TNF-α Signaling Research Articles

M2 macrophages-derived exosomes regulate osteoclast differentiation by the CSF2/TNF-α axis

BackgroundOsteoclast-mediated bone resorption cause bone loss in several bone diseases. Exosomes have been reported to regulate osteoclast differentiation. M2-polarized macrophages exhibit anti-inflammatory activity. This study aimed to explore the effect of exosomes from M2 polarized macrophages (M2-exos) on osteoclastogenesis and molecular mechanisms.MethodsM2-exos were isolated from IL-4-induced Raw264.7 cells (M2 macrophages) and used to treat osteoclasts (RANKL-induced Raw264.7 cells). Osteoclast differentiation was visualized using tartrate resistant acid phosphatase staining. Quantitative real-time PCR (qPCR) was conducted to measure the levels of osteoclastogenesis-related genes. The underlying mechanisms of M2-exos were evaluated using qPCR and western blotting.ResultsM2-exos suppressed osteoclast differentiation induced by RANKL. Additionally, CSF2 was highly expressed in M2 macrophages, and knockdown of CSF2 further enhanced the effects of M2-exos on osteoclast differentiation. Moreover, CSF2 positively regulated TNF-α signaling, which inhibition promoted differentiation of M2-exo-treated osteoclasts.ConclusionM2-exos inhibited RANKL-induced osteoclast differentiation by downregulating the CSF2 expression through inactivating the TNF-α signaling, suggesting the potential application of exosomes in bone disease therapy.

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol prevents TNF-α-mediated impairment of mineralization in mouse osteoblastic MC3T3-E1 cells: Impact of macrophage activation

The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in the Pacific oyster Crassostrea gigas, is a superior peroxyl radical scavenger compared to other materials, including Trolox. DHMBA may play an important role in the prevention of health disorders. This study elucidates whether DHMBA prevents the impairment of mineralization of mouse osteoblastic MC3T3-E1 cells under inflammatory conditions by using mouse macrophage RAW264.7 cells in vitro. Culturing with DHMBA (1–100 μM) did not affect the proliferation and death of MC3T3-E1 cells. DHMBA stimulated osteoblastic mineralization. DHMBA blocked the decrease in mineralization of MC3T3-E1 cells caused by culture with the inflammatory cytokine TNF-α. DHMBA inhibited the production of TNF-α by stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The growth of MC3T3-E1 cells was suppressed by coculture with macrophages under LPS stimulation through the crosstalk of both cells. Interestingly, the growth of MC3T3-E1 cells was suppressed by culturing with the conditioned medium obtained by culturing macrophages with LPS. The effect of the conditioned medium was blocked by the presence of DHMBA or Bay 11–7082, an inhibitor of the TNF-α pathway. The blocking effect of DHMBA was not further enhanced in the presence of Bay 11–7082. Mechanistically, DHMBA was found to decrease the levels of NF-κB p65 and the activity of NF-κB reporter expression in MC3T3-E1 cells. DHMBA was shown to prevent the impairment of osteoblastic mineralization via TNF-α signaling involved in macrophage activation in the bone marrow microenvironment. This study may provide a novel strategy for the therapy of osteoblastic impairment.

Enhanced CT-based radiomics model to predict natural killer cell infiltration and clinical prognosis in non-small cell lung cancer.

Natural killer (NK) cells are crucial for tumor prognosis; however, their role in non-small-cell lung cancer (NSCLC) remains unclear. The current detection methods for NSCLC are inefficient and costly. Therefore, radiomics represent a promising alternative. We analyzed the radiogenomics datasets to extract clinical, radiological, and transcriptome data. The effect of NK cells on the prognosis of NSCLC was assessed. Tumors were delineated using a 3D Slicer, and features were extracted using pyradiomics. A radiomics model was developed and validated using five-fold cross-validation. A nomogram model was constructed using the selected clinical variables and a radiomic score (RS). The CIBERSORTx database and gene set enrichment analysis were used to explore the correlations of NK cell infiltration and molecular mechanisms. Higher infiltration of NK cells was correlated with better overall survival (OS) (P = 0.002). The radiomic model showed an area under the curve of 0.731, with 0.726 post-validation. The RS differed significantly between high and low infiltration of NK cells (P < 0.01). The nomogram, using RS and clinical variables, effectively predicted 3-year OS. NK cell infiltration was correlated with the ICOS and BTLA genes (P < 0.001) and macrophage M0/M2 levels. The key pathways included TNF-α signaling via NF-κB and Wnt/β-catenin signaling. Our radiomic model accurately predicted NK cell infiltration in NSCLC. Combined with clinical characteristics, it can predict the prognosis of patients with NSCLC. Bioinformatic analysis revealed the gene expression and pathways underlying NK cell infiltration in NSCLC.

Nitisinone attenuates cartilage degeneration and subchondral osteoclastogenesis in osteoarthritis and concomitantly inhibits the cGAS/STING/NF-κB pathway

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and subchondral bone remodelling. Currently, conservative treatment strategies cannot effectively alleviate the progression of OA. In this study, we used computer network analysis to show that Nitisinone (NTBC) is closely related to extracellular matrix degradation in OA and mainly interferes with the TNF-α signaling pathway. NTBC is an orphan drug used to treat hereditary type I tyrosinemia by altering phenylalanine/tyrosine metabolic flow. In this study, we found that NTBC effectively reduced chondrocyte inflammation and extracellular matrix degradation induced by TNF-α. Mechanistically, NTBC inhibited the cGAS/STING signaling pathway and reduced activation of the STING-dependent NF-κB pathway to alleviate inflammation. In addition, NTBC inhibited osteoclastogenesis and delayed the occurrence of subchondral bone remodelling. In mice with ACLT-induced osteoarthritis, intra-articular injection of NTBC significantly reduced cartilage degradation and subchondral bone remodelling. NTBC showed impressive therapeutic efficacy as a potential pharmaceutical intervention for the treatment of OA.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn’s Disease

Background and aimsThe incidence of Crohn’s disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. Here, we aim to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. MethodsWe performed single cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of CD patients compared to healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signalling networks. We overlapped our differentially expressed genes with publicly available microarray datasets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD TREGs and to test predicted therapeutics. ResultsWe identified five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to TNFα signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid co-culture system. As predicted in silico, the histone deacetylase inhibitor Vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. ConclusionWe identified a novel, proinflammatory FOXP3+ T cell subpopulation in CD patients and developed a pipeline to specifically target these cells using the FDA-approved drug Vorinostat.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

Elucidating T cell dynamics and molecular mechanisms in syngeneic and allogeneic islet transplantation through single-cell RNA sequencing.

Islet transplantation is a promising therapy for diabetes treatment. However, the molecular underpinnings governing the immune response, particularly T-cell dynamics in syngeneic and allogeneic transplant settings, remain poorly understood. Understanding these T cell dynamics is crucial for enhancing graft acceptance and managing diabetes treatment more effectively. This study aimed to elucidate the molecular mechanisms, gene expression differences, biological pathway alterations, and intercellular communication patterns among T-cell subpopulations after syngeneic and allogeneic islet transplantation. Using single-cell RNA sequencing, we analyzed cellular heterogeneity and gene expression profiles using the Seurat package for quality control and dimensionality reduction through t-SNE. Differentially expressed genes (DEGs) were analyzed among different T cell subtypes. GSEA was conducted utilizing the HALLMARK gene sets from MSigDB, while CellChat was used to infer and visualize cell-cell communication networks. Our findings revealed genetic variations within T-cell subpopulations between syngeneic and allogeneic islet transplants. We identified significant DEGs across these conditions, highlighting molecular discrepancies that may underpin rejection or other immune responses. GSEA indicated activation of the interferon-alpha response in memory T cells and suppression in CD4+ helper and γδ T cells, whereas TNFα signaling via NFκB was particularly active in regulatory T cells, γδ T cells, proliferating T cells, and activated CD8+ T cells. CellChat analysis revealed complex communication patterns within T-cell subsets, notably between proliferating T cells and activated CD8+ T cells. In conclusion, our study provides a comprehensive molecular landscape of T-cell diversity in islet transplantation. The insights into specific gene upregulation in xenotransplants suggest potential targets for improving graft tolerance. The differential pathway activation across T-cell subsets underscores their distinct roles in immune responses posttransplantation.

TNFSF12 is associated with breast cancer prognosis and immune cell infiltration.

Breast cancer (BRCA) is one of the most common cancers in women and is the leading cause of cancer-related deaths in women. TNFSF12, originally a member of the TNF superfamily, is considered a key molecule that is associated with poor prognosis of many cancers. However, its role in progression of BRCA remains unclear. In this study, the expression profile and clinical information of TNFSF12 across various cancers were obtained from The Cancer Genome Atlas (TCGA) database. Differences in TNFSF12 expression levels between carcinoma and paraneoplastic cancers were compared, and its association with prognosis was examined. Functional enrichment analysis was conducted to explore the potential signaling pathways and biological functions linked with TNFSF12. Moreover, the correlation between TNFSF12 and immune cell infiltration, response to immune checkpoint inhibitors (ICIs), and response to chemotherapy were evaluated. TNFSF12 level in BRCA and normal serum was detected by ELISA. TNFSF12 was lowly expressed in BRCA and is significantly associated with PAM50. TNFSF12 low expression correlates with poor overall survival, particularly among HER2-positive patients. Patients with high level of TNFSF12 expression are usually accompanied with elevated levels of various immune cells, including CD8 T cells, cytotoxic cells, DCs, eosinophils, iDCs, mast cells, neutrophils, NK CD56bright cells, NK cells, pDC, T cells, Tem, and TFH Th17 cells, and exhibit sensitivity to immune checkpoint inhibitors. Functional enrichment analysis indicates significant activation of KRAS signaling, TNFA signaling via NFKB, and epithelial-mesenchymal transition (EMT) in the high TNFSF12 expression group, while MTORC1 signaling, MYC, G2M checkpoint, and E2F targets are inhibited. Furthermore, patients in the low expression group demonstrate higher sensitivity to paclitaxel and rapamycin, whereas those in the high expression group show increased sensitivity to erlotinib and foretinib. ELISA analysis also confirmed a significant decrease of TNFSF12 protein levels in BRCA patients. This study presents a comprehensive analysis of the close correlation between TNFSF12 and prognosis, immune response, as well as the effectiveness of chemotherapeutic agents in BRCA patients.

Nanoarchitectonics of Injectable Biomimetic Conjugates for Cartilage Protection and Therapy Based on Degenerative Osteoarthritis Progression.

Osteoarthritis (OA) is a common age-related degenerative disease characterized by changes in the local tissue environment as inflammation progresses. Inspired by the wind-dispersal mechanism of dandelion seeds, this study develops responsive biomimetic microsphere-drug conjugate for OA therapy and protection. The conjugate integrates dibenzaldehyde polyethylene glycol (DFPEG) with chitosan and polyethylene glycol diacrylate (PEGDA) through dynamic covalent bonds to form a dual-network hydrogel microsphere. Based on the progression of OA, the conjugate with the surface-anchored cyclic peptide cortistatin-14 (CST-14) achieves targeted drug therapy and a self-regulating hydrogel network. In cases of progressing inflammation (pH < 5), CST-14 dissociates from the microsphere surface (viz. the drug release rate increased) and inhibits TNF-α signaling to suppress OA. Concurrently, the monomer DFPEG responsively detaches from the hydrogel network and scavenges reactive oxygen species (ROS) to protect the cartilage tissue. The ROS scavenging of DFPEG is comparable to that of coenzyme Q10 and vitamin C. The degraded PEGDA microspheres provide tissue lubrication through reused conjugates. The rat OA model successfully achieved a synergistic therapeutic effect greater than the additive effect (1 + 1 > 2). This strategy offers an approach for anchoring amine-containing drugs and has marked potential for OA treatment and protection.

Mechanisms underlying the therapeutic effects of Semen cuscutae in treating recurrent spontaneous abortion based on network pharmacology and molecular docking.

Background: This paper aims to analyse the active components of Semen cuscutae (SC) by network pharmacology and screen the most stable compounds with tumour necrosis factor-alpha (TNF-α) by molecular docking to explore the mechanisms of SC treatment of recurrent spontaneous abortion (RSA) and provide a theoretical basis for drug development. Methods: The active compounds of SC and the potential inflammatory targets of RSA were obtained from the Traditional Chinese Medicine Systems Pharmacology database and GeneCards, respectively. The RSA-SC target gene interaction network was obtained and visualized using the STRING database and Cytoscape software. GO and KEGG pathway enrichment analyses were obtained from DAVID to further explore the RSA mechanism and therapeutic effects of SC. Interactions between TNF-α and drugs were analysed by molecular docking. Treatment of human trophoblast cells with sesamin and TNF-α was carried out to detect their proliferative and apoptotic abilities, and WB assay was carried out to detect EGFR, PTGS2, and CASP3 protein expression. Results: Ten compounds and 128 target genes were screened from SC, of which 79 overlapped with RSA target inflammatory genes, which were considered potential therapeutic targets. Network pharmacological analysis showed that sesamin, matrine, matrol, and other SC compounds had a good correlation with the inflammatory target genes of RSA. Related genes included PGR, PTGS1, PTGS2, TGFB1, and CHRNA7. Several signalling pathways are involved in the pathogenesis of RSA, such as the TNF-α signalling pathway, HIF-1 signalling pathway, oestrogen signalling pathway, proteoglycans in cancer cells, and FoxO signalling pathway. Molecular docking results suggested that sesamin was the most suitable natural tumour necrosis factor inhibitor (TNFi). Sesamin can promote proliferation and inhibit apoptosis in human trophoblasts by downregulating EGFR and CASP3 expression and upregulating PTGS2 expression. Conclusion: Our findings play an important role and basis for further research into the molecular mechanism of SC treatment of RSA and drug development of TNFi.

Implantable Epigallocatechin Gallate Sustained-Release Nanofibers for the Prevention of Immobilization-Induced Muscle Atrophy.

Long-term immobilization of joints can lead to disuse atrophy of the muscles in the joints. Oral nutrients are used clinically for rehabilitation and therapeutic purposes, but bioavailability and targeting are limited. Here, we report tea polyphenols (dietary polyphenols), sustained-release nanofilms that release tea polyphenols through slow local degradation of core-shell nanofibers in muscles. This dietary polyphenol does not require gastrointestinal consumption and multiple doses and can directly remove inflammatory factors and superoxide generated in muscle tissue during joint fixation. The quality of muscles is increased by 30%, and muscle movement function is effectively improved. Although nanofibers need to be implanted into muscles, they can improve bacterial infections after joint surgery. To investigate the biological mechanism of this core-shell nanomembrane prevention, we conducted further transcriptomic studies on muscle, confirming that in addition to achieving antioxidation and anti-inflammation by inhibiting TNF-α and NF-κB signaling pathways, tea polyphenol core-shell nanofibers can also promote muscle formation by activating the p-Akt signaling pathway.

Treatment with β-Adrenoceptor Agonist Isoproterenol Reduces Non-parenchymal Cell Responses in LPS/D-GalN-Induced Liver Injury

There is an increasing evidence indicating the involvement of the sympathetic nervous system (SNS) in liver disease development. To achieve an extensive comprehension of the obscure process by which the SNS alleviates inflammatory damage in non-parenchymal liver cells (NPCs) during acute liver failure (ALF), we employ isoproterenol (ISO), a beta-adrenoceptor agonist, to mimic SNS signaling. ISO was administered to C57BL/6J mice to establish an acute liver failure (ALF) model using LPS/D-GalN, which was defined as ISO + ALF. Non-parenchymal cells (NPCs) were isolated from liver tissues and digested for tandem mass tag (TMT) labeled proteomics to identify differentially expressed proteins (DEPs). The administration of ISO resulted in a decreased serum levels of pro-inflammatory cytokines, e.g., TNF-α, IL-1β, and IL-6 in ALF mice, which alleviated liver damage. By using TMT analysis, it was possible to identify 1587 differentially expressed proteins (DEPs) in isolated NPCs. Notably, over 60% of the DEPs in the ISO + ALF vs. ALF comparison were shared in the Con vs. ALF comparison. According to enrichment analysis, the DEPs influenced by ISO in ALF mice were linked to biological functions of heme and fatty acid metabolism, interferon gamma response, TNFA signaling pathway, and mitochondrial oxidation function. Protein-protein interaction network analysis indicated Mapk14 and Caspase3 may serve as potentially valuable indicators of ISO intervention. In addition, the markers on activated macrophages, such as Mapk14, Casp1, Casp8, and Mrc1, were identified downregulated after ISO initiation. ISO treatment increased the abundance of anti-inflammatory markers in mouse macrophages, as evidenced by the immunohistochemistry (IHC) slides showing an increase in Arg + staining and a reduction in iNOS + staining. Furthermore, pretreatment with ISO also resulted in a reduction of LPS-stimulated inflammation signaling markers, Mapk14 and NF-κB, in human THP-1 cells. Prior treatment with ISO may have the potential to modify the biological functions of NPCs and could serve as an innovative pharmacotherapy for delaying the pathogenesis and progression of ALF.

Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and β-catenin signaling to promote adipocyte dedifferentiation

BackgroundRecent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming.MethodsTo induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of β-catenin and applying BML-284, an agonist of β-catenin.ResultsHypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-β-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-β-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment.ConclusionsHypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/β-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/β-catenin signaling using BML-284.

Retracted: Inhibition of TNF-α and JNK Signaling Pathway Can Reduce Paclitaxel-Induced Apoptosis of Mouse Cardiomyocytes.

[This retracts the article DOI: 10.1155/2022/8460121.].

The Potential Utility of Salivary and Tear Proteomics to Discriminate Sjögren's Disease from Non-Sjögren's Sicca.

Sjögren's Disease (SjD) is an autoimmune disorder associated with decreased saliva and/or tear secretions, resulting in patients reporting dryness in the mouth and eyes. Serum autoantibodies directed against the Ro60/SS-A and La/SS-B autoantigens are a distinctive feature of the disease. Analysis of the saliva and tear proteomes represents one promising alternative method of both classifying and monitoring the condition, and research into salivary and tear proteomics in patients with SjD, with and without sicca, has shown its efficacy and practicality in both clinical and research settings. Studies analyzing the saliva proteomics of SjD patients have generally shown an overexpression of proteins involved in T-cell activation, the immune response, β-2 microglobulin, and the recruitment of pro-inflammatory agents. These studies also show a decrease in or downregulation of proteins involved in salivary secretion. Studies analyzing the tear proteomics of patients with SjD have generally indicated an upregulation of proteins involved with TNF-α signaling, B-cell survival, and the recruitment of pro-inflammatory agents. Studies also note the differential expression of tear protein folding as a hallmark of ocular involvement in this condition. These findings help to elucidate the biochemical relationship between the proteomes of saliva/tear fluids and the general pathophysiology of the gland involved with the pathogenesis of this condition, giving further credence to the potential role of salivary and tear proteomics in the future of diagnosis and treatment for patients with SjD.

Adalimumab Effectively Decreases Inflammation Downstream of TNFα Signaling in Synoviocytes from Extended Oligoarticular Juvenile Idiopathic Arthritis.

Fibroblast-like synoviocytes (FLS) play a critical role in inflammation that contributes to disease progression in juvenile idiopathic arthritis (JIA). In rheumatoid arthritis (RA), FLS express tumor necrosis factor alpha (TNFα). TNFα signaling has been shown to be upstream of transforming growth factor beta (TGFβ) signaling. Overexpression of TNFα and TGFβ, as well as related proteins, can cause increased inflammation in RA. In this study, we examine the effects of inhibiting TNFα signaling with adalimumab on FLS isolated from synovial fluid of patients with JIA. Synovial fluid samples were selected from 41 patients in our repository. Of these samples, 23 were diagnosed with persistent oligoarticular JIA and 18 were diagnosed with extended oligoarticular JIA. All samples were taken prior to patients extending to a polyarticular course, or what we termed extended-to-be (ETB), and were on no medications or nonsteroidal anti-inflammatory drugs (NSAIDs) only at the time of arthrocentesis. For cell studies, FLS were isolated from synovial fluid and treated with adalimumab for 24h. Protein antibody arrays were performed by RayBiotech, Inc. according to their protocols. In persistent FLS, TNFα (fold change [FC] = 1.2 p = 0.001), TGFβ (FC = 1.5 p = 0.001), lymphotoxin alpha (LTα) (FC = 4.3 p = 0.015), soluble tumor necrosis factor receptor1 (sTNFRI) (FC = 5.1 p = 0.008), and soluble tumor necrosis factor receptor2 (sTNFRII) (FC = 3.8 p = 0.025) were significantly elevated in adalimumab treated cells compared to untreated cells. In ETB FLS, TNFα was significantly elevated (FC = 1.04 p = 0.023) while TGFβ (FC = 1.03 p = 0.037) was significantly decreased in adalimumab-treated cells compared to untreated cells. This data suggests that, after only 24h, adalimumab is effective at decreasing inflammation that occurs downstream of initial TNFα signaling in extended-to-be fibroblast-like synoviocytes.

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.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=&amp;lt;0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=&amp;lt;0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

The Gene Expression Landscape of BTK Inhibitor Treatment in Chronic Lymphocytic Leukemia Exposed By Shallow Depth RNA Sequencing

Efforts to understand chronic lymphocytic leukemia (CLL) subgroups have focused on multi-omics profiling of patient cohorts (in steady-state) and linking biomarkers to responses. Here, we aim to combine ex-vivo drug screening and gene expression profiling (GEP) in primary CLL samples to dissect disease biology based on the observed phenotype as a qualitative and quantitative function of steady-state features and drug perturbation effects. As most differential gene expression changes in CLL are retrieved from highly expressed genes, we hypothesized that a relevant set of genes could still be reliably captured by reduced sequencing coverage. We profiled five samples with traditional whole transcript TruSeq and 3'-end QuantSeq (Moll et al., Nat Methods, 2014) library preparation with deep and shallow depth sequencing, respectively. We obtained 43.48-72.68 million mappable reads with TruSeq and 2.24-3.78 million with QuantSeq. Although the read coverage was reduced, QuantSeq was able to recover 71.17 ± 0.87% of all genes that were detected by TruSeq with reproducible quantification ( R &amp;gt; 0.85, P &amp;lt; 0.001 for all samples). Shallow sequencing of CLL samples reliably profiled intermediate to highly abundant genes with reduced sensitivity only for lowly expressed genes. We then selected 105 representative CLL patient samples from a well-characterized cohort, treated them ex-vivo with 100 nM ibrutinib for 48h and performed GEP using shallow depth QuantSeq. Sample viability was measured by flow cytometry before and after perturbation. Library preparation and sequencing was successful for 91% of samples. With a median coverage of 2.19 million mappable reads per sample, the principal components (PC) of transcriptional variation in DMSO-treated samples included IGHV (12.0%), methylation subgroup and trisomy 12 status (7.1%). These data show that the screening method reliably captured the transcriptional features of CLL at steady-state and thus support the use of shallow depth RNA sequencing. We were interested in the landscape of transcriptional change after perturbation of the B-cell receptor (BCR) pathway. Samples from the same patients clustered together and spread along a gradient of sample viability in the first PC. After correlation of gene counts from DMSO-treated samples with sample viability and subsequent gene set enrichment analysis, we found activation of the canonical apoptosis pathway via NFκB-mediated TNF-α signaling and cell stress to be associated with this signature of cell death. BTKi-treated samples showed the same signature of spontaneous apoptosis and additionally strong suppression of MYC and OXPHOS activity. To identify the ibrutinib-regulated transcriptional signature, we performed differential gene expression analysis between ibrutinib and DMSO. BTKi treatment induced 904 differentially expressed genes (DEGs) ( FDR &amp;lt; 0.005). These genes were negatively enriched in the hallmarks allograft rejection, MYC targets, TNF-α signaling via NFκB and KEGG pathways associated with BCR signaling, and immune cell response. Top DEGs included e. g. FCRL5, PTPN6, FILIP1L, LRMP and LAPTM5 ( FDR &amp;lt; 0.001) which were also significantly downregulated after in-vivo ibrutinib exposure (Rendeiro et al. Nat Commun, 2020). Inhibition of the BCR induces apoptosis in CLL in an IGHV status-dependent manner. Samples with unmutated IGHV (UM-CLL) were more sensitive to BTKi compared to samples with mutated IGHV (M-CLL) (83.1 vs. 92.3% relative viability, P &amp;lt; 0.001). On gene expression level, we observed more DEGs within UM-CLL (387) compared to M-CLL (182) ( FDR &amp;lt; 0.005). Response to BTKi was largely similar although the effect size in M-CLL tended to be smaller. To identify BCR-dependent genes that were significantly modulated by IGHV, we tested for the interaction between BTKi treatment and IGHV status in the differential expression models. We found the effects on the expression of CA12, FIG RPL5 and P2RY11 to be different upon BTK inhibition between UM- and M-CLL ( FDR &amp;lt; 0.1). In conclusion, we establish shallow depth sequencing in CLL and show it can be used to reliably capture disease biology and drug effects. By treating patient samples with ibrutinib, we show that abrogating BTK strongly suppresses BCR-dependent processes which are largely independent of IGHV status. By adding more pathway perturbations, this approach could be used for further functional dissection of CLL biology.