstalk. In ERα-expressing cells, E2 and TNFα synergistically regulated many genes that were not typically responsive to either E2 or TNFα. Functional analysis of microarray data inferred that E2/TNFα-induced transcriptomic changes improved cell survival and movement. Viability and colony formation assays validated that E2 and TNFα together increased cisplatin tolerance of ERα-expressing cells. Wound healing assays also confirmed that E2/TNFα cotreatment increased cell migration in an ERα-dependent manner. E2/TNFα-induced dysregulation of genes such as cell survival and movement-associated genes, proto-oncogenes, metallothioneins and histone core genes was correlated with poor overall survival in patients. In summary, E2 and TNFα engaged in an ERα-dependent positive crosstalk in lung adenocarcinoma cells, consequently increasing NFκB activation, cisplatin tolerance and cell migration and worsening prognosis.

Spent Hen Muscle Protein-Derived RAS Regulating Peptides Show Antioxidant Activity in Vascular Cells.

Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)—upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR-dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.

Respiratory Burst and TNF-α Receptor Expression of Neutrophils after Sepsis and Severe Injury-Induced Inflammation in Children.

Systemic inflammatory response syndrome (SIRS) is defined as the systemic host response to infection or a non-infectious factor. The purpose of this study was to evaluate the involvement of reactive oxygen species (ROS) in severe inflammation and to assess the discrimination strength of the neutrophil BURSTTEST assay regarding its etiology in three groups of patients (sepsis, burns, and bone fractures) who met the SIRS criteria. The neutrophil activation (respiratory burst of granulocytes as well as p55 and p75 tumor necrosis factor (TNF-α) receptor expression) was evaluated twice using flow cytometry, and the results were compared with healthy controls and among SIRS subjects. A decreased oxygen metabolism in neutrophils after E. coli stimulation and increased TNF-α receptor expression were found in septic and burned patients on admission, while ROS production augmented and TNF-α receptor expression diminished with the applied therapy. The significant differences in neutrophil respiratory burst intensity among septic and burned patients and those with sepsis and bone fractures were found (however, there were not any such differences between patients with thermal and mechanical injuries). This study indicates that the neutrophil BURSTTEST evaluation might be a clinically reliable marker for differentiating the SIRS etiology.

In vitro Evaluation of the Safety of Adalimumab for the Eye Under HTLV-1 Infection Status: A Preliminary Study.

Adalimumab (ADA), a fully human monoclonal tumor necrosis factor (TNF)-α antibody, is one of the most widely used biologics in the treatment of inflammatory diseases. However, ADA can exacerbate infectious conditions, induce paradoxical reactions such as inflammation, and cause neoplasia. Human T-cell leukemia virus type 1 (HTLV-1) is an infectious agent that induces inflammation and neoplastic infiltration in the eye. To date, numerous HTLV-1 carriers have been treated with adalimumab to suppress inflammation out of necessity, when standard anti-inflammatory drugs such as steroids and immunosuppressive agents have proven inadequate to control the inflammation. Here, we clarify the safety of adalimumab for the eye under HTLV-1 infectious conditions in vitro. We used the adult retinal pigment epithelial cell line (ARPE)-19 cell line as ocular resident cells, and used MT2 and TL-Om1 as HTLV-1-infected cells. ARPE-19 and MT2/TL-Om1 were co-cultured, and then adalimumab was administered. Production of cytokines and chemokines, TNF-α receptor (TNF-R), HTLV-1 proviral load (PVL), and apoptosis were measured to assess the effects of adalimumab. Contact between ARPE-19 and MT2/TL-Om1 produced inflammatory cytokines such as TNF, interleukin (IL)-6, IL-8 and IL-10, and transduced chemokines such as interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP-1), monokine induced by interferon-γ (MIG), and regulated on activation, normal T cell expressed and secreted (RANTES). No inflammatory cytokines and chemokines were exacerbated by adalimumab. Expression of TNF-R on ARPE-19 and MT2/TL-Om1 cells, HTLV-1 PVLs of MT2/TL-Om1 cells, and cell growth rate and apoptotic rate of ARPE-19 were unaffected by adalimumab. In conclusion, adalimumab does not appear to exacerbate HTLV-1-associated inflammatory conditions in the eye or increase PVL in HTLV-1-infected T cells. These data suggest that adalimumab could be used safely for the eye under HTLV-1 infectious conditions from the perspective of in vitro assessment.

Oxidative Stress and Inflammatory Modulation of Ca2+ Handling in Metabolic HFpEF-Related Left Atrial Cardiomyopathy.

Metabolic syndrome-mediated heart failure with preserved ejection fraction (HFpEF) is commonly accompanied by left atrial (LA) cardiomyopathy, significantly affecting morbidity and mortality. We evaluate the role of reactive oxygen species (ROS) and intrinsic inflammation (TNF-α, IL-10) related to dysfunctional Ca2+ homeostasis of LA cardiomyocytes in a rat model of metabolic HFpEF. ZFS-1 obese rats showed features of HFpEF and atrial cardiomyopathy in vivo: increased left ventricular (LV) mass, E/e’ and LA size and preserved LV ejection fraction. In vitro, LA cardiomyocytes exhibited more mitochondrial-fission (MitoTracker) and ROS-production (H2DCF). In wildtype (WT), pro-inflammatory TNF-α impaired cellular Ca2+ homeostasis, while anti-inflammatory IL-10 had no notable effect (confocal microscopy; Fluo-4). In HFpEF, TNF-α had no effect on Ca2+ homeostasis associated with decreased TNF-α receptor expression (western blot). In addition, IL-10 substantially improved Ca2+ release and reuptake, while IL-10 receptor-1 expression was unaltered. Oxidative stress in metabolic syndrome mediated LA cardiomyopathy was increased and anti-inflammatory treatment positively affected dysfunctional Ca2+ homeostasis. Our data indicates, that patients with HFpEF-related LA dysfunction might profit from IL-10 targeted therapy, which should be further explored in preclinical trials.

Effect of nicotine on placental inflammation and apoptosis in preeclampsia-like model

AimsPlacental tissues from patients with preeclampsia (PE) and in the lipopolysaccharide (LPS)-induced PE-like model were used to investigate the implication of placental inflammation and apoptosis in PE. Whether the beneficial effects of nicotine are related to inhibition of placental inflammation and apoptosis in the PE-like model were investigated. Main methodsPlacental apoptosis was detected in PE patients and the PE-like rat model by TUNEL staining. Changes in the number of CD68+ macrophages in placental tissues from PE patients were detected by immunofluorescent staining. The mRNA expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL-1β), MCP-1, and proteins involved in extrinsic or intrinsic apoptosis signaling in the PE-like model was determined by qRT-PCR; immunofluorescent staining was used to detect the expression of TNF-α receptor (TNFR1), MCP-1 and apoptosis-related proteins. Key findingsPlacental apoptosis was increased in both PE patients and the PE-like model, more macrophages infiltrated into placenta in PE patients. A significant upregulation in mRNA expression of TNF-α, IL-1β, MCP-1, and caspase 3, caspase 8, caspase 9 was found in the PE-like rats compared to the control animals, the immunoreactivity of placental MCP-1, TNFR1, and apoptosis-related proteins (caspase 3, caspase 8, caspase 9, Bax) was also enhanced; nicotine treatment significantly reversed those changes. SignificanceOur data suggests that the protective effects of nicotine are associated with inhibiting placenta inflammation and apoptosis, and nicotine might be a potentially therapeutic candidate for preventing preeclampsia.

Triglyceride is a Good Biomarker of Increased Injury Severity on a High Fat Diet Rat After Traumatic Brain Injury.

Injury severity is correlated with poor prognosis after traumatic brain injury (TBI). It is not known whether triglycerides (TGs) or total cholesterol (TC) is good biomarker of increased injury of neuroinflammation and apoptosis in a high fat diet (HFD)-treated rat after TBI episodes. Five-week-old male Sprague-Dawley (SD) rats were fed a HFD for 8weeks. The anesthetized male SD rats were divided into three sub-groups: sham-operated and TBI with 1.6atm or with 2.4atm fluid percussion injury (FPI). Cell infarction volume (triphenyltetrazolium chloride stain), tumor necrosis factor-alpha (TNF-α) expression in the microglia (OX42 marker) and astrocytes (Glial fibrillary acidic protein marker), TNF-α receptor expression in the neurons (TNFR1 and TNFR2 markers), and the extent of neuronal apoptosis (TUNEL marker) were evaluated by immunofluorescence, and the functional outcome was assessed by an inclined plane test. These tests were performed 72h after TBI. Serum triglyceride and cholesterol levels were measured at 24, 48 and 72h after TBI. The FPI with 2.4atm significantly increased body weight loss, infarction volume, neuronal apoptosis and TNF-α expression in the microglia and astrocytes, and it decreased the maximum grasp degree and TNFR1 and TNFR2 expression in neurons at the 3rd day following TBI. The serum TG level was positively correlated with FPI force, infarction volume, Neu-N-TUNEL, GFAP-TNFα, and OX42-TNFα Simultaneously; the serum TG level was negatively correlated with Neu-N-TNFR1 and Neu-N-TNFR2. TG is a good biomarker of increased injury for neuroinflammation and apoptosis at the 3rd day after TBI in HFD rats.

Restraint stress of male mice triggers apoptosis in spermatozoa and spermatogenic cells via activating the TNF-α system.

Studies have indicated that psychological stress impairs human fertility and that various stressors can induce apoptosis of testicular cells. However, the mechanisms by which psychological stress on males reduces semen quality and stressors induce apoptosis in testicular cells are largely unclear. Using a psychological (restraint) stress mouse model, we tested whether male psychological stress triggers apoptosis of spermatozoa and spermatogenic cells through activating tumour necrosis factor (TNF)-α signalling. Wild-type or TNF-α-/- male mice were restrained for 48 h before examination for apoptosis and expression of TNF-α and TNF receptor 1 (TNFR1) in spermatozoa, epididymis, seminiferous tubules and spermatogenic cells. The results showed that male restraint significantly decreased fertilization rate and mitochondrial membrane potential, while increasing levels of malondialdehyde, active caspase-3, TNF-α and TNFR1 in spermatozoa. Male restraint also increased apoptosis and expression of TNF-α and TNFR1 in caudae epididymides, seminiferous tubules and spermatogenic cells. Sperm quality was also significantly impaired when spermatozoa were recovered 35 days after male restraint. The restraint-induced damage to spermatozoa, epididymis and seminiferous tubules was significantly ameliorated in TNF-α-/- mice. Furthermore, incubation with soluble TNF-α significantly reduced sperm motility and fertilizing potential. Taken together, the results demonstrated that male psychological stress induces apoptosis in spermatozoa and spermatogenic cells through activating the TNF-α system and that the stress-induced apoptosis in spermatogenic cells can be translated into impaired quality in future spermatozoa.

Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets.

Introduction: Tumor necrosis factor (TNFα) is an important proinflammatory cytokine in rheumatoid arthritis (RA) immune processes. However, TNFα activity and functions may be regulated by soluble receptors, which act as decoys, and by number, density, and co-expression of its membrane-bound receptors type 1 and 2 (TNFR1 and TNFR2). The aim of this study was to reveal associations between TNFR1/2 co-expression profile parameters and RA disease activity indicators. Methods: PBMC were analyzed from 46 healthy donors and 64 patients with RA using flow cytometry. Patients were divided according to the disease activity score (DAS) 28 index into groups with high (n = 22, 34.4%), moderate (n = 30, 46.9%), and low (n = 12, 18.8%) disease activity. Co-expression of TNFR1 and TNFR2 was studied by evaluating the percentage of cells, with different receptors, and by counting the number of receptors of each type per cell, using QuantiBritePE beads. Associations between disease severity and activity indicators and parameters of TNFα receptor expression in subpopulations of immune cells were studied. Results: T cell subsets from RA patients were characterized by co-expression of TNFR1 and TNFR2, and were found to differ significantly compared with healthy donors. Memory cells both among T helper cells and cytotoxic T cells demonstrated the most significant differences in TNFR-expression profile. Multivariable logistic regression revealed model to identified RA patients from healthy individual based on the TNFR1/2 co-expression parameters. Conclusion: The profile of TNFR1\\2 co-expression differs in RA comparing with health. Proportion of TNFR1+TNFR2- cells increased significantly among memory T helper cells and activated cytotoxic T cells, and decreased significantly among naïve cytotoxic T cells and T regulatory cells as compared with health. The parameters of TNFR1\\2 co-expression in RA are associated with clinical and laboratory indicators of disease activity.

Abstract B072: MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer

Abstract Background: Activating mutations in BRAF, a key mediator of RAS signaling, are present in ~50% of melanoma patients. Pharmacological inhibition of BRAF or the downstream MAP kinase MEK are highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Immunotherapeutics, especially those targeting checkpoint receptors on T cells, have revolutionized treatment of many cancer types. It has been proposed that MEK inhibitors(MEKi) may help generate a tumor microenvironment that enhances response to immunotherapy. The aim of this study was to investigate a novel cross-talk mechanism between MEKi and cytokine signaling response which could be used to enhance therapeutic efficacy against cancer types that are minimally responsive to MEKi. Methods: The association between MEK inhibition, NF-κB signaling and clinical response was analyzed using publicly available RNA-sequencing data from pre-treatment and on-treatment biopsies of patients (EGA S00001000992). To identify clinical and pre-clinical compounds that enhance cytokine response, we performed drug library screening for CCL5 and CXCL10 expression. 289 different agents (0.1 mM and 1 mM) were incubated with A549 cells in 96 well plates, with or without the presence of 0.2 ng/ml TNFα and 1 ng/ml IFNγ. Chemokine levels in supernatant were detected using Bead-Based Multiplex Assays. To investigate cytokine response modulatory mechanisms of MEKi, qPCR, Western Blot and flow cytometry were used to determine NF-κB activity and targeting gene expressions in lung cancer cells lines. The anti-tumor effect of MEKi in combination with TNFα and IFNγ was assessed using viable cell counting, cell cycle assay and apoptosis marker (cleaved caspase 3) expression in vitro. For in vivo studies, we performed combinatory therapies using MEKi plus intratumoral cytokine injection, MEKi plus PD-1 blockade in LKR subcutaneous tumor model. Results: We show that treatment of melanoma patients with BRAF and MEK inhibitors activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. MEKi increased cell surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Drug screening results demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the anti-tumor efficacy of MEKi while the administration of TNFα and IFNγ in MEKi-treated mice enhanced the anti-tumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. Conclusions:These findings define a novel cytokine response modulatory function of MEKi which can be therapeutically exploited. We show that lung cancer cells are rendered sensitive to MEKi by TNFα and IFNγ, providing strong mechanistic rationale for combining MEKi with immunotherapeutics, such as checkpoint blockers, in lung cancer. Citation Format: Mengyu Xie, Hong Zheng, Ranjna Madan-Lala, Wenjie Dai, Nicholas T. Gimbrone, Zhihua Chen, Fumi Kinose, Sarah A. Blackstone, Keiran S. M. Smalley, Douglas Cress, Eric B. Haura, Uwe Rix, Amer A. Beg. MEK inhibition modulates cytokine response to mediate therapeutic efficacy in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B072. doi:10.1158/1535-7163.TARG-19-B072

Transcriptomic Analysis of Stem Cells Treated with Moringin or Cannabidiol: Analogies and Differences in Inflammation Pathways.

Inflammation is a common feature of many neurodegenerative diseases. The treatment of stem cells as a therapeutic approach to repair damage in the central nervous system represents a valid alternative. In this study, using Next-Generation Sequencing (NGS) technology, we analyzed the transcriptomic profile of human Gingival Mesenchymal Stem Cells (hGMSCs) treated with Moringin [4-(α-l-ramanosyloxy)-benzyl isothiocyanate] (hGMSCs-MOR) or with Cannabidiol (hGMSCs-CBD) at dose of 0.5 or 5 µM, respectively. Moreover, we compared their transcriptomic profiles in order to evaluate analogies and differences in pro- and anti-inflammatory pathways. The hGMSCs-MOR selectively downregulate TNF-α signaling from the beginning, reducing the expression of TNF-α receptor while hGMSCs-CBD limit its activity after the process started. The treatment with CBD downregulates the pro-inflammatory pathway mediated by the IL-1 family, including its receptor while MOR is less efficient. Furthermore, both the treatments are efficient in the IL-6 signaling. In particular, CBD reduces the effect of the pro-inflammatory JAK/STAT pathway while MOR enhances the pro-survival PI3K/AKT/mTOR. In addition, both hGMSCs-MOR and hGMSCs-CBD improve the anti-inflammatory activity enhancing the TGF-β pathway.

MEK Inhibition Modulates Cytokine Response to Mediate Therapeutic Efficacy in Lung Cancer.

Activating mutations in BRAF, a key mediator of RAS signaling, are present in approximately 50% of melanoma patients. Pharmacologic inhibition of BRAF or the downstream MAP kinase MEK is highly effective in treating BRAF-mutant melanoma. In contrast, RAS pathway inhibitors have been less effective in treating epithelial malignancies, such as lung cancer. Here, we show that treatment of melanoma patients with BRAF and MEK inhibitors (MEKi) activated tumor NF-κB activity. MEKi potentiated the response to TNFα, a potent activator of NF-κB. In both melanoma and lung cancer cells, MEKi increased cell-surface expression of TNFα receptor 1 (TNFR1), which enhanced NF-κB activation and augmented expression of genes regulated by TNFα and IFNγ. Screening of 289 targeted agents for the ability to increase TNFα and IFNγ target gene expression demonstrated that this was a general activity of inhibitors of MEK and ERK kinases. Treatment with MEKi led to acquisition of a novel vulnerability to TNFα and IFNγ-induced apoptosis in lung cancer cells that were refractory to MEKi killing and augmented cell-cycle arrest. Abolishing the expression of TNFR1 on lung cancer cells impaired the antitumor efficacy of MEKi, whereas the administration of TNFα and IFNγ in MEKi-treated mice enhanced the antitumor response. Furthermore, immunotherapeutics known to induce expression of these cytokines synergized with MEKi in eradicating tumors. These findings define a novel cytokine response modulatory function of MEKi that can be therapeutically exploited. SIGNIFICANCE: Lung cancer cells are rendered sensitive to MEK inhibitors by TNFα and IFNγ, providing a strong mechanistic rationale for combining immunotherapeutics, such as checkpoint blockers, with MEK inhibitor therapy for lung cancer.See related commentary by Havel, p. 5699.

The Influence of Photoperiod on the Action of Exogenous Leptin on Gene Expression of Proinflammatory Cytokines and Their Receptors in the Thoracic Perivascular Adipose Tissue (PVAT) in Ewes.

Leptin resistance is either a condition induced by human obesity or a natural phenomenon associated with seasonality in ruminants. In the cardiovascular system, the leptin resistance state presence is a complex issue. Moreover, the perivascular adipose tissue (PVAT) appears to be crucial as a source of proinflammatory cytokines and as a site of interaction for leptin contributing to endothelium dysfunction and atherosclerosis progression. So the aim of this study was to examine the influence of the photoperiod on the action of exogenous leptin on gene expression of selected proinflammatory cytokines and their receptors in thoracic PVAT of ewe with or without prior lipopolysaccharide (LPS) stimulation. The experiment was conducted on 48 adult, female ewes divided into 4 group (n = 6 in each): control, with LPS intravenous (iv.) injection (400 ng/kg of BW), with leptin iv. injection (20 μg/kg BW), and with LPS and 30-minute-later leptin injection, during short-day (SD) and long-day (LD) seasons. Three hours after LPS/control treatment, animals were euthanized to collect the PVAT adherent to the aorta wall. The leptin injection enhanced IL1B gene expression only in the LD season; however, in both seasons leptin injection intensified LPS-induced increase in IL1B gene expression. IL1R2 gene expression was increased by leptin injection only in the SD season. Neither IL6 nor its receptor and signal transducer gene expressions were influenced by leptin administration. Leptin injection increased TNFA gene expression regardless of photoperiodic conditions. Only in the SD season did leptin treatment increase the gene expression of both TNFα receptors. To conclude, leptin may modulate the inflammatory reaction progress in PVAT. In ewe, the sensitivity of PVAT on leptin action is dependent upon the photoperiodic condition with stronger effects stated in the SD season.

Electroacupuncture alleviated incisional neck pain possibly by suppressing TNF-α expression and increasing IL-4/IL-4 receptor signaling in cervical dorsal part of spinal cord in incisio-nal neck pain rats

To observe the effect of electroacupuncture (EA) on incisional pain and expression of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin-4 (IL-4) of cervical dorsal part of spinal cord in rats with incisional neck pain, so as to explore its analgesic mechanisms. Eighty-four male SD rats were randomly divided into normal control, model, EA-Futu(LI18) and EA-Zusanli(ST36)-Yanglingquan(GB34, EA-ST36-GB34) groups (n=21 in each group). The incisional neck pain model was established by making a longitudinal incision along the bilateral cervical thyroid regions and repeated mechanical separation stimulation. For rats of the EA groups, EA (2 Hz/100 Hz, 1 mA) was applied to bilateral LI18 or ST36-GB34 for 30 min/ time during the surgery, and 20 and 44 h after surgery, respectively. The thermal pain threshold (TPT) of the incisional region was detected. The immunoactivity of TNF-α and IL-10 of the dorsal portion of the cervical spinal cord (C2-C5) was detected by immunofluorescence, and the expression of TNF-α, IL-10, IL-4 and IL-4 receptor (IL-4R) mRNAs was determined by quantitative real-time PCR. Compared with the normal group, the TPT of the incisional area was significantly decreased at 4, 24 and 48 h after neck-incision (P<0.05), the levels of TNF-α mRNA, IL-10 mRNA and TNF-α IL-10 immunoactivity at 24 h were remarkably increased (P<0.05), and the expression of IL-4R mRNA was considerably decreased at 24 h in the model group (P<0.05). Following EA intervention, the TPT, and expression levels of IL-4 mRNA and IL-4R mRNA were significantly increased at 24 h after surgery in the EA-LI18 group relevant to the model group (P<0.05), while the expression level of TNF-α(coexpressed with microgliacytes) in the EA-LI18 group, and TNF-α mRNA expression at 24 h in both EA-LI18 and EA-ST36-GB34 groups, as well as the expression of IL-10 and IL-10 mRNA at 24 h in both EA-LI18 and EA-ST36-GB34 groups were significantly decreased (P<0.05). The effect of EA LI18 was significantly superior to that of EA ST36-GB34 in up-regulating TPT and expression of IL-4 mRNA and IL-4R mRNA at 24 h (P<0.05). EA of LI18 has an analgesic effect in incisional neck pain rats, which may be related to its effect in down-regulating the expression of TNF-α, IL-10 and promoting IL-4 /IL-4R signaling in dorsal portions of the cervical spinal cord. The analgesic effect of EA LI18 is better than that of EA ST36-GB34.

Safety of Infliximab for the Eye Under Human T-Cell Leukemia Virus Type 1 Infectious Conditions in vitro.

Use of biologics has been widely advocated for inflammatory diseases recently. Anti-tumor necrosis factor (TNF)-α antibody therapy is reportedly effective against ocular inflammation. However, side effects of TNF-α inhibition have been reported, particularly in the form of exacerbation of infections such as tuberculosis. Paradoxical reactions such as exacerbated inflammation are also well known. Around 20 million humans are infected with human T-cell leukemia virus type 1 (HTLV-1) globally, and this virus can cause adult T-cell leukemia, HTLV-1-associated myelopathy and HTLV-1 uveitis. As for ophthalmic concerns, it has not been identified whether anti-TNF-α antibody stimulates HTLV-1-infected cells and ocular cells to induce HTLV-1 uveitis in HTLV-1 carriers. Here we investigated the effects of anti-TNF-α antibody on ocular status under HTLV-1 infectious conditions using ocular cells and HTLV-1-infected cells in vitro. We used the ARPE-19 human retinal pigment epithelial cell line as ocular cells considered to play an important role in the blood-ocular barrier, and the MT2 HTLV-1-infected cell line. Jurkat cells were used as controls. Infliximab (IFX) was used as an anti-TNF-α antibody to achieve TNF-α inhibition. We evaluated the production of inflammatory cytokines and intercellular adhesion molecule (ICAM)-1, proliferation of ARPE-19, expression of TNF-α receptor (TNF-R) and HTLV-1 proviral DNA, and the percentage of apoptotic ARPE-19. Inflammatory cytokines such as interleukin (IL)-6, IL-8, TNF, and ICAM-1 were significantly elevated through contact between ARPE-19 and MT2. Treatment with IFX tented to inhibit TNF production, although the level of production was low, but changes in IL-6, IL-8, and ICAM-1 remained unaffected. Expression of TNFR was unaltered by IFX treatment. HTLV-1 proviral DNA was not significantly changed with treatment. No change in cell growth rate or apoptotic rate of ARPE-19 was seen with the addition of IFX. In conclusion, IFX did not exacerbate production of inflammatory cytokines, and did not affect expression of TNFR, proliferation of ARPE-19, HTLV-1 proviral load, or apoptosis of ARPE-19. These results suggest that IFX does not exacerbate HTLV-1-related inflammation in the eye and represents an acceptable treatment option under HTLV-1 infectious conditions.