Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages.

TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.

Maintenance of Lognormal-Like Skewed Dendritic Spine Size Distributions in Dentate Granule Cells of TNF, TNF-R1, TNF-R2, and TNF-R1/2-Deficient Mice.

Dendritic spines are sites of synaptic plasticity and their head size correlates with the strength of the corresponding synapse. We recently showed that the distribution of spine head sizes follows a lognormal-like distribution even after blockage of activity or plasticity induction. As the cytokine tumor necrosis factor (TNF) influences synaptic transmission and constitutive TNF and receptor (TNF-R)-deficiencies cause changes in spine head size distributions, we tested whether these genetic alterations disrupt the lognormality of spine head sizes. Furthermore, we distinguished between spines containing the actin-modulating protein synaptopodin (SP-positive), which is present in large, strong and stable spines and those lacking it (SP-negative). Our analysis revealed that neither TNF-deficiency nor the absence of TNF-R1, TNF-R2 or TNF-R 1 and 2 (TNF-R1/R2) degrades the general lognormal-like, skewed distribution of spine head sizes (all spines, SP-positive spines, SP-negative spines). However, TNF, TNF-R1 and TNF-R2-deficiency affected the width of the lognormal distribution, and TNF-R1/2-deficiency shifted the distribution to the left. Our findings demonstrate the robustness of the lognormal-like, skewed distribution, which is maintained even in the face of genetic manipulations that alter the distribution of spine head sizes. Our observations are in line with homeostatic adaptation mechanisms of neurons regulating the distribution of spines and their head sizes.

Mts1 (S100A4) and Its Peptide Demonstrate Cytotoxic Activity in Complex with Tag7 (PGLYRP1) Peptide.

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10-8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.

RIPK1 protects naive and regulatory T cells from TNFR1-induced apoptosis.

The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1-/- double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.

TNFR1/p38αMAPK signaling in Nex + supraspinal neurons regulates estrogen-dependent chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Sesquiterpene Lactones Containing an α-Methylene-γ-Lactone Moiety Selectively Down-Regulate the Expression of Tumor Necrosis Factor Receptor 1 by Promoting Its Ectodomain Shedding in Human Lung Adenocarcinoma A549 Cells.

Alantolactone is a eudesmane-type sesquiterpene lactone containing an α-methylene-γ-lactone moiety. Previous studies showed that alantolactone inhibits the nuclear factor κB (NF-κB) signaling pathway by targeting the inhibitor of NF-κB (IκB) kinase. However, in the present study, we demonstrated that alantolactone selectively down-regulated the expression of tumor necrosis factor (TNF) receptor 1 (TNF-R1) in human lung adenocarcinoma A549 cells. Alantolactone did not affect the expression of three adaptor proteins recruited to TNF-R1. The down-regulation of TNF-R1 expression by alantolactone was suppressed by an inhibitor of TNF-α-converting enzyme. Alantolactone increased the soluble forms of TNF-R1 that were released into the culture medium as an ectodomain. The structure-activity relationship of eight eudesmane derivatives revealed that an α-methylene-γ-lactone moiety was needed to promote TNF-R1 ectodomain shedding. In addition, parthenolide and costunolide, two sesquiterpene lactones with an α-methylene-γ-lactone moiety, increased the amount of soluble TNF-R1. Therefore, the present results demonstrate that sesquiterpene lactones with an α-methylene-γ-lactone moiety can down-regulate the expression of TNF-R1 by promoting its ectodomain shedding in A549 cells.

Cellular heterogeneity in TNF/TNFR1 signalling: live cell imaging of cell fate decisions in single cells.

Cellular responses to TNF are inherently heterogeneous within an isogenic cell population and across different cell types. TNF promotes cell survival by activating pro-inflammatory NF-κB and MAPK signalling pathways but may also trigger apoptosis and necroptosis. Following TNF stimulation, the fate of individual cells is governed by the balance of pro-survival and pro-apoptotic signalling pathways. To elucidate the molecular mechanisms driving heterogenous responses to TNF, quantifying TNF/TNFR1 signalling at the single-cell level is crucial. Fluorescence live-cell imaging techniques offer real-time, dynamic insights into molecular processes in single cells, allowing for detection of rapid and transient changes, as well as identification of subpopulations, that are likely to be missed with traditional endpoint assays. Whilst fluorescence live-cell imaging has been employed extensively to investigate TNF-induced inflammation and TNF-induced cell death, it has been underutilised in studying the role of TNF/TNFR1 signalling pathway crosstalk in guiding cell-fate decisions in single cells. Here, we outline the various opportunities for pathway crosstalk during TNF/TNFR1 signalling and how these interactions may govern heterogenous responses to TNF. We also advocate for the use of live-cell imaging techniques to elucidate the molecular processes driving cell-to-cell variability in single cells. Understanding and overcoming cellular heterogeneity in response to TNF and modulators of the TNF/TNFR1 signalling pathway could lead to the development of targeted therapies for various diseases associated with aberrant TNF/TNFR1 signalling, such as rheumatoid arthritis, metabolic syndrome, and cancer.

ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1.

Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Introduction: Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. Methods: We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. Results: More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1ß, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. Discussion: These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Retinal response to systemic inflammation differs between sexes and neurons.

Background: Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. Methods: A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. Results: In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1ß. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Conclusions: Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

Progesterone modulates TNF receptors expression by Jurkat cells cultured with plasma from pregnant women with preeclampsia.

Pregnant women with preeclampsia (PE) present a shift in the immune response to an inflammatory profile. This deviation could be due to the interaction of tumor necrosis factor (TNF) with TNFR1 and TNFR2 receptors, besides the failure in modulation of inflammation regulatory mechanisms. This study evaluated the effects of progesterone on the expression of TNFR1 and TNFR2 by Jurkat cells after stimulation with plasma from PE and normotensive (NT) pregnant women. Jurkat cells were cultured with or without progesterone in a medium containing 20% (v/v) plasma from PE or NT women. The expression of TNF receptors was evaluated by flow cytometry. The concentration of soluble forms of TNF receptors and cytokines was determined in culture supernatant and plasma by ELISA. The plasma of PE women showed significantly higher concentrations of sTNFR1 and TNF and lower concentrations of sTNFR2 compared to the NT group. TNFR1 receptor expression was increased in Jurkat cells, while TNFR2 was decreased after culture with PE plasma when compared with Jurkat cells cultured with progesterone and plasma from NT women. The concentration of sTNFR1, TNF, and IL-10 in the culture supernatant of Jurkat cells was increased after culture with PE plasma, while the sTNFR2 receptor was decreased when compared to the NT group. Results demonstrate that in preeclamptic women a systemic inflammation occurs with an increase of inflammatory molecules, and progesterone may have a modulating effect on the expression of TNF receptors, shifting Jurkat cells towards an anti-inflammatory profile with greater expression of TNFR2.

Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate.

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate. Video Abstract.

Identification of TNFRSF1A as a potential biomarker for osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a relatively rare malignant bone tumor in teenagers; however, its molecular mechanisms are not yet understood comprehensively. OBJECTIVE: The study aimed to use necroptosis-related genes (NRGs) and their relationships with immune-related genes to construct a prognostic signature for OS. METHODS: TARGET-OS was used as the training dataset, and GSE 16091 and GSE 21257 were used as the validation datasets. Univariate regression, survival analysis, and Kaplan-Meier curves were used to screen for hub genes. The immune-related targets were screened using immune infiltration assays and immune checkpoints. The results were validated using nomogram and decision curve analyses (DCA). RESULTS: Using univariate Cox regression analysis, TNFRSF1A was screened from 14 NRGs as an OS prognostic signature. Functional enrichment was analyzed based on the median expression of TNFRSF1A. The prognosis of the TNFRSF1A low-expression group in the Kaplan-Meier curve was notably worse. Immunohistochemistry analysis showed that the number of activated T cells and tumor purity increased considerably. Furthermore, the immune checkpoint lymphocyte activation gene 3 (LAG-3) is a possible target for intervention. The nomogram accurately predicted 1-, 3-, and 5-year survival rates. DCA validated the model (C = 0.669). Conclusion: TNFRSF1A can be used to elucidate the potential relationship between the immune microenvironment and NRGs in OS pathogenesis.

Bisphenol A Regulates the TNFR1 Pathway and Excessive ROS Mediated by miR-26a-5p/ADAM17 Axis to Aggravate Selenium Deficiency-Induced Necroptosis in Broiler Veins.

Selenium (Se) deficiency can affect the expression of microRNA (miRNA) and induce necroptosis, apoptosis, etc., resulting in damage to various tissues and organs. Bisphenol A (BPA) exposure can cause adverse consequences such as oxidative stress, endothelial dysfunction, and atherosclerosis. The toxic effects of combined treatment with Se-deficiency and BPA exposure may have a synergistic effect. We replicated the BPA exposure and Se-deficiency model in broiler to investigate whether the combined treatment of Se-deficiency and BPA exposure induced necroptosis and inflammation of chicken vascular tissue via the miR-26A-5p/ADAM17 axis. We found that Se deficiency and BPA exposure significantly inhibited the expression of miR-26a-5p and increased the expression of ADAM17, thereby increasing reactive oxygen species (ROS) production. Subsequently, we discovered that the tumor necrosis factor receptor (TNFR1), which was highly expressed, activated the necroptosis pathway through receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like (MLKL), and regulated the heat shock proteins-related genes expressions and inflammation-related genes expressions after exposure to BPA and selenium deficiency. In vitro, we found that miR-26a-5p knockdown and increased ADAM17 can induce necroptosis by activating the TNFR1 pathway. Similarly, both N-Acetyl-L-cysteine (NAC), Necrostatin-1 (Nec-1), and miR-26a-5p mimic prevented necroptosis and inflammation caused by BPA exposure and Se deficiency. These results suggest that BPA exposure activates the miR-26a-5p/ADAM17 axis and exacerbates Se deficient-induced necroptosis and inflammation through the TNFR1 pathway and excess ROS. This study lays a data foundation for future ecological and health risk assessments of nutrient deficiencies and environmental toxic pollution.

Photoaffinity labeling coupled with proteomics identify PDI-ADAM17 module is targeted by (-)-vinigrol to induce TNFR1 shedding and ameliorate rheumatoid arthritis in mice.

Various biological agents have been developed to target tumor necrosis factor alpha (TNF-α) and its receptor TNFR1 for the rheumatoid arthritis (RA) treatment, whereas small molecules modulating such cytokine receptors are rarely reported in comparison to the biologicals. Here, by revealing the mechanism of action of vinigrol, a diterpenoid natural product, we show that inhibition of the protein disulfide isomerase (PDI, PDIA1) by small molecules activates A disintegrin and metalloprotease 17 (ADAM17) and then leads to the TNFR1 shedding on mouse and human cell membranes. This small-molecule-induced receptor shedding not only effectively blocks the inflammatory response caused by TNF-α in cells, but also reduces the arthritic score and joint damage in the collagen-induced arthritis mouse model. Our study indicates that targeting the PDI-ADAM17 signaling module to regulate the shedding of cytokine receptors by the chemical approach constitutes a promising strategy for alleviating RA.

Chronic restraint stress and social transfer of stress produce tactile allodynia mediated by the HMGB1/TNFα/TNFR1 pathway in female and male rats.

Previous studies have shown the relevance of high mobility group box 1 protein (HMGB1) and tumor necrosis factor α (TNFα) in nerve or tissue injury-induced nociception. However, the role of these proteins in chronic stress and social transfer of stress (STS)-induced dysfunctional pain is not entirely known. The aim of this study was to determine the participation of the spinal HMGB1-TNFα signaling pathway and TNFα receptor 1 (TNFR1) in rats subjected to chronic restraint stress (CRS) and STS. Non-stressed female and male rats in contact with CRS rats increased sniffing behavior of the anogenital area, behavior related to STS. Rats subjected to CRS and STS reduced 50 % withdrawal threshold and reached the value of tactile allodynia after 21 days of stress. Rats return to the basal withdrawal threshold after 30 days without stress and return to allodynia values in only 5 days of stress sessions (priming). Female and male rats subjected to 28 days of CRS or STS were intrathecal injected with glycyrrhizin (inhibitor of HMGB1), thalidomide (inhibitor of the TNFα synthesis), and R7050 (TNFR1 antagonist), in all the cases, an antiallodynic effect was observed. Rats under CRS or STS enhanced HMGB1 and TNFR1 protein expression in DRG and dorsal spinal cord. Data suggest that the spinal HMGB1/TNFα/TNFR1 signaling pathway plays a relevant role in the maintenance of CRS and STS-induced nociceptive hypersensitivity in rats. These proteins could be helpful in developing pain treatments for fibromyalgia in humans.

Knockdown of ADAMDEC1 ameliorates ox-LDL-induced endothelial cell injury and atherosclerosis progression.

This study was designed to investigate the role of a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC-1) in atherosclerosis (AS). The Gene Expression Omnibus (GEO) database was utilized to identify differentially expressed genes (DEGs) between carotid atheroma plaque and carotid tissue adjacent atheroma plaque obtained from AS patients. Gene functional enrichment analysis was conducted on DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). QRT-PCR was employed to quantify mRNAs expression. AS animal model was established using ApoE-/- mice; serum triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were detected. Aortic sinus atherosclerotic lesions were observed using H&E staining and Oil Red O staining. ADAMDEC-1 was silenced using small interfering RNAs (siRNAs) in human vascular smooth muscle cells (HVSMCs). Cell proliferation, migration, and cell cycle progression were detected by cell count kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EDU), wound scratch healing assay, transwell assay, and flow cytometry, respectively. Western blot was used to evaluate various protein expression levels. Our results showed that ADAMDEC-1 was highly expressed in the serum of AS patients, consistent with the in silico results. The elevated TG, LDL-C, and HDL-C levels along with H&E and Oil Red O staining confirmed the successful establishment of the AS mouse model. ADAMDEC-1 expression was also elevated in AS mice. ADAMDEC-1 knockdown in HVSMCs suppressed cell proliferation, inhibited the expression of proliferating cell nuclear antigen (PCNA), and reduced the levels of matrix metalloproteinases (MMP2 and MMP9) proteins. Protein-protein interaction (PPI) analysis indicated that ADAMDEC-1 was associated with CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50. The expression levels of CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50 increased, while ADAMDEC-1 knockdown attenuated the expression of these proteins. Our study findings substantiate that ADAMDEC-1 may represent a novel target for AS.

Knockouts of TNFRSF1A and TNFRSF1B Genes in K562 Cell Line Lead to Diverse Long-Lasting Responses to TNF-α.

This research delves into the intricate landscape of tumor necrosis factor-alpha (TNF-α) signaling, a multi-functional cytokine known for its diverse cellular effects. Specifically, we investigate the roles of two TNF receptors, TNFR1 and TNFR2, in mediating TNF-α-induced transcriptional responses. Using human K562 cell lines with TNFR1 and TNFR2 knockouts, we explore changes in gene expression patterns following TNF-α stimulation. Our findings reveal distinct transcriptional profiles in TNFR1 and TNFR2 knockout cells, shedding light on the unique contributions of these receptors to TNF-α signaling. Notably, several key pathways associated with inflammation, apoptosis, and cell proliferation exhibit altered regulation in the absence of TNFR1 or TNFR2. This study provides valuable insights into the intricate mechanisms governing TNF-α signaling and its diverse cellular effects, with potential implications for targeted therapeutic strategies.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Erythropoietin relieves neuronal apoptosis in epilepsy rats via TGF-ß/Smad signaling pathway.

This study aimed to investigate the influence of recombinant human erythropoietin (rHuEPO) on pentylenetetrazol (PTZ)-induced neuronal apoptosis in epilepsy rats, and to explore the signaling pathways related to the action. Healthy Sprague-Dawley rats aged 8 weeks old were randomly divided into 5 groups, namely, control group, PTZ model group, PTZ + rHuEPO intervention group, PTZ + SB431542 + rHuEPO intervention group and PTZ + SB431542 (TGF-ß/Smad inhibitor) intervention group. The expressions of apoptotic proteins [tumor necrosis factor receptor 1 (TNFR1) and caspase-3] and the transforming growth factor-beta (TGF-ß)/Smad signaling pathway-related proteins [phosphorylated smad3 (p-smad3) and TGF-ß1] in the brain tissues were determined via Western blotting (WB). Epilepsy was successfully induced by PTZ in the rats. The results of the TUNEL assay showed that the intervention with rHuEPO could remarkably reduce the number of PTZ-induced apoptotic neurons in the hippocampus, while SB431542 inhibitor could attenuate the protective effect of rHuEPO against neuronal apoptosis (P<0.05). In addition, the intraperitoneal injection of 50 µg/kg rHuEPO could activate the TGF-ß/Smad signaling pathway, markedly up-regulate the expressions of TGF-ß1 and p-smad3 (P<0.05), down-regulate the expressions of apoptotic proteins TNFR1 and caspase-3 (P<0.01) and reduce neuronal apoptosis. Moreover, SB431542 was able to notably repress the protective effect of rHuEPO against neuronal apoptosis, and down-regulate the expressions of p-smad3 and TGF-ß1 (P<0.01). In conclusion, the inhibitory effect of rHuEPO on nerve cell apoptosis in epilepsy rats may be realized by activating the TGF-ß/Smad signaling pathway, thus relieving neuronal apoptosis and ameliorating the symptoms of epilepsy.