TNF-α Signaling Research Articles

Clinical and molecular characterization of FAP and SPP1 in colorectal cancer (CRC), CALGB (Alliance)/SWOG 80405 and real-world data.

276 Background: FAP and SPP1 contribute to immune modulation in the CRC tumor microenvironment (TME). FAP is expressed by cancer associated fibroblasts, aiding in tissue remodeling and tumor invasion. SPP1 is an integrin-binding protein expressed by tumor associated macrophages that promotes tumor growth, adhesion, and metastasis. We present a clinical and molecular characterization of FAP and SPP1 in CRC. Methods: We analyzed 24,257 CRC samples tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq), NextGen DNA sequencing (NextSeq), and PD-L1 expression (SP142, positive ≥ 2+, 5%). RNA deconvolution analysis estimated cell infiltration in the TME. Data from the phase 3 CALGB/SWOG 80405 trial (NCT00265850) on 433 metastatic CRC patients treated with bevacizumab (Bev, n = 226) or cetuximab (Cet, n = 207) in combination with first-line chemotherapy were also evaluated. RNA isolated from FFPE tumor samples were sequenced with HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared using Cox regression in categorical gene expression tertiles (high (T3), medium (T2), and low (T1)) for FAP and SPP1 . Results: FAP -T3 and SPP1 -T3 had increased PD-L1 positivity (q<0.05), while SPP1 -T3 also demonstrated increased MSI-H (8.4% vs 5.3%) and TMB-H(>10 mt/mb, 15.0% vs 10.1%) status relative SPP1 -T1 (all q<0.001). T3 of both genes of correlated with increased M1/M2 macrophages, NK cells and T cell inflamed score (q<0.001); dendritic cells and neutrophils were increased in SPP1 -T3 and FAP -T1 tumors (q<0.05). FAP -T3 and SPP1 -T3 had increased pathway activation of epithelial-mesenchymal transition (EMT), inflammatory response, TNF-a signaling, angiogenesis and KRAS signaling (all q < .005). In Caris cohorts, FAP -T3 demonstrated worse OS in Cet/panitumumab treated CRC (T3: 23.6 vs T1: 21.0 months [mo], P = .005; HR 0.85, 95% CI [0.77-0.95]), but SPP1 did not correlate with OS. In 80405, FAP -T3 showed shorter PFS (T3: 9.5 vs T2: 11.5 vs T1: 12.6 mo, T3 vs T1 (reference) adjusted HR 1.27 [1.07-1.51]) and OS (25.2 vs 29.4 vs 35.5 mo, adjusted HR 1.31 [1.10-1.57]). Similarly, SPP1 -T3 demonstrated worse PFS (9.0 vs 12.7 vs 14.0 mo, adjusted HR 1.29 [1.12-1.48]) and OS (20.9 vs 34.0 vs 36.3 mo, HR 1.24 [1.07-1.44], P < 0.001). Treatment interaction tests noted FAP -T1 CRC benefited from Cet over Bev with respect to PFS ( P = 0.003) and OS ( P = 0.044), while SPP1-T1 tumors demonstrated a PFS benefit with Cet ( P = 0.009). Conclusions: Our results indicate that increased FAP and SPP1 expression is associated with immune cell infiltration, EMT, and inflammatory signaling. Additionally, their expression may be prognostic and predictive of targeted therapy. These data support the evaluation of FAP and SPP1 as predictive markers and therapeutic targets in CRC.

P0078 The role of epithelial STING in a genetic IBD Model of autophagy deficiency and its interplay with the Integrated stress response

Abstract Background Stimulator of interferon genes (STING) regulates intestinal homeostasis and inflammation by detecting cytoplasmic double-stranded DNA, driving type-1 interferon (IFN) responses.1 The autophagy-related gene ATG16L1, a major risk factor for inflammatory bowel disease (IBD), links to STING overactivation in intestinal epithelial cells (IEC), causing inflammation via IFN1, NF-κB, and TNFα signaling.2 Excessive STING activity is associated with autoinflammatory disorders1, whereas a full-body Sting knockout (Tmem173gt/gt) protects against dextran sodium sulfate (DSS)-induced colitis.3 STING and ATG16L1, key regulators of cellular stress and inflammation, may interact with the integrated stress response (ISR). Activating transcription factor 4 (ATF4), a central ISR mediator, is induced by intestinal inflammation4 and could be affected by STING activation through mitochondrial stress and autophagy dysfunction caused by ATG16L1 deficiency. This study investigates the so far unknown role of STING in IEC and its interplay with autophagy deficiency and ATF4-mediated ISR. Methods Murine IEC (ModeK cells) were treated with DNA fractions, pro-inflammatory stimuli, and STING agonists to analyse responses via WB and RT-qPCR in vitro. In vivo, we used epithelial STING deficient mice (Tmem173dIEC) for clinical and histopathological analysis in DSS-induced colitis and steady-state, the latter also with combined Atg16l1 deficiency. Mice were analyzed at 12 and 52 weeks of age. Protein and RNA analyses of crypt cells were performed via WB and RT-qPCR. Results Unlike Tmem173gt/gt mice, Tmem173dIEC mice were not protected from DSS-induced colitis, suggesting that epithelial STING alone is less critical to immune responses. However, aged untreated Tmem173dIEC mice showed reduced epithelial inflammation and cell death compared to wildtype mice. In Atg16l1-deficient ModeK cells, silencing Sting reduced pro-inflammatory responses to STING agonists and pro-inflammatory stimulants. To assess if STING deficiency ameliorates inflammation in Atg161ldIEC mice, we created Atg161ldIEC/Tmem173dIEC mice, observing reduced relative liver and spleen weights in young animals. We observed increased Atf4 expression in DSS-treated colitis, while Atf4 was absent in Sting-deficient ModeK cells, indicating STING regulates proinflammatory cytokine induction via ATF4. Silencing Atf4 decreased the activation of Sting, supporting a bidirectional link between STING signalling and the ISR, which will be further explored in aged and DSS-treated Tmem173dIEC and Atg161ldIEC/Tmem173dIEC mice. Conclusion We show that STING essentially coordinates pro-inflammatory cytokine induction in response to Atg16l1 deficiency in intestinal epithelial cells via a mechanism involving ATF4.

DOP132 An integrated single-cell atlas enables the discovery and validation of a novel candidate therapeutic target for Inflammatory Bowel Disease

Abstract Background Single-cell technologies enable the fine mapping of disease and treatment mechanisms in inflammatory bowel disease (IBD). We sought to leverage these insights to discover novel drug targets with precise therapeutic hypotheses. To this end, we previously constructed one of the largest integrated single-cell atlas of IBD patient-derived tissue samples1. We then used Immunai’s ImmunoDynamics EngineTM, a proprietary machine learning (ML) framework, to identify transcriptional signatures of inflammation and non-response to anti-TNF treatment in specific cell populations, and extracted a ranked gene list that was highly enriched in targets of currently marketed IBD therapies. We selected top-ranked genes for in vitro functional genomic validation and leveraged the IBD single-cell atlas to support clinical relevance of in vitro observations. Here, we present a target case study demonstrating this approach. Methods The top 400 genes were triaged to choose 20 candidate targets based on novelty, druggability, genetic association with IBD, and experimental feasibility. Each of these 20 targets was individually deleted under optimized culture conditions in the primary human cell type(s) in which it showed a significant disease association in the IBD single-cell atlas, including monocyte-derived macrophages (MDM), CD8, CD4 helper and/or regulatory T cells, and intestinal fibroblasts. Knockout (KO) cells were characterized by flow cytometry, secreted proteome profiling, and bulk transcriptomics, with suitable positive and negative controls per cell type. KO-induced transcriptomic changes were interpreted via gene-set enrichment of canonical pathways and of IBD-associated transcriptional signatures derived from orthogonal clinical cohorts. KO-induced transcriptomic signatures were also computed in each sample in the IBD single-cell atlas. Results Deletion of one selected target in MDMs led to a reduction in TNFα and IFNγ signaling and in the expression of IBD inflammation-associated signatures from orthogonal patient cohorts. Genes downregulated by the deletion of this target in MDMs were enriched in macrophages from inflamed tissue in the IBD single-cell atlas. Conclusion The functional genomic data indicate that deletion of the selected target shifts MDMs away from a disease-associated inflammatory state, suggesting that its pharmacologic inhibition in macrophages may have a therapeutic benefit. These findings highlight the relevance of cell type-specific methods to understand disease mechanisms and characterize putative novel targets. Applying these experimental and clinical contextualization methods to additional candidate genes will further expand the basis for novel therapeutic strategies in IBD.

Exploring the protective role of caffeine against Taraxacum-Induced ribotoxic stress mediated through autophagy and mitochondrial depolarization

The ribotoxic stress response is a pathway that gets activated when ribosomes get impaired, leading to disruptions in protein synthesis, increased inflammatory signaling, and cell death if left unresolved. Taraxacum can induce apoptosis-associated ribosomal RNA (rRNA) cleavage, however, the exact working mechanism of Taraxacum-induced rRNA cleavage remains unclear. In this study, we used the RNA integrity (RIN) value and 28S/18S ratio to confirm the integrity of experiments. Our RNA sequencing data showed that Taraxacum formosanum (T. formosanum) upregulated 893 genes and downregulated 509 genes and triggered hallmark genes of spliceosomes, TNF-α signaling via NF-κB, inflammatory response, and IL6-JAK-STAT3 signaling. Additionally, T. formosanum imbalanced the levels of ribosomal proteins of the large and small subunits. We found that caffeine was the only screening agent that could rescue the cleavage of 28S and 18S rRNA induced by T. formosanum. However, caffeine failed to rescue T. formosanum-targeted mRNAs when the RIN values were relatively lower. T. formosanum induced the N-terminal clipping of histone H3, which was observed not only in human HeLa cervical cancer cells but also in human Huh6 and HepG2 liver cancer cells. Our study revealed that caffeine could reverse the effects of T. formosanum on the reduction of autophagy and the disruption of mitochondrial membrane potential. However, caffeine could only change the populations of necrotic and apoptotic cells but not T. formosanum-induced cell death. By providing detailed information on Taraxacum-induced rRNA cleavage and N-truncated histone H3’s mechanisms of gene regulation, we hope to understand their respective cellular death and survival stresses.

Cumulus cells and the TNF-alpha signaling facilitate aging of ovine oocytes.

Post-maturation oocyte aging (PMOA) is known to significantly impair the developmental potential of oocytes; however, comprehensive studies on ovine PMOA remain limited. In mice, cumulus cells (CCs) accelerate oocyte aging by releasing cytokines, but the roles of CCs and cytokines in PMOA of domestic animals are poorly understood. This study aimed to elucidate the involvement of CCs and tumor necrosis factor (TNF)-α in the PMOA of ovine oocytes. Our findings reveal that PMOA significantly reduced blastocyst rates and the expression of development-promoting genes, while increasing oocyte degeneration and activation rates, along with expression of development-inhibiting genes, compared to newly matured oocytes. These detrimental effects were more pronounced in oocytes aged as cumulus-oocyte complexes than as cumulus-denuded oocytes. Additionally, PMOA led to increased apoptotic rates, TNF-α production, and TNF receptor 1 (TNFR1) expression in CCs, coupled with a significant reduction in the expression of anti-apoptotic genes. Mature oocytes expressed TNFR1, with levels decreasing significantly during PMOA. Importantly, the addition of the TNF-α antagonist Etanercept to the aging medium markedly improved parthenogenetic embryo development and the expression of competence-related genes, while mitigating CC apoptosis during PMOA of COCs. In conclusion, PMOA compromises developmental potential while heightening oocyte degeneration and activation sensitivity in ovine oocytes. Cumulus cells exacerbate PMOA through increased TNF-α signaling activity, highlighting the potential of TNF-α antagonists as therapeutic agents to counteract the deleterious effects of PMOA.

The role of MRO as an M2 macrophage-associated gene in non-small cell lung cancer: insights into immune infiltration, prognostic significance, and therapeutic implications

BackgroundNon-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), is influenced by tumor-immune interactions. M2 macrophages play a significant role in the tumor microenvironment. This study explores the role of MRO, an M2 macrophage-associated gene, in NSCLC, focusing on immune infiltration, prognostic significance, and therapeutic potential.MethodsNSCLC samples from The Cancer Genome Atlas (TCGA) were analyzed using the CIBERSORT algorithm to quantify immune cell compositions. Differential gene expression and correlation studies examined MRO’s association with M2 macrophages. Univariate Cox regression and Kaplan–Meier analyses assessed prognostic significance. Single-cell RNA sequencing data from TISCH2 evaluated MRO expression in different cell types. The ESTIMATE algorithm analyzed correlations between MRO expression and immune scores, while TIDE and Submap analyses predicted immunotherapy responses.ResultsMRO was highly expressed in NSCLC, particularly in LUAD and LUSC, and associated with M2 macrophages. MRO correlated with key immune pathways, including TNFα signaling via NFκB, inflammatory response, and IL6 JAK STAT3 signaling. High MRO expression correlated with poorer overall survival (OS) and disease-specific survival (DSS). Single-cell analysis confirmed MRO expression in macrophages. The ESTIMATE algorithm showed positive correlations between MRO expression and immune scores. TIDE and Submap analyses suggested low MRO expression in LUSC patients might predict better immunotherapy responses.ConclusionsMRO is a critical M2 macrophage-associated gene in NSCLC, influencing immune infiltration and prognosis. It may serve as a biomarker for prognostication and a target for therapeutic intervention in NSCLC.

High Expression of GPR183 Predicts Poor Survival in Cytogenetically Normal Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with a normal karyotype (CN-AML) constitutes approximately 50% of all AML cases, presenting significant prognostic variability, and highlighting the urgent need for the identification of novel molecular biomarkers. In this study, we systematically assessed GPR183 expression levels using qRT-PCR in our clinical follow-up study which included 283 CN-AML patients. Using Kaplan-Meier analysis, we found that patients with high GPR183 expression levels exhibited significantly worse overall survival (OS) (P = 0.046) and event-free survival (EFS) (P = 0.030) compared to those with low GPR183 expression. Comprehensive univariate and multivariate Cox regression analyses confirmed that GPR183 expression is a prognostic factor for OS and EFS (P < 0.05). To further validate these findings, we analyzed an independent cohort of 104 CN-AML patients from the GSE71014 dataset, corroborating our primary results, and indicating that high GPR183 expression is associated with poorer survival outcomes. Additionally, RNA-seq data from the GSE71014 dataset were analyzed by Gene Set Enrichment Analysis (GSEA). The results suggested that GPR183 may influence disease progression through the activation of the "TNFa Signaling Via NF-κB" pathway. Collectively, these findings suggested that GPR183 could serve as a valuable prognostic biomarker in CN-AML, offering insights into the underlying mechanisms of disease progression.

The Anaphylactic and Anti-allergenic Properties of Shuanghuanglian: A Review.

Shuanghuanglian (SHL) and its primary constituents have demonstrated protective effects against allergenic diseases. This review examines the anaphylactic and anti-allergenic activities of SHL and its constituents. We also discuss potential avenues for future research, particularly regarding the expansion of the clinical applications of SHL formulations (oral or nebulized) for the treatment of allergenic disorders. For this review, we searched the PubMed, Web of Science, and China National Knowledge Infrastructure databases for relevant publications. Additionally, details of the essential active components and target genes of SHL were obtained from the Traditional Chinese Medicine Systems Pharmacology database (TCMSP), and information on allergy-related genes was collected from the GeneCards and Online Mendelian Inheritance in Man(OMIM) databases. Lists of both the SHL target and disease-related genes were imported into the 'Draw Venn Diagram' tool on the website (http://bioinformatics.psb.ugen /web tools/Venn/). A protein-protein interaction network for SHL and disease targets was constructed with reference to the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the potential pathways were identified based on Kyoto Encyclopaedia of Genes and Genome enrichment analyses. The allergenic reactions induced by SHL injection (intravenous) and its main constituents (intraperitoneal or intravenous injection) have been verified in animal experiments. Furthermore, the protective effects of SHL injection (intraperitoneal) and its individual Chinese herb components (intragastric administration), namely, Flos Lonicerae, Radix Scutellariae, and Fructus Forsythiae, as well as their main constituents (intraperitoneal or intragastric administration), have been verified in asthma, rhinitis, atopic dermatitis, and both IgE- and non-IgE-mediated systemic allergic responses. The network pharmacology analysis revealed that the therapeutic effects of SHL might be primarily mediated through the regulation of the IL-17 and TNF-α signalling pathways and Th17 cell differentiation. Accumulated research data provide a theoretical basis for the clinical application of SHL (via extravascular routes) in the treatment of allergenic diseases.

Neurotrophomodulatory effect of TNF-α through NF-κB in rat cortical astrocytes.

Tumor necrosis factor alpha (TNF-α) is a well-known pro-inflammatory cytokine originally recognized for its ability to induce apoptosis and cell death. However, recent research has revealed that TNF-α also plays a crucial role as a mediator of cell survival, influencing a wide range of cellular functions. The signaling of TNF-α is mediated through two distinct receptors, TNFR1 and TNFR2, which trigger various intracellular pathways, including NF-κB, JNK, and caspase signaling cascades. Both TNFR1 and TNFR2 are expressed in astrocytes, which are specialized glial cells essential for maintaining the structural and functional integrity of the central nervous system (CNS). Astrocytes support neuronal function by regulating brain homeostasis, maintaining synaptic function, and supplying metabolic substrates. In addition, astrocytes are known to secrete a variety of growth factors and neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/5. These neurotrophins play a critical role in supporting neuronal survival, synaptic plasticity, and myelination within the brain. The present study focuses on the role of TNF-α in modulating neurotrophin expression and secretion in rat cortical astrocytes. We demonstrate that TNF-α induces the upregulation of neurotrophins, particularly NGF and BDNF, in cultured astrocytes. This effect is accompanied by an increase in the expression of their respective receptors (TrkA & TrkB), further suggesting a functional modulation of neurotrophic signaling pathways. Notably, we show that the modulation of neurotrophin expression by TNF-α is mediated via the NF-κB signaling pathway. Additionally, we observed that TNF-α also regulates the secretion levels of NGF and BDNF into the culture media of astrocytes in a dose-dependent manner, indicating that TNF-α can modulate both the production and release of these growth factors. Taken together, our findings highlight a previously underexplored neuroprotective role of TNF-α in astrocytes. Specifically, we propose that TNF-α, through the upregulation of neurotrophins, may contribute to maintaining neuronal health and supporting neuroprotection under disease conditions.

ScRNA-seq reveals elevated interferon responses and TNF-α signaling via NFkB in monocytes in children with uncomplicated malaria.

Malaria causes significant morbidity and mortality worldwide, disproportionately impacting sub-Saharan Africa. Disease phenotypes associated with Plasmodium falciparum infection can vary widely, from asymptomatic to life-threatening. To date, prevention efforts, particularly those related to vaccine development, have been hindered by an incomplete understanding of which factors impact host immune responses resulting in these divergent outcomes. Here, we conducted a field study of 224 individuals to determine host-parasite factors associated with symptomatic malaria "patients" compared to asymptomatic malaria-positive "controls" at both the community and healthy facility levels. We further performed comprehensive immune profiling to obtain deeper insights into differences in response between the pair. First, we determined the relationship between host age and parasite density in patients (n = 134/224) compared to controls (n = 90/224). Then, we applied single-cell RNA sequencing to compare the immunological phenotypes of 18,176 peripheral blood mononuclear cells isolated from a subset of the participants (n = 11/224), matched on age, sex, and parasite density. Patients had higher parasite densities compared to the controls, although the levels had a negative correlation with age in both groups, suggesting that they are key indicators of disease pathogenesis. On average, patients were characterized by a higher fractional abundance of monocytes and an upregulation of innate immune responses, including those to type I and type II interferons and tumor necrosis factor-alpha signaling via NFκB. Further, in the patients, we identified more putative interactions between antigen-presenting cells and proliferating CD4 T cells, and naïve CD8 T cells driven by MHC-I and MHC-II signaling pathways, respectively. Together, these findings highlight transcriptional differences between immune cell subsets associated with disease phenotypes that may help guide the development of improved malaria vaccines and new therapeutic interventions.

Sustained hypoxia but not intermittent hypoxia induces HIF-1α transcriptional response in human aortic endothelial cells.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxic environments at the cellular level and is an independent risk factor for the development of cardiovascular disease. Endothelial cell (EC) dysfunction precedes the development of cardiovascular disease; however, the mechanisms by which ECs respond to these intermittent hypoxic events are poorly understood. To better understand EC responses to hypoxia, we examined the effects of sustained hypoxia (SH) and intermittent hypoxia (IH) on the activation of HIF-1α in ECs. While SH stabilized HIF-1α and led to its nuclear localization, IH did not activate HIF-1α and the expression of its target genes. Using RNA-sequencing, we evaluated transcriptional responses of ECs to hypoxia. SH induced the expression of HIF-1α and hypoxia response genes, while IH affected cell-cycle regulation genes. A cytoscape protein-protein interaction network for EC response to hypoxia was created with differentially expressed genes. The network comprises cell-cycle regulation, inflammatory signaling via NF-κB and response to VEGF stimulus subnetworks on which SH and IH had distinct activities. As OSA is associated with elevated catecholamines, we investigated the effect of epinephrine on the EC response to SH and IH. Transcriptomic responses under IH and epinephrine revealed protein-protein interaction networks emphasizing distinct subnetworks, including cytokine-mediated TNFα signaling via NF-κB, Wnt/LRP/DKK signaling and cell cycle regulation. This study reveals differential transcriptomic responses under SH and IH characterised by HIF-1α transcriptional response induced only by SH, but not by IH. The study also features the potential molecular events that may occur at the vascular level in OSA.

Overexpression of RGPR-p117 reveals anticancer effects by regulating multiple signaling pathways in bone metastatic human breast cancer MDA-MB-231 cells.

The role of RGPR-p117, a transcription factor, which binds to the TTGGC motif in the promoter region of the regucalcin gene, in cell regulation remains to be investigated. This study elucidated whether RGPR-p117 regulates the activity of triple-negative human breast cancer MDA-MB-231 cells in vitro. The wild-type and RGPR-p117-overexpressing cancer cells were cultured in DMEM supplemented with fetal bovine serum. RGPR-p117 overexpression suppressed colony formation and growth of cancer cells. Stimulatory effects of epidermal growth factor on cell growth were blocked by RGPR-p117 overexpression. Wild-type cell proliferation was repressed by cell cycle and intracellular signaling inhibitors. These effects were not potentiated in transfectants. Overexpressed RGPR-p117 protected cancer cells against apoptosis inducers. Mechanistic results showed that RGPR-p117 overexpression decreased the expression of Ras, PI3-kinase, Akt, mitogen-activated protein kinase, and mTOR, which are involved in cell growth, while it elevated the levels of the cancer cell suppressor p53, Rb, p21, and regucalcin. Overexpression of RGPR-p117 suppressed cancer cell migration and adhesion. Interestingly, osteoblastic MC3T3-E1 cells or macrophage RAW264.7 cells involved in the bone microenvironment were impaired by coculture with MDA-MB-231 cells. The effects of cancer cells were blocked by transfection. Coculture with conditioned medium obtained from breast cancer cells repressed proliferation and enhanced the death of osteoblastic cells and macrophages. A TNF-α signaling inhibitor blocked these effects. Thus, overexpressed RGPR-p117 was found to suppress the activity of breast cancer cells by regulating various signaling processes, providing new insight into cellular signaling regulation.

Short-term exposure to low doses of aflatoxin B1 aggravates nonalcoholic steatohepatitis by TLR4-mediated necroptosis.

Aflatoxin B1 (AFB1), a worldwide mycotoxin found in food and foodstuffs, is a potent hepatotoxin in humans and animals. Non-alcoholic fatty liver disease (NAFLD), a widespread disease, could progress from simple steatosis to non-alcoholic steatohepatitis (NASH), hepatic cirrhosis, and even hepatocellular carcinoma (HCC). To date, little is known concerning the relationship between AFB1 and the progression of NAFLD. The effects of low doses of AFB1 on the development of NASH and their mechanism were investigated in vivo and in vitro. The results in vivo showed that AFB1 at 20 and 40 μg/kg.bw aggravated CDAHFD-induced NASH in mice as demonstrated by increasing the serum and liver lipid accumulation, liver inflammation and injury. The results in vitro showed that AFB1 at 1.0 μM aggravated FFA-induced lipid accumulation, inflammation and cell damage in HepG2 cells. In addition, RNA-seq indicated that necroptosis, Toll like receptor signaling and TNF-α signaling showed a significant change in KEGG pathway enrichment in AFB1 at 40 μg/kg.bw. AFB1 significantly upregulated the mRNA and protein levels of TLR4, RIPK3, p-RIPK3, MLKL and p-MLKL, and increased TUNEL positive cells. Also, immunofluorescence results showed that TUNEL, TLR4, TNF-α had co-localized with RIPK3, respectively. Necroptosis inhibitor (GSK-872) attenuated the aggravating effects of AFB1 on NASH. Knockout of TLR4 inhibited necroptosis and rescued the aggravating effects of AFB1 on NASH. These data indicate that low dose of AFB1 aggravated NASH via TLR4-mediated necroptosis. This suggests that low dose of AFB1 is potentially harmful to animals and humans, as they exacerbate NASH.

Phytochemical investigation of Jie-Geng-Tang and regulatory role in the TNF-α pathway in mitigating pulmonary fibrosis using UPLC-Q-TOF/MS.

Jie-Geng-Tang (JGT), composed of Platycodon grandiflorus (Jacq.) A. DC and Glycyrrhiza uralensis Fisch, is widely used in traditional Chinese medicine for its potential effects in preventing pulmonary fibrosis (PF). This study systematically explored the effects of JGT's water and 70% EtOH extracts in bleomycin (BLM)-induced PF models. In vitro, the 70% EtOH extract significantly reversed BLM-induced reductions in cell viability and apoptosis, whereas the water extract had limited impact. In vivo, the EtOH extract markedly reduced fibrosis markers, such as α-SMA and collagen-I, alleviating lung tissue damage and collagen deposition. UPLC-Q-TOF/MS analysis revealed that the EtOH extract contained a higher abundance of flavonoids compared to the water extract. Through network pharmacology analysis of the EtOH extract, four key flavonoids-apigenin, kaempferol, kaempferol 3-glucuronoside, and quercetin-were identified as crucial compounds. These flavonoids were found to reverse BLM-induced cell viability loss, with apigenin showing the most pronounced effect by modulating the TNF-α signaling pathway and inhibiting caspase-3 activation. Apigenin, as a primary active component derived from JGT, holds significant potential as a preventive agent against pulmonary fibrosis.

Analgesic effect of Dahuang Fuzi Decoction in neuropathic pain through inhibiting TNF-α and PI3K-AKT signaling.

Neuropathic pain (NeP) presents considerable challenges in terms of effective management and significantly impacts the quality of life for affected patients. The current treatment options for NeP are limited, highlighting the need for alternative therapeutic approaches. Dahuang Fuzi Decoction (DF), a formula from traditional Chinese medicine, has shown potential in relieving pain symptoms associated with various types of NeP. However, the mechanisms through which DF exerts its effects remain largely unknown. In this study, we employed ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) to analyze the chemical composition of DF. A chronic sciatic nerve compression injury (CCI) rat mode was used to assess the analgesic efficacy of DF for NeP. Network pharmacology analysis was performed to identify the potential signaling pathways affected by DF. DF treatment significantly increased the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in CCI rats, indicating its analgesic effect. Network pharmacology analysis suggested that DF potentially modulated TNF-α and PI3K-AKT signaling pathways. Furthermore, DF treatment decreased the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in spinal cord tissues of CCI rats, suggesting an anti-inflammatory effect. Western blot analysis revealed that DF treatment reduced the expression of TNF-α, TNFR1, and phosphorylated forms of PI3K, AKT, IKKα/β, IKBα, and NF-κB in the spinal cord of CCI rats. Immunofluorescence analysis confirmed significant reductions in TNF-α and TNFR1 expression, as well as in AKT and NF-κB phosphorylation within astrocytes following DF administration. Our findings characterize the chemical constituents of DF and elucidate its underlying mechanism for relieving NeP. The analgesic effect of DF involves the inhibition of TNF-α and PI3K-AKT signaling pathways, providing a potential therapeutic approach for NeP management.

Multi-omic integration with human DRG proteomics highlights TNFα signalling as a relevant sexually dimorphic pathway.

The peripheral nervous system has been widely implicated in pathological conditions that exhibit distinct clinical presentations in men and women, most notably in chronic pain disorders. Here, we explored this sexual dimorphism at a molecular level. We expanded the available omics landscape in the PNS to include quantitative proteomics of the human dorsal root ganglia (hDRG) and nerve. Using data-independent acquisition mass spectrometry, we uncovered an extensive protein landscape, validated against tissue-specific differences between the nerve and hDRG. Using a combination of multi-omic analyses and in vitro functional support, we then examined sex-differences, highlighting TNFα signalling as a relevant sexually dimorphic pathway in males. These results support a functional sexually dimorphism in the periphery, which is of particular importance to sensory- and pain-related clinical translation.

Programmed Cell Death Protein 1 Contributes to Oral Cancer Pain via Regulating Tumor Necrosis Factor Alpha in the Spinal Trigeminal Nucleus Caudalis.

Oral cancer causes intense pain at the primary site, and such pain can impair oral functions. However, the underlying mechanisms for oral cancer pain are still not fully understood. In the present study, it is investigated whether programmed cell death protein 1 (PD-1) is involved in the development of oral cancer pain. RMP1-14, a specific anti-PD-1 antibody, was injected into spinal trigeminal nucleus caudalis (Sp5C) and measured pain behaviors using von Frey filaments and dolognawmeter. Western blotting and immunofluorescence staining were performed to analyze the expression of PD-1 and tumor necrosis factor alpha (TNFα) in the Sp5C. It was observed that the PD-1 antibody significantly inhibited mechanical hypersensitivity and functional allodynia in our oral cancer pain mouse model. Moreover, we found that TNFα was highly upregulated in the Sp5C following the induction of oral cancer pain and that intra-Sp5C injection of the PD-1 antibody diminished the upregulation of TNFα. It was found that genetic deletion of TNFα or its receptor antagonism synergized the analgesic effect of PD-1 antibody on oral cancer pain. Our results suggest that PD-1 in the Sp5C contributes to oral cancer pain by altering TNFα signaling in the trigeminal nociceptive system, and PD-1 could be targeted to develop a novel approach for oral cancer pain management.

TNF-α exacerbates SARS-CoV-2 infection by stimulating CXCL1 production from macrophages.

Since most genetically modified mice are C57BL/6 background, a mouse-adapted SARS-CoV-2 that causes lethal infection in young C57BL/6 mice is useful for studying innate immune protection against SARS-CoV-2 infection. Here, we established two mouse-adapted SARS-CoV-2, ancestral and Delta variants, by serial passaging 80 times in C57BL/6 mice. Although young C57BL/6 mice were resistant to infection with the mouse-adapted ancestral SARS-CoV-2, the mouse-adapted SARS-CoV-2 Delta variant caused lethal infection in young C57BL/6 mice. In contrast, MyD88 and IFNAR1 KO mice exhibited resistance to lethal infection with the mouse-adapted SARS-CoV-2 Delta variant. Treatment with recombinant IFN-α/β at the time of infection protected mice from lethal infection with the mouse-adapted SARS-CoV-2 Delta variant, but intranasal administration of recombinant IFN-α/β at 2 days post infection exacerbated the disease severity following the mouse-adapted ancestral SARS-CoV-2 infection. Moreover, we showed that TNF-α amplified by type I IFN signals exacerbated the SARS-CoV-2 infection by stimulating CXCL1 production from macrophages and neutrophil recruitment into the lung tissue. Finally, we showed that intravenous administration to mice or hamsters with TNF protease inhibitor 2 alleviated the severity of SARS-CoV-2 and influenza virus infection. Our results uncover an unexpected mechanism by which type I interferon-mediated TNF-α signaling exacerbates the disease severity and will aid in the development of novel therapeutic strategies to treat respiratory virus infection and associated diseases such as influenza and COVID-19.

P72 The role of the transcription factor Bcl11b in IL-17, IL-23 and/or TNF-induced activation of the canonical and non-canonical NF-κB pathways in psoriatic keratinocytes

Abstract Bcl11b (CTIP2) is a crucial transcription factor involved in the differentiation processes across various cell types, including T cells and keratinocytes. It functions both as a transcriptional repressor and activator, interacting with complexes such as NuRD and proteins like p300. Bcl11b is significantly expressed in skin and immune cells suggesting its potential involvement in diseases like psoriasis, which is characterized by hyperproliferation and aberrant differentiation of keratinocytes alongside immune cell infiltration and cytokine release. However, the mechanism underlying the role of Bcl11b in the pathogenesis of psoriasis remains unidentified. To elucidate Bcl11b’s role in psoriasis by analysing its expression and correlated pathways through meta-analysis of sequencing-based transcriptome profiles and by functional assays in normal and genetically engineered keratinocytes under inflammatory conditions. RNA-sequencing datasets from psoriatic and healthy skin samples were processed and analysed for differential expression of transcription factors, with a focus on Bcl11b. Pearson correlation analysis identified genes correlated with Bcl11b expression. Enrichment and pathway analyses were conducted using Enrichr. Functional studies involved Bcl11b knockdown in HaCaT keratinocytes via CRISPRi, followed by cytokine stimulation including IL-17A, IL-23, TNF-α and the combinations. The activation of canonical and non-canonical NF-κB pathways was assessed. Downregulation of Bcl11b in psoriatic lesions was confirmed. Correlated genes were enriched in pathways involving IL-17A, IL-23 and TNF-α signalling. Knockdown of Bcl11b in keratinocytes resulted in morphological changes indicative of incomplete differentiation and reduced proliferation. Under cytokine-driven inflammatory conditions, canonical NF-κB pathway activation was impaired in Bcl11b KD cells, while non-canonical NF-κB pathway activation was dependent on IL-17A and the combination of IL-23/TNF-α in these KD cells. These data highlight the pivotal role of Bcl11b in the proinflammatory cytokine-induced regulation of the NF-κB signalling pathways. Absence of Bcl11b results in abnormal keratinocyte behaviour associated with psoriasis. Our data suggest that key proinflammatory cytokines in psoriasis act differently on Bcl11b associated activation of the NF-kB pathways.

The Impact of Resident Adipose Tissue Macrophages on Adipocyte Homeostasis and Dedifferentiation.

Obesity is concurrent with immunological dysregulation, resulting in chronic low-grade inflammation and cellular dysfunction. In pancreatic islets, this loss of function has been correlated with mature β-cells dedifferentiating into a precursor-like state through constant exposure to inflammatory stressors. As mature adipocytes likewise have the capability to dedifferentiate in vitro and in vivo, we wanted to analyze this cellular change in relation to adipose tissue (AT) inflammation and adipose tissue macrophage (ATM) activity. Using our organotypic AT explant culture method combined with a double-reporter mouse model for labeling ATMs and mature adipocytes, we were able to visualize and quantify dedifferentiated fat (DFAT) cells in AT explants. Preliminary testing showed increased dedifferentiation after tamoxifen (TAM) stimulation, making TAM-dependent lineage-tracing models unsuitable for quantification of naturally occurring DFAT cells. The regulatory role of ATMs in adipocyte dedifferentiation was shown through macrophage depletion using Plexxicon 5622 or clodronate liposomes, which significantly increased DFAT cell levels. Subsequent bulk RNA sequencing of macrophage-depleted explants revealed enrichment of the tumor necrosis factor α (TNFα) signaling pathway as well as downregulation of associated genes. Direct stimulation with TNFα decreased adipocyte dedifferentiation, while application of a TNFα-neutralizing antibody did not significantly alter DFAT cell levels. Our findings suggest a regulatory role of resident ATMs in maintaining the mature adipocyte phenotype and preventing excessive adipocyte dedifferentiation. The specific regulatory pathways as well as the impact that DFAT cells might have on ATMs, and vice versa, are subject to further investigation.