C-C Motif Chemokine Ligand 2 Research Articles

Suberanilohydroxamic acid (SAHA), a HDAC inhibitor, suppresses the effect of Treg cells by targeting the c-Myc/CCL1 pathway in glioma stem cells and improves PD-L1 blockade therapy.

A strong immunosuppressive tumor microenvironment (TME) represents the major barrier responsible for the failure of current immunotherapy approaches in treating Glioblastoma Multiforme (GBM). Within the TME, the regulatory T cells (Tregs) exert immunosuppressive effects on CD8+ T cell - mediated anti-cancer immune killing. Consequently, targeting and inhibiting their immunosuppressive function emerges as an effective therapeutic strategy for GBM. The present study aimed to investigate the mechanisms and effects of Suberanilohydroxamic Acid (SAHA), a histone deacetylase inhibitor, on immunosuppressive Tregs. The tumor-infiltrating immune cells in the immunocompetent GBM intracranial implanted xenograft mouse model were analyzed by immunohistochemistry and flow cytometry techniques. The mRNA expressions were assessed through the RT-qPCR method, while the related protein expressions were determined using western blot, ELISA, immunofluorescence (IF), and flow cytometry techniques. The relationship between c-Myc and C-C motif Chemokine Ligand 1 (CCL1) promotor was validated through a dual-luciferase reporter assay system and chromatin immunoprecipitation. SAHA suppressed effectively tumor growth and extended significantly overall survival in the immunocompetent GBM intracranial xenograft mouse model. Additionally, it promoted the infiltration of CD8+ T lymphocytes while suppressed the infiltration of CD4+ CD25+ Tregs. Furthermore, SAHA enhanced anti-PD-L1 immune therapy in the intracranial xenograft of mice. Mechanistically, SAHA exerted its effects by inhibiting histone deacetylase 2 (HDAC2), thereby suppressing the binding between c-Myc and the CCL1 promotor. SAHA inhibited the binding of c-Myc with the CCL1 promoter and then suppressed the transcription of CCL1.Additionally, it effectively blocked the interplay of CCL1-CCR8, resulting in reduced activity of Tregs and alleviation of tumor immunosuppression.

Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells.

Inflammation processes of the central nervous system (CNS) play a vital role in the pathogenesis of several neurological and psychiatric disorders like depression. These processes are characterized by the activation of glia cells, such as microglia. Clinical studies showed a decrease in symptoms associated with the mentioned diseases after the treatment with anti-inflammatory drugs. Therefore, the investigation of novel anti-inflammatory drugs could hold substantial potential in the treatment of disorders with a neuroinflammatory background. In this in vitro study, we report the anti-inflammatory effects of a novel hexacyclic peptide-peptoid hybrid in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The macrocyclic compound X15856 significantly suppressed Interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), c-c motif chemokine ligand 2 (CCL2), CCL3, C-X-C motif chemokine ligand 2 (CXCL2), and CXCL10 expression and release in LPS-treated BV2 microglial cells. The anti-inflammatory effects of the compound are partially explained by the modulation of the phosphorylation of p38 mitogen-activated protein kinases (MAPK), p42/44 MAPK (ERK 1/2), protein kinase C (PKC), and the nuclear factor (NF)-κB, respectively. Due to its remarkable anti-inflammatory properties, this compound emerges as an encouraging option for additional research and potential utilization in disorders influenced by inflammation, such as depression.

Spatiotemporally Controlled T-Cell Combination Therapy for Solid Tumor.

Due to multidimensional complexity of solid tumor, development of rational T-cell combinations and corresponding formulations is still challenging. Herein, a triple combination of T cells are developed with Indoleamine 2,3-dioxygenase inhibitors (IDOi) and Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). To maximize synergism, a spatiotemporally controlled T-cell engineering technology to formulate triple drugs into one cell therapeutic, is established. Specifically, a sequentially responsive core-shell nanoparticle (SRN) encapsulating IDOi and CDK4/6i is anchored onto T cells. The yielded SRN-T cells migrated into solid tumor, and achieved a 1st release of IDOi in acidic tumor microenvironment (TME). Released IDOi restored tryptophan supply in TME, which activated effector T cells and inhibited Tregs. Meanwhile, 1st released core is internalized by tumor cells and degraded by glutathione (GSH), to realize a 2nd release of CDK4/6i, which induced up-regulated expression of C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine ligand 5 (CCL5), and thus significantly increased tumor infiltration of T cells. Together, with an enhanced recruitment and activation, T cells significantly suppressed tumor growth, and prolonged survival of tumor-bearing mice. This study demonstrated rationality and superiority of a tri-drug combination mediated by spatiotemporally controlled cell-engineering technology, which provides a new treatment regimen for solid tumor.

CCL2 regulated by the CTBP1-AS2/miR-335-5p axis promotes hemangioma progression and angiogenesis

Context Hemangioma (HA) is a benign vascular neoplasm that can lead to permanent scarring. C-C motif chemokine ligand 2 (CCL2) plays a crucial role in facilitating growth and angiogenesis during HA progression. However, the mechanism regulating CCL2 in HA remains poorly elucidated. Objective To elucidate the mechanism regulating CCL2 in HA. Methods Quantitative real-time polymerase chain reaction (RT-qPCR) was employed to determine the expression levels of CCL2, long noncoding RNA (lncRNA) CTBP1 divergent transcript (CTBP1-AS2), and microRNAs (miRNAs). Proliferation, migration, invasion, and angiogenic abilities of human HA endothelial cells (HemECs) were assessed using cell counting kit-8 (CCK-8), colony formation, flow cytometry, transwell, and tube formation assays. Bioinformatics analysis, RNA pull-down, and luciferase reporter assays were conducted to investigate whether CCL2 targets miR-335-5p. Additionally, rescue experiments were performed in this study. Results CCL2 expression was markedly upregulated in HemECs. CCL2 promoted HA cell proliferation, migration, invasion, and angiogenesis while inhibiting apoptosis. CCL2 was directly targeted by miR-335-5p. Additionally, we found that CTBP1-AS2 could function as a competing endogenous RNA (ceRNA) to sponge miR-335-5p, thereby upregulating CCL2. Conclusion Our findings suggest that targeting the CTBP1-AS2/miR-335-5p/CCL2 axis may hold promise as a therapeutic strategy for HA.

Deregulation in adult IgA vasculitis skin as the basis for the discovery of novel serum biomarkers

IntroductionImmunoglobulin A vasculitis (IgAV) in adults has a variable disease course, with patients often developing gastrointestinal and renal involvement and thus contributing to higher mortality. Due to understudied molecular mechanisms in IgAV currently used biomarkers for IgAV visceral involvement are largely lacking. Our aim was to search for potential serum biomarkers based on the skin transcriptomic signature.MethodsRNA sequencing analysis was conducted on skin biopsies collected from 6 treatment-naïve patients (3 skin only and 3 renal involvement) and 3 healthy controls (HC) to get insight into deregulated processes at the transcriptomic level. 15 analytes were selected and measured based on the transcriptome analysis (adiponectin, lipopolysaccharide binding protein (LBP), matrix metalloproteinase-1 (MMP1), C-C motif chemokine ligand (CCL) 19, kallikrein-5, CCL3, leptin, C-X-C motif chemokine ligand (CXCL) 5, osteopontin, interleukin (IL)-15, CXCL10, angiopoietin-like 4 (ANGPTL4), SERPIN A12/vaspin, IL-18 and fatty acid-binding protein 4 (FABP4)) in sera of 59 IgAV and 22 HC. Machine learning was used to assess the ability of the analytes to predict IgAV and its organ involvement.ResultsBased on the gene expression levels in the skin, we were able to differentiate between IgAV patients and HC using principal component analysis (PCA) and a sample-to-sample distance matrix. Differential expression analysis revealed 49 differentially expressed genes (DEGs) in all IgAV patient’s vs. HC. Patients with renal involvement had more DEGs than patients with skin involvement only (507 vs. 46 DEGs) as compared to HC, suggesting different skin signatures. Major dysregulated processes in patients with renal involvement were lipid metabolism, acute inflammatory response, and extracellular matrix (ECM)-related processes. 11 of 15 analytes selected based on affected processes in IgAV skin (osteopontin, LBP, ANGPTL4, IL-15, FABP4, CCL19, kallikrein-5, CCL3, leptin, IL-18 and MMP1) were significantly higher (p-adj < 0.05) in IgAV serum as compared to HC. Prediction models utilizing measured analytes showed high potential for predicting adult IgAV.ConclusionSkin transcriptomic data revealed deregulations in lipid metabolism and acute inflammatory response, reflected also in serum analyte measurements. LBP, among others, could serve as a potential biomarker of renal complications, while adiponectin and CXCL10 could indicate gastrointestinal involvement.

Structure-activity relationship of anti-inflammatory meroterpenoids isolated from Dictyopteris polypodioides in RAW264 cells.

In this study, we explored anti-inflammatory compounds from the brown alga Dictyopteris polypodioides and isolated 7 meroterpenoids. Their anti-inflammatory activities were evaluated using the lipopolysaccharide-stimulated mouse macrophage cell line, RAW264. Yahazunol (1) exhibited similar nitric oxide (NO) production inhibitory activity as zonarol (2), which has previously been shown to be an anti-inflammatory compound. Yahazunol (1), zonarol (2), and isozonarol (3) inhibited not only NO production but also inducible nitric oxide synthase, interleukin-6, and C-C motif chemokine ligand 2 mRNA expression in RAW264 cells. The structure-activity relationships of the 11 compounds, including their synthetic analogs, revealed the significance of the hydroquinone moiety in the anti-inflammatory activity of these sesquiterpenoids in RAW264 cells. Diacetylated zonarol (9) exhibited an activity comparable to that of zonarol as a result of intracellular deacetylation. These results provide new insights into the anti-inflammatory activity of hydroquinone-containing natural products.

Abstract 2021: The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes approximately 50% of affected individuals to develop plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs) in approximately 10% of patients. NF1 is caused by mutations in the tumor suppressor gene NF1, which encodes for neurofibromin, a negative regulator of RAS activity. Selumetinib, a specific inhibitor of MEK1/2, is the only FDA-approved drug for NF1-associated PNFs. However, the anti-tumor effects of selumetinib are limited in MPNSTs and have dose-limiting side effects. Deficiency of the NF1 gene not only promotes tumorigenesis but also has broad effects on the immune cells and cytokine signaling driven by hyperactive RAS signaling. Because macrophages account for almost half of cells in NF1 lesions and their infiltration correlates with disease progression, we hypothesized that targeting tumor-promoting immune cells could be an alternative approach for treating NF1. The novel retinoid X receptor (RXR) agonist MSU-42011 reduces tumor growth in experimental Kras-driven cancers by decreasing pERK expression, reducing tumor-promoting immune cells like CD206+ macrophages and regulatory T cells, and increasing activated cytotoxic T cells. Here, we treated NF1-deficient cells and macrophages with MSU-42011 and selumetinib, either alone or in combination, using monoculture and conditioned media (CM) conditions. In human PNF cells and mouse MPNSTs, treatment with 200 nM MSU-40211 or 50 nM selumetinib for 3 hours reduced pERK protein levels compared with untreated controls, and the combination treatment enhanced this reduction in pERK protein levels. Additionally, there was a trend toward reduction in cell viability with increasing drug concentrations after 72 hours of the combination treatment. Moreover, CM from human and mouse PNF cells increased the mRNA expression of monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2) and the secretion of IL-6 and TNFα in human THP1 monocytes/macrophages and bone marrow derived macrophages (BMDM). Notably, MSU-42011 and selumetinib alone inhibited CCL2 mRNA expression in THP1 macrophages and BMDM stimulated with CM from human and mouse PNF cells, respectively, and the inhibition of CCL2 mRNA expression was greatest with combination treatment. The combination of MSU42011 and selumetinib also significantly reduced tumor burden in a LL2 model of lung cancer driven by an activating Kras mutation. Based on the similarities in RAS activation and immune cell infiltration in NF1 and lung cancer, our next step is to confirm the immunomodulatory and anti-tumor effects of MSU-42011 and selumetinib in a syngeneic model of PNF and MPNST. Citation Format: Pei-Yu Hung, Jessica A. Moerland, Karen T. Liby. The RXR agonist MSU-42011 and the MEK inhibitor selumetinib reduce pERK levels in NF1-deficient cells and inhibit cytokine production in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2021.

Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease

Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling invitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

Prior Exposure to Experimental Preeclampsia Increases Atherosclerotic Plaque Inflammation in Atherogenic Mice-Brief Report.

Women with a history of preeclampsia have evidence of premature atherosclerosis and increased risk of myocardial infarction and stroke compared with women who had a normotensive pregnancy. Whether this is due to common risk factors or a direct impact of prior preeclampsia exposure has never been tested in a mouse atherosclerosis model. Pregnant LDLR-KO (low-density lipoprotein receptor knockout; n=35) female mice were randomized in midgestation to sFlt1 (soluble fms-like tyrosine kinase 1)-expressing adenovirus or identical control adenovirus. Postpartum, mice were fed high-fat diet for 8 weeks to induce atherogenesis. Comparison between the control and preeclampsia models was made for metabolic parameters, atherosclerosis burden and composition by histology, plaque inflammation by flow cytometry, and aortic cytokines and inflammatory markers using a cytokine array. In pregnant LDLR-KO mice, sFlt1 adenovirus significantly induced serum sFlt1, blood pressure, renal endotheliosis, and decreased pup viability. After 8 weeks of postpartum high fat feeding, body weight, fasting glucose, plasma cholesterol, HDL (high-density lipoprotein), and LDL (low-density lipoprotein) were not significantly different between groups with no change in aortic root plaque size, lipid content, or necrotic core area. Flow cytometry demonstrated significantly increased CD45+ aortic arch leukocytes and CD3+T cells and aortic lysate contained more CCL (CC motif chemokine ligand) 22 and fetuin A and decreased expression of IGFBP6 (insulin-like growth factor-binding protein 6) and CCL21 in preeclampsia-exposed mice compared with controls. In atherogenic LDLR-KO mice, exposure to sFlt1-induced preeclampsia during pregnancy increases future atherosclerotic plaque inflammation, supporting the concept that preeclampsia directly exacerbates atherosclerotic inflammation independent of preexisting risk factors. This mechanism may contribute to ischemic vascular disease in women after preeclampsia pregnancy.

C-Cmotif chemokine ligand 2 regulates prostaglandin synthesis and embryo attachment of the bovine endometrium during implantation.

C-C motif chemokine ligand 2 (CCL2) has been reported to be expressed in the bovine endometrium during pregnancy. However, the details of its functions involved in the implantation mechanism are still not clear. The purpose of this study is to analyze the functional properties of CCL2 in the bovine endometrium and embryos. The expression of CCR2 was not different between the luteal phase and implantation phase of their endometrial tissues, but was significantly high in IFNa treated bovine endometrial stromal (BES) cells in vitro. The expressions of PGES1, PGES2, AKR1C4, and AKR1C4 were high at the implantation stage compared with the luteal stage. On the other hand, PGES2 and AKR1B1 in BEE and PGES3 and AKR1A1 in BES were significantly increased by CCL2 treatment, respectively. The expressions of PCNA and IFNt were found significantly high in the bovine trophoblastic cells (BT) treated with CCL2 compared to the control. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The expression of OPN and ICAM-1 increased in BEE, and ICAM-1 increased in BT by CCL2 treatment, respectively. The present results indicate that CCL2 has the potential to regulate the synthesis of PGs in the endometrium and the embryo growth. In addition, CCL2 has the possibility to regulate the process of bovine embryo attachment to the endometrium by modulation of binding molecules expression.

Lactate dehydrogenase A regulates tumor-macrophage symbiosis to promote glioblastoma progression

Abundant macrophage infiltration and altered tumor metabolism are two key hallmarks of glioblastoma. By screening a cluster of metabolic small-molecule compounds, we show that inhibiting glioblastoma cell glycolysis impairs macrophage migration and lactate dehydrogenase inhibitor stiripentol emerges as the top hit. Combined profiling and functional studies demonstrate that lactate dehydrogenase A (LDHA)-directed extracellular signal-regulated kinase (ERK) pathway activates yes-associated protein 1 (YAP1)/ signal transducer and activator of transcription 3 (STAT3) transcriptional co-activators in glioblastoma cells to upregulate C-C motif chemokine ligand 2 (CCL2) and CCL7, which recruit macrophages into the tumor microenvironment. Reciprocally, infiltrating macrophages produce LDHA-containing extracellular vesicles to promote glioblastoma cell glycolysis, proliferation, and survival. Genetic and pharmacological inhibition of LDHA-mediated tumor-macrophage symbiosis markedly suppresses tumor progression and macrophage infiltration in glioblastoma mouse models. Analysis of tumor and plasma samples of glioblastoma patients confirms that LDHA and its downstream signals are potential biomarkers correlating positively with macrophage density. Thus, LDHA-mediated tumor-macrophage symbiosis provides therapeutic targets for glioblastoma.

Mechanic evaluation of Wu-Mei-Pill on colitis-associated colorectal cancer: An integrated transcriptomics, metabolomics, and experimental validation study

BackgroundChronic intestinal inflammatory diseases play a crucial role in the onset of colorectal cancer (CRC). Effectively impeding the progression of colitis-associated colorectal cancer (CAC) can be instrumental in hindering CRC development. Wu-Mei-Pill (WMP), a formulation comprising various herbal extracts, is clinically employed for CAC treatment, yet the underlying mechanism of WMP's efficacy in CAC remains unclear. Our study firstly demonstrated the effects and mechanisms of WMP on transcriptional and metabolic levels based on integrated transcriptomics and untargeted metabolomics and relative experimental validations. Materials and methodsA CAC mouse model was established through a single injection of azoxymethane (AOM) followed by intermittent dextran sodium sulfate (DSS) intervention, with subsequent WMP administration. Initially, the therapeutic impact of WMP on the CAC model was assessed by observing survival rate, body weight change, colon length, tumor number, tumor load, and pathological changes in the colon tissue of CAC mice post-WMP intervention. Subsequently, differential genes and metabolites in the colorectal tissue of CAC mice following WMP intervention were identified through transcriptomics and non-targeted metabolomics. Finally, the influence of WMP on the peroxisome proliferator activated receptor (PPAR) pathway, Wnt pathway, and CC motif chemokine ligand 3 (CCL3)/ CC motif chemokine receptor 1 (CCR1) axis in CAC mice was verified through western blot, immunofluorescence, and ELISA based on the results of transcriptomics and non-targeted metabolomics. ResultsWMP intervention enhanced survival, alleviated body weight loss, shortened colon length, tumor occurrence, and pathological changes in the colorectal tissue of CAC mice, such as glandular damage, tumourigenesis, and inflammatory cell infiltration. Transcriptomic and non-targeted metabolomic results revealed that WMP intervention up-regulated the expression of key regulatory mechanisms of fatty acid oxidation PPAR pathway-related genes (Pparg, Ppara, Cpt1a, and Acadm) and metabolites (L-carnitine and L-palmitoylcarnitine). Additionally, it down-regulated Wnt pathway-related genes (Wnt3, Axin2, Tcf7, Mmp7, Lgr5, Wnt5a, Fzd6, Wnt7b, Lef1, and Fzd10 etc.) and pro-inflammatory related genes (Il1b, Il6, Il17a, Ccl3, and Ccr1 etc.). Experimental validation demonstrated that WMP up-regulated PPAR pathway-related proteins [PPARγ, PPARα, carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA dehydrogenase medium chain (ACADM)] in the colorectal tissue of CAC mice. It also down-regulated Wnt pathway-related proteins [β-catenin, T-cell factor (TCF), lymphoid enhancer-binding factor (LEF), and matrix metallopeptidase 7 (MMP7)], inhibited the nuclear translocation of the key transcription factor β-catenin in the Wnt pathway, and suppressed epithelial-to-mesenchymal transition (EMT) activation induced by the Wnt pathway (up-regulated E-cadherin and down-regulated Vimentin). Furthermore, WMP intervention reduced pro-inflammatory factors [interleukin (IL)-6, IL-1β, and IL-17A] and decreased CCL3/CCR1 axis factors, including CCL3 protein levels and diminished F4/80+CCR1+ positive expressed cells. ConclusionWMP significantly inhibits CAC tumorigenesis by up-regulating PPARα-mediated fatty acid oxidation, inhibiting the Wnt signaling pathway-mediated EMT, and suppressing CCL3/CCR1-mediated inflammatory responses.

C-C Motif Chemokine Ligand 5 (CCL5): A Potential Biomarker and Immunotherapy Target for Osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, which has an insidious onset and is difficult to detect early, and few early diagnostic markers with high specificity and sensitivity. Therefore, this study aims to identify potential biomarkers that can help diagnose OS in its early stages and improve the prognosis of patients. The data sets of GSE12789, GSE28424, GSE33382 and GSE36001 were combined and normalized to identify Differentially Expressed Genes (DEGs). The data were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genome (KEGG) and Disease Ontology (DO). The hub gene was selected based on the common DEG that was obtained by applying two regression methods: the Least Absolute Shrinkage and Selection Operator (LASSO) and Support vVector Machine (SVM). Then the diagnostic value of the hub gene was evaluated in the GSE42572 data set. Finally, the correlation between immunocyte infiltration and key genes was analyzed by CIBERSORT. The regression analysis results of LASSO and SVM are the following three DEGs: FK501 binding protein 51 (FKBP5), C-C motif chemokine ligand 5 (CCL5), complement component 1 Q subcomponent B chain (C1QB). We evaluated the diagnostic performance of three biomarkers (FKBP5, CCL5 and C1QB) for osteosarcoma using receiver operating characteristic (ROC) analysis. In the training group, the area under the curve (AUC) of FKBP5, CCL5 and C1QB was 0.907, 0.874 and 0.676, respectively. In the validation group, the AUC of FKBP5, CCL5 and C1QB was 0.618, 0.932 and 0.895, respectively. It is noteworthy that these genes were more expressed in tumor tissues than in normal tissues by various immune cell types, such as plasma cells, CD8+ T cells, T regulatory cells (Tregs), activated NK cells, activated dendritic cells and activated mast cells. These immune cell types are also associated with the expression levels of the three diagnostic genes that we identified. We found that CCL5 can be considered an early diagnostic gene of osteosarcoma, and CCL5 interacts with immune cells to influence tumor occurrence and development. These findings have important implications for the early detection of osteosarcoma and the identification of novel therapeutic targets.

Caspase-8 activation regulates enterovirus D68 infection-induced inflammatory response and cell death

Enterovirus D68 (EV-D68) infection causes severe acute respiratory infection and severe neurological complications, such as acute flaccid myelitis (AFM), in children. However, although EV-D68 has pandemic potential, no effective drugs or vaccines are currently clinically available. Furthermore, EV-D68 infection-induced inflammatory response and cell death are not fully understood. In this study, we demonstrated that several inflammatory cytokines were upregulated in a multiplicity of infection (MOI) dependent manner in EV-D68-infected human rhabdomyosarcoma (RD) cells. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) confirmed that tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), C-C motif chemokine ligand-5 (CCL-5), and CXC motif chemokine ligand-5 (CXCL-5) mRNA levels were highly upregulated after EV-D68 infection. IL-1β processing and maturation mediated by caspase-8 was inhibited by the caspase-8 inhibitor Z-IETD-FMK. EV-D68 infection activates caspase-8 to mediate IL-1β maturation and secretion. Additionally, EV-D68 activated cell death-related proteins such as caspase-3, poly (ADP-ribose) polymerase 1 (PARP-1), phosphorylation of Mixed Lineage Kinase domain-like protein (pMLKL), and gasdermin E (GSDME). Thus, EV-D68 infection activates caspase-8, which triggers the necroptosis and apoptosis pathways. Overall, our data suggest that caspase-8 activation is associated with the inflammatory response and cell death in EV-D68-infected RD cells. This mechanism represents a novel target for the treatment of EV-D68 infection by inhibiting caspase-8 activation.

Directing the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells represent a promising cancer therapy platform because the implementation of allogenic, off-the-shelf product candidates is possible. However, intravenous administration of human Vγ9Vδ2 T cells manufactured under good manufacturing practice (GMP)-compliant, serum-free conditions are not tested easily in most mouse models, mainly because they lack the ability to migrate from the blood to tissues or tumors. We demonstrate that these T cells do not migrate from the circulation to the mouse bone marrow (BM), the site of many malignancies. Thus, there is a need to better characterize human γδ T-cell migration in vivo and develop strategies to direct these cells to in vivo sites of therapeutic interest. To better understand the migration of these cells and possibly influence their migration, NSG mice were conditioned with agents to clear BM cellular compartments, i.e., busulfan or total body irradiation (TBI), or promote T-cell migration to inflamed BM, i.e., incomplete Freund's adjuvant (IFA), prior to administering γδ T cells. Conditioning with TBI, unlike busulfan or IFA, increases the percentage and number of γδ T cells accumulating in the mouse BM, and cells in the peripheral blood (PB) and BM display identical surface protein profiles. To better understand the mechanism by which cells migrate to the BM, mice were conditioned with TBI and administered γδ T cells or tracker-stained red blood cells. The mechanism by which γδ T cells enter the BM after radiation is passive migration from the circulation, not homing. We tested if these ex vivo-expanded cells can migrate based on chemokine expression patterns and showed that it is possible to initiate homing by utilizing highly expressed chemokine receptors on the expanded γδ T cells. γδ T cells highly express CCR2, which provides chemokine attraction to C-C motif chemokine ligand 2 (CCL2)-expressing cells. IFNγ-primed mesenchymal stromal cells (MSCs) (γMSCs) express CCL2, and we developed in vitro and in vivo models to test γδ T-cell homing to CCL2-expressing cells. Using an established neuroblastoma NSG mouse model, we show that intratumorally-injected γMSCs increase the homing of γδ T cells to this tumor. These studies provide insight into the migration of serum-free, ex vivo-expanded Vγ9Vδ2 T cells in NSG mice, which is critical to understanding the fundamental properties of these cells.

Development of a CCR2 targeted 18F-labeled radiotracer for atherosclerosis imaging with PET

Atherosclerosis is a chronic inflammatory disease and the leading cause of morbidity and mortality worldwide. CC motif chemokine ligand 2 and its corresponding cognate receptor 2 (CCL2/CCR2) signaling has been implicated in regulating monocyte recruitment and macrophage polarization during inflammatory responses that plays a pivotal role in atherosclerosis initiation and progression. In this study, we report the design and synthesis of a novel 18F radiolabeled small molecule radiotracer for CCR2-targeted positron emission tomography (PET) imaging in atherosclerosis. The binding affinity of this radiotracer to CCR2 was evaluated via in vitro binding assay using CCR2+ membrane and cells. Ex vivo biodistribution was carried out in wild type mice to assess radiotracer pharmacokinetics. CCR2 targeted PET imaging of plaques was performed in two murine atherosclerotic models. The sensitive detection of atherosclerotic lesions highlighted the potential of this radiotracer for CCR2 targeted PET and warranted further optimization.

Oxidized low-density lipoproteins impair the pro-atherosclerotic effect of granulocyte-macrophage-colony-stimulating factor-producing T helper cells on macrophages.

T cells contribute to the pathogenesis of atherosclerosis. However, the presence and function of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in atherosclerosis development is unknown. This study aims to characterize the phenotype and function of ThGM cells in experimental atherosclerosis. Atherosclerosis was induced by feeding apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet. Aortic ThGM cells were detected and sorted by flow cytometry. The effect of oxidized low-density lipoprotein (oxLDL) on ThGM cells and the impact of ThGM cells on macrophages were evaluated by flow cytometry, quantitative RT-PCR, oxLDL binding/uptake assay, immunoblotting and foam cell formation assay. We found that GM-CSF+IFN-γ- ThGM cells existed in atherosclerotic aortas. Live ThGM cells were enriched in aortic CD4+CCR6-CCR8-CXCR3-CCR10+ T cells. Aortic ThGM cells triggered the expression of interleukin-1β (IL-1β), tumour necrosis factor (TNF), interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in macrophages. Besides, aortic ThGM cells expressed higher CD69 than other T cells and bound to oxLDL. oxLDL suppressed the cytokine expression in ThGM cells probably via inhibiting the signal transducer and activator of transcription 5 (STAT5) signalling. Furthermore, oxLDL alleviated the effect of ThGM cells on inducing macrophages to produce pro-inflammatory cytokines and generate foam cells. The nuclear receptor subfamily 4 group A (NR4A) members NR4A1 and NR4A2 were involved in the suppressive effect of oxLDL on ThGM cells. Collectively, oxLDL suppressed the supportive effect of ThGM cells on pro-atherosclerotic macrophages.

Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway

Lower back pain (LBP), which is a primary cause of disability, is largely attributed to intervertebral disc degeneration (IDD). Macrophages (MΦs) in degenerated intervertebral discs (IVDs) form a chronic inflammatory microenvironment, but how MΦs are recruited to degenerative segments and transform into a proinflammatory phenotype remains unclear. We evaluated chemokine expression in degenerated nucleus pulposus cells (NPCs) to clarify the role of NPCs in the establishment of an inflammatory microenvironment in IDD and explored the mechanisms. We found that the production of C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 7 (CCL7) was significantly increased in NPCs under inflammatory conditions, and blocking CCL2/7 and their receptor, C-C chemokine receptor type 2(CCR2), inhibited the inductive effects of NPCs on MΦ infiltration and proinflammatory polarization. Moreover, activation of the integrated stress response (ISR) was obvious in IDD, and ISR inhibition reduced the production of CCL2/7 in NPCs. Further investigation revealed that activating Transcription Factor 3 (ATF3) responded to ISR activation, and ChIP-qPCR verified the DNA-binding activity of ATF3 on CCL2/7 promoters. In addition, we found that Toll-like receptor 4 (TLR4) inhibition modulated ISR activation, and TLR4 regulated the accumulation of mitochondrial reactive oxygen species (mtROS) and double-stranded RNA (dsRNA). Downregulating the level of mtROS reduced the amount of dsRNA and ISR activation. Deactivating the ISR or blocking CCL2/7 release alleviated inflammation and the progression of IDD in vivo. Moreover, MΦ infiltration and IDD were inhibited in CCR2-knockout mice. In conclusion, this study highlights the critical role of TLR4/mtROS/dsRNA axis-mediated ISR activation in the production of CCL2/7 and the progression of IDD, which provides promising therapeutic strategies for discogenic LBP.

Bombesin-like receptor 3 expression induced by bisphenol A is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing inflammation in liver cells.

Bisphenol A (BPA) is an exogenous endocrine disruptor mimicking hormones closely associated with health complications, such as cancer progression. BPA is also related to an increase in the prevalence of obesity-related diseases due to its obesogenic action. Bombesin-like receptor 3 (BRS3) is an important factor that should be considered in the adipogenic gene network, as depletion of this gene alters adiposity. Therefore, the present study aimed to investigate the messenger ribonucleic acid (mRNA) expression of BRS3 in human liver THLE-2 cells post-BPA treatment by real-time polymerase chain reaction. The effects of BPA on the levels of pro-inflammatory proteins, interleukin 6 (IL6) and CC motif chemokine ligand 2 (CCL2), in conditioned media of BPA-treated THLE-2 cells and deoxyribonucleic acid (DNA) synthesis in replicating BPA-treated THLE-2 cells during the cell cycle were also examined by enzyme-linked immunosorbent assay(ELISA) and flow cytometry, respectively. The study found that the mRNA expression of BRS3 was increased in THLE-2 cells treated with BPA. The study also showed that the expression levels of IL6 and CCL2 reached an optimum level in the conditioned media of BPA-treated THLE-2 cells after 48h of treatment. Subsequently, the DNA synthesis analysis showed that bromodeoxyuridine/propidium iodide(BrdU/PI) stained positive cells were decreased in BPA-treated THLE-2 cells at 72h of treatment. The study demonstrates that BRS3 expression induced by BPA is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing cellular inflammation in liver cells.

OP30 Endothelial cell-mediated smooth muscle hyperplasia in Crohn’s disease intestinal strictures: Caveolin 1 as a potential therapeutic target

Abstract Background Intestinal stricture is a severe and common complication of Crohn’s disease (CD). Current therapies offer limited success and surgery is often needed. Smooth muscle hyperplasia and hypertrophy play a key role in CD stricture development. This study explores the transcriptomic features of fibrostenotic CD, focusing on the endothelial contribution to smooth muscle changes within intestinal strictures. Methods 9 CD patients undergoing surgery for fibrotic strictures were prospectively enrolled. Dissected samples from the stricture and the adjacent proximal and distal non-strictured areas were obtained from fresh specimens collected directly from the operating theatre. RNA was extracted from all samples for bulk-transcriptomic analysis. Differentially expressed genes (adj. p-value &amp;lt;0.05) were identified and pathway enrichment analysis pinpointed relevant pathways. Subsequently, single-cell RNA sequencing (scRNAseq) analysis was performed using the 10x Chromium Controller® platform. A total of 31.195 cells were sequenced and bioinformatic analysis revealed 13 distinct clusters based on the expression of key lineage target genes. To better characterise the nature of clusters expressing stromal like markers and positioned closely in the UMAP plot, a further subclustering was performed. This resulted in 5 groups, including plasma cells, endothelial cells, fibroblasts, epithelial cells and dendritic like cells. Results Bulk-transcriptomic analysis identified 81 genes differentially expressed in strictures compared to both proximal and distal regions. Notably, genes associated with leukocyte and macrophage recruitment and chemotaxis, namely Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), C-C motif Chemokine Ligand 2 (CCL2), and Endothelin Receptor Type B (EDNRB), resulted overexpressed in strictures. scRNAseq identified a distinct subset of endothelial cells (CD34+, PECAM1+). Caveolin 1 (CAV1), a gene encoding for a structural protein predominantly expressed in smooth muscle cell plasma membrane invaginations, was exclusively up-regulated in endothelial cells of ileal stricture samples. Previous research linked CAV1 to smooth muscle proliferation and fibrosis in various organs. Conclusion This study highlights the pivotal role of endothelial cells in CD strictures. The transcriptomic analysis shows the involvement of inflammatory targets featured genes involved in transendothelial immune cell migration. Furthermore, the up-regulation of CAV1 in endothelial cells within stricture segments suggests a mechanistic connection between endothelial cells and smooth muscle hyperplasia in intestinal strictures. CAV1 holds promise as a potential therapeutic target in fibrostenotic CD.