An atlas of genetic effects on cellular composition of the tumor microenvironment.

Deciphering the composition of the tumor microenvironment (TME) is critical for understanding tumorigenesis and to design immunotherapies. In the present study, we mapped genetic effects on cell-type proportions using single-cell and bulk RNA sequencing data, identifying 3,494 immunity quantitative trait loci (immunQTLs) across 23 cancer types from The Cancer Genome Atlas. Functional annotation revealed regulatory potential and we further assigned 1,668 genes that regulate TME composition. We constructed a combined immunQTL map by integrating data from European and Chinese colorectal cancer (CRC) samples. A polygenic risk score that incorporates these immunQTLs and hits on a genome-wide association study outperformed in CRC risk stratification within 447,495 multiethnic individuals. Using large-scale population cohorts, we identified that the immunQTL rs1360948 is associated with CRC risk and prognosis. Mechanistically, the rs1360948-G-allele increases CCL2 expression, recruiting regulatory T cells that can exert immunosuppressive effects on CRC progression. Blocking the CCL2-CCR2 axis enhanced anti-programmed cell death protein 1 ligand therapy. Finally, we have established a database (CancerlmmunityQTL2) to serve the research community and advance our understanding of immunogenomic interactions in cancer pathogenesis.

Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool.

Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages.

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.

Sex chromosomes and hormones independently influence healthy brain development but act similarly after cranial radiation.

The course of normal development and response to pathology are strongly influenced by biological sex. For instance, female childhood cancer survivors who have undergone cranial radiation therapy (CRT) tend to display more pronounced cognitive deficits than their male counterparts. Sex effects can be the result of sex chromosome complement (XX vs. XY) and/or gonadal hormone influence. The contributions of each can be separated using the four-core genotype mouse model (FCG), where sex chromosome complement and gonadal sex are decoupled. While studies of FCG mice have evaluated brain differences in adulthood, it is still unclear how sex chromosome and sex hormone effects emerge through development in both healthy and pathological contexts. Our study utilizes longitudinal MRI with the FCG model to investigate sex effects in healthy development and after CRT in wildtype and immune-modified Ccl2-knockout mice. Our findings in normally developing mice reveal a relatively prominent chromosome effect prepubertally, compared to sex hormone effects which largely emerge later. Spatially, sex chromosome and hormone influences were independent of one another. After CRT in Ccl2-knockout mice, both male chromosomes and male hormones similarly improved brain outcomes but did so more separately than in combination. Our findings highlight the crucial role of sex chromosomes in early development and identify roles for sex chromosomes and hormones after CRT-induced inflammation, highlighting the influences of biological sex in both normal brain development and pathology.

ALS iPSC-derived microglia and motor neurons respond to astrocyte-targeted IL-10 and CCL2 modulation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs). The loss of MNs in ALS leads to muscle weakness and wasting, respiratory failure, and death often within two years of diagnosis. Glial cells in ALS show aberrant expression of pro-inflammatory and neurotoxic proteins associated with activation and have been proposed as ideal therapeutic targets. In this study, we examined astrocyte-targeted treatments to reduce glial activation and neuron pathology using cells differentiated from ALS patient-derived iPSC carrying SOD1 and C9ORF72 mutations. Specifically, we tested the ability of increasing interleukin 10 (IL-10) and reducing C-C motif chemokine ligand 2 (CCL2/MCP-1) signaling targeted to astrocytes to reduce activation phenotypes in both astrocytes and microglia. Overall, we found IL10/CCL2NAb treated astrocytes to support anti-inflammatory phenotypes and reduce neurotoxicity, through different mechanisms in SOD1 and C9ORF72 cultures. We also found altered responses of microglia and motor neurons to astrocytic influences when cells were cultured together rather than in isolation. Together these data support IL-10 and CCL2 as non-mutation-specific therapeutic targets for ALS and highlight the role of glial-mediated pathology in this disease.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Hdac3 deficiency limits periosteal reaction associated with Western diet feeding in female mice.

Diet-induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti-inflammatory agents. Prior reports show that myeloid progenitor-directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high-fat/high-sugar (HFD) diet. Micro-CT analyses demonstrated that HFD-feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3-ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3-ablated cells exhibited a four-fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2-positve cells within bone defects was significantly higher in Hdac3-deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet-induced obesity, possibly through increased production of CCL2 by macrophages within the defect.

A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2.

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Spinal nerve transection-induced upregulation of KDM4A in the dorsal root ganglia contributes to the development and maintenance of neuropathic pain via promoting CCL2 expression in rats.

Studies indicate that the lysine-specific demethylase 4A (KDM4A), acts as a key player in neuropathic pain, driving the process through its involvement in promoting neuroinflammation. Emerging evidence reveals that C-C Motif Chemokine Ligand 2 (CCL2) participates in neuroinflammation, which plays an important role in the development and maintenance of neuropathic pain. However, it remains unclear if KDM4A plays a role in regulating CCL2 in neuropathic pain. This study found that following spinal nerve transection (SNT) of the lumbar 5 nerve root in rats, the expression of KDM4A and CCL2 increased in the ipsilateral L4/5 dorsal root ganglia (DRG). Injecting KDM4A siRNA into the DRGs of rats post-SNT resulted in a higher paw withdrawal threshold (PWT) and paw-withdrawal latency (PWL) compared to the KDM4A scRNA group. In addition, prior microinjection of AAV-EGFP-KDM4A shRNA also alleviates the decrease in PWT and PWL caused by SNT. Correspondingly, microinjection of AAV-EGFP-KDM4A shRNA subsequent to SNT reduced the established mechanical and thermal hyperalgesia. Furthermore, AAV-EGFP-KDM4A shRNA injection decreased the expression of CCL2 in DRGs. ChIP-PCR analysis revealed that increased binding of p-STAT1 with the CCL2 promoter induced by SNT was inhibited by AAV-EGFP-KDM4A shRNA treatment. These findings suggest that KDM4A potentially influences neuropathic pain by regulating CCL2 expression in DRGs.

Lysophosphatidic acid promotes ESCC progression by increasing the level of CCL2 secreted by esophageal epithelial cells.

BACKGROUND: Lysophosphatidic acid (LPA) is a small bioactive lipid which acts as a potent regulator in various tumor progressions through six G-protein-coupled receptors (LPA1-LPA6). Our previous study demonstrated that the LPA-producing enzyme, autotaxin (ATX), was upregulated in esophageal squamous cell carcinoma (ESCC) and ATX high expression levels indicated a poor prognosis. Esophageal squamous cell carcinoma is a type of malignant tumor which originates from epithelial cells. Its progression can be affected by the interaction between cancer cells and normal cells. However, the impact of LPA on the interaction between esophageal epithelial cells and cancer cells in the development of ESCC remains uncertain. METHODS: MTS and Edu assays were performed to determine ESCC cell proliferation in culture medium (CM) derived from LPA-stimulated esophageal epithelial cells (Het-1a). A wound healing assay, transwell migration and an invasion assay were performed to assess the metastatic ability of ESCC cells. Cytokine array analysis was conducted to detect the differentially secreted cytokines in CM. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized to uncover the pathways and cytokines that are influenced by LPA in ESCC. Immunohistochemical staining was employed to measure the expression of ATX and CCL2 in early-stage ESCC. Quantitative real-time PCR, western blot, enzyme-linked immunosorbent assay and an antibody neutralization assay were employed to measure the mechanism of LPA-mediated communication between epithelial cells and cancer cells. RESULTS: Functional experiments showed that exposing ESCC cancer cells to CM from LPA-treated Het-1a results in promoting proliferation, migration, invasion and epithelial-mesenchymal transition processes. Using cytokine array analysis, we discovered that LPA triggers the release of multiple cytokines from epithelial cells. After screening of the TCGA and GEO databases, CCL2 was identified and found to be correlated with ATX expression in ESCC. Furthermore, CCL2 levels in both mRNA expression and secretion were observed to be upregulated in epithelial cells upon stimulation with LPA. Blocking CCL2 effectively reduced the pro-migration influence of CM derived from LPA-treated Het-1a. Mechanism studies have demonstrated that LPA activated the NF-κB signaling pathway through LPA1/3, ultimately causing an increase in CCL2 expression and secretion in Het-1a. CONCLUSIONS: Our findings, taken together, demonstrate that CM from LPA-treated esophageal epithelial cells plays a significant role in promoting the progression of ESCC, with CCL2 acting as the primary regulator.

Causal effect of circulating cytokines on MRI markers of cerebral small vessel disease: A mendelian randomization study.

BACKGROUND: Previous observational studies have reported the correlation between circulating inflammatory cytokines and cerebral small vessel disease (CSVD). However, the causality of this association is uncertain. This study used Mendelian randomization to investigate the causal effect of circulating inflammatory cytokines on neuroimaging changes in CSVD. METHODS: This study utilized genetic variances of 41 inflammatory cytokines and 3 neuroimaging markers of CSVD from genome-wide association studies to assess the causal effects in a two-sample Mendelian randomization approach. Inverse variance weighted analysis was used as the main analytical method, and sensitivity analysis was used to further validate the robustness of the results. RESULTS: Increased IL-18 was associated with increased white matter hyperintensity (WMH) and mean diffusivity (MD) (ß = 0.034, 95 % CI 0.002, 0.065, P=0.038, ß = 0.157, 95 % CI 0.015, 0.299, P=0.030). However, increased IL-18 was associated with decreased fractional anisotropy (FA) (ß = -0.141, 95 % CI -0.279, -0.002, P=0.047). Increased monocyte chemotactic protein-1(MCP-1) was associated with decreased FA (ß = -0.278, 95 % CI -0.502, -0.054, P=0.015). Increased IL-10 levels and IL-2ra levels were associated with decreased risks of MD (ß = -0.228, 95 % CI -0.448, -0.009, p = 0.041; ß = -0.204, 95 % CI=-0.377, -0.031, p = 0.021). CONCLUSIONS: This study revealed that increased levels of IL-18 and MCP-1 were associated with white matter microstructural injury, and increased levels of IL-10 and IL-2ra were associated with decreased MD.

SLC11A1 promotes kidney renal clear cell carcinoma (KIRC) progression by remodeling the tumor microenvironment.

Kidney renal clear cell carcinoma (KIRC) is a highly immune-infiltrated kidney cancer with the highest mortality rate and the greatest potential for invasion and metastasis. Solute carrier family 11 member1 (SLC11A1) is a phagosomal membrane protein located in monocytes and plays a role in innate immunity, autoimmune diseases, and infection, but its expression and biological role in KIRC is still unknown. In this study, we sought to investigate the potential value of SLC11A1 according to tumor growth and immune response in KIRC. TIMER and UALCAN database was used to analyze the expression feature and prognostic significance of SLC11A1 and its correlation with immune-related biomarkers in KIRC. Proliferation, migration, and invasion were measured using colony formation, EdU, and transwell assays. Role of SLC11A1 on KIRC tumor growth was examined by the xenograft tumor model in vivo. Effects of KIRC cells on macrophage polarization and the proliferation and apoptosis of CD8+ T cells were analyzed using flow cytometry assays. Herein, SLC11A1 was highly expressed in KIRC tissues and cell lines. SLC11A1 downregulation repressed KIRC cell proliferation, migration, invasion, macrophage, and lymphocyte immunity in vitro, as well as hindered tumor growth in vivo. SLC11A1 is significantly correlated with immune cell infiltration and immune-related biomarkers. In KIRC patients, SLC11A1 is highly expressed and positively correlated with the immune-related factors CCL2 and PD-L1. SLC11A1 induced CCL2 and PD-L1 expression, thereby activating the JAK/STAT3 pathway. SLC11A1 deficiency constrained KIRC cell malignant phenotypes and immune response via regulating CCL2 and PD-L1-mediated JAK/STAT3 pathway, providing a promising therapeutic target for KIRC treatment.

Palmitic acid-activated GPRs/KLF7/CCL2 pathway is involved in the crosstalk between bone marrow adipocytes and prostate cancer.

BACKGROUND: Obesity-induced abnormal bone marrow microenvironment is one of the important risk element for bone metastasis in prostate cancer (PCa). The present study aimed to determine whether obesity-induced elevation in palmitic acid (PA), which is the most abundant of the free fatty acids (FFAs), increased CCL2 via the GPRs/KLF7 pathway in bone marrow adipocytes (BMA) to facilitate PCa growth and metastasis. METHODS: We constructed a bone-tumor bearing mouse model with obesity through high-fat diet, and observed the tumor formation ability of PCa cells. In vitro, observe the effect of PA on the expression level of CCL2 in BMA through GPRs/KLF7 signaling pathway. After co-culture of BMA and PCa cells, CCK8 assay and transwell experiment were used to detect the changes in biological behavior of PCa cells stimulated by BMA. RESULTS: The BMA distribution in the bone marrow cavity of BALB/c nude mice fed with the high-fat diet (HFD) was evidently higher than that in the mice fed with the normal diet (ND). Moreover, HFD-induced obesity promoted KLF7/CCL2 expression in BMA and PCa cell growth in the bone marrow cavity of the mice. In the vitro experiment, a conditioned medium with increased CCL2 obtained from the BMA cultured with PA (CM-BMA-PA) was used for culturing the PCa cell lines, which evidently enhanced the proliferation, invasion, and migration ability. KLF7 significantly increased the CCL2 expression and secretion levels in BMA by targeting the promoter region of the CCL2 gene. In addition, GPR40/120 engaged in the PA-induced high KLF7/CCL2 levels in BMA to facilitate the malignant progression of PC-3 cells. CONCLUSIONS: PA-activated GPRs/KLF7/CCL2 pathway in BMA facilitates prostate cancer growth and metastasis.

Targeting CCL2-CCR2 signaling pathway alleviates macrophage dysfunction in COPD via PI3K-AKT axis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) remains a leading cause of morbidity and mortality worldwide, characterized by persistent respiratory symptoms and airflow limitation. The involvement of C-C motif chemokine ligand 2 (CCL2) in COPD pathogenesis, particularly in macrophage regulation and activation, is poorly understood despite its recognized role in chronic inflammation. Our study aims to elucidate the regulatory role and molecular mechanisms of CCL2 in the pathogenesis of COPD, providing new insights for therapeutic strategies. METHODS: This study focused on the CCL2-CCR2 signaling pathway, exploring its role in COPD pathogenesis using both Ccl2 knockout (KO) mice and pharmacological inhibitors. To dissect the underlying mechanisms, we employed various in vitro and in vivo methods to analyze the secretion patterns and pathogenic effects of CCL2 and its downstream molecular signaling through the CCL2-CCR2 axis. RESULTS: Elevated Ccl2 expression was confirmed in the lungs of COPD mice and was associated with enhanced recruitment and activation of macrophages. Deletion of Ccl2 in knockout mice, as well as treatment with a Ccr2 inhibitor, resulted in protection against CS- and LPS-induced alveolar injury and airway remodeling. Mechanistically, CCL2 was predominantly secreted by bronchial epithelial cells in a process dependent on STAT1 phosphorylation and acted through the CCR2 receptor on macrophages. This interaction activated the PI3K-AKT signaling pathway, which was pivotal for macrophage activation and the secretion of inflammatory cytokines, further influencing the progression of COPD. CONCLUSIONS: The study highlighted the crucial role of CCL2 in mediating inflammatory responses and remodeling in COPD. It enhanced our understanding of COPD's molecular mechanisms, particularly how CCL2's interaction with the CCR2 activates critical signaling pathways. Targeting the CCL2-CCR2 axis emerged as a promising strategy to alleviate COPD pathology.

STC1 competitively binding ßPIX enhances melanoma progression via YAP nuclear translocation and M2 macrophage recruitment through the YAP/CCL2/VEGFA/AKT feedback loop.

This study investigates the role of Stanniocalcin-1 (STC1) in melanoma progression, with a focus on its impact on metastasis, angiogenesis, and immune evasion. Systematic bioinformatics analysis revealed the potential influence of STC1 dysregulation on prognosis, immune cell infiltration, response to immune therapy, and cellular functions. In vitro assays were conducted to assess the proliferation, invasion, migration, and angiogenesis capabilities of A375 cells. In vivo experiments utilizing C57BL/6 J mice established a lung metastasis model using B16-F10 cells to evaluate macrophage infiltration and M2 polarization. A Transwell co-culture system was employed to explore the crosstalk between melanoma and macrophages. Molecular interactions among STC1, YAP, ßPIX, and CCL2 are investigated using mass spectrometry, Co-Immunoprecipitation, Dual-Luciferase Reporter Assay, and Chromatin Immunoprecipitation experiments. STC1 was found to enhance lung metastasis by promoting the recruitment and polarization of M2 macrophages, thereby fostering an immunosuppressive microenvironment. Mechanistically, STC1 competes with YAP for binding to ßPIX within the KER domain in melanoma cells, leading to YAP activation and subsequent CCL2 upregulation. CCL2-induced M2 macrophages secrete VEGFA, which enhances tumor vascularization and increases STC1 expression via the AKT signaling pathway in melanoma cells, establishing a pro-metastatic feedback loop. Notably, STC1-induced YAP activation increases PD-L1 expression, promoting immune evasion. Silencing STC1 enhances the efficacy of PD-1 immune checkpoint therapy in mice. This research elucidates STC1's role in melanoma metastasis and its complex interactions with tumor-associated macrophages, proposing STC1 as a potential therapeutic target for countering melanoma metastasis and augmenting the efficacy of PD-1 immunotherapy.

The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues.

PURPOSE: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. METHODS: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. RESULTS: We established a flow cytometry gating strategy for identifying and isolating FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells. CONCLUSION: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.

Alleviating iatrogenic effects of paclitaxel via antiinflammatory treatment.

BACKGROUND: Paclitaxel (PTX) is touted as an essential medicine due to its extensive use as a chemotherapeutic agent for various cancers and an antiproliferative agent for endovascular applications. Emerging studies in cardio-oncology implicate various vascular complications of chemotherapeutic agents. METHODS: We evaluated the inflammatory response induced by the systemic administration of PTX. The investigation included RNAseq analysis of primary human endothelial cells (ECs) treated with PTX to identify transcriptional changes in pro-inflammatory mediators. Additionally, we used dexamethasone (DEX), a well-known antiinflammatory compound, to assess its effectiveness in counteracting these PTX-induced changes. Further, we studied the effects of PTX on monocyte chemoattractant protein-1 (MCP-1) levels in the media of ECs. The study also extended to in vivo analysis, where a group of mice was injected with PTX and subsequently harvested at different times to assess the immediate and delayed effects of PTX on inflammatory mediators in blood and aortic ECs. RESULTS: Our RNAseq analysis revealed that PTX treatment led to significant transcriptional perturbations in pro-inflammatory mediators such as MCP-1 and CD137 within primary human ECs. These changes were effectively abrogated when DEX was administered. In vitro experiments showed a marked increase in MCP-1 levels in EC media following PTX treatment, which returned to baseline upon treatment with DEX. In vivo, we observed a threefold increase in MCP-1 levels in blood and aortic ECs 12 h post-PTX administration. Similar trends were noted for CD137 and other downstream mediators like tissue factor, vascular cell adhesion molecule 1, and E-selectin in aortic ECs. CONCLUSION: Our findings illustrate that PTX exposure induces an upregulation of atherothrombotic mediators, which can be alleviated with concurrent administration of DEX. Considering these observations, further long-term investigations should focus on understanding the systemic implications associated with PTX-based therapies and explore the clinical relevance of DEX in mitigating such risks.

Group IVA Phospholipase A2 in Collagen-Producing Cells Promotes High-Fat Diet-Induced Infiltration of Inflammatory Cells into the Liver by Upregulating the Expression of MCP-1.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic inflammation and fibrosis due to excessive fat accumulation. Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that infiltrates inflammatory cells into the liver during the development of NASH. Our previous studies demonstrated that a systemic deficiency of group IVA phospholipase A2 (IVA-PLA2), an enzyme that contributes to the production of lipid inflammatory mediators, protects mice against high-fat diet-induced hepatic fibrosis and markedly suppresses the CCl4-induced expression of MCP-1 in the liver. However, it remains unclear which cell types harboring IVA-PLA2 are involved in the elevated production of MCP-1. Hence, the present study assessed the types of cells responsible for IVA-PLA2-mediated production of MCP-1 using cultured hepatic stellate cells, endothelial cells, macrophages, and hepatocytes, as well as cell-type specific IVA-PLA2 deficient mice fed a high-fat diet. A relatively specific inhibitor of IVA-PLA2 markedly suppressed the expression of MCP-1 mRNA in cultured hepatic stellate cells, but the suppression of MCP-1 expression was partial in endothelial cells and not observed in monocytes/macrophages or hepatocytes. In contrast, a deficiency of IVA-PLA2 in collagen-producing cells (hepatic stellate cells), but not in other types of cells, reduced the high-fat diet-induced expression of MCP-1 and inflammatory cell infiltration in the liver. Our results suggest that IVA-PLA2 in hepatic stellate cells is critical for hepatic inflammation in the high-fat diet-induced development of NASH. This supports a potential therapeutic approach for NASH using a IVA-PLA2 inhibitor targeting hepatic stellate cells.

Non-Cell-Autonomous Regulation of Prostate Epithelial Homeostasis by Androgen Receptor.

Prostate inflammation has been suggested as an etiology for benign prostatic hyperplasia (BPH). We show that decreased expression of the androgen receptor (AR) in luminal cells of human BPH specimens correlates with a higher degree of regional prostatic inflammation. However, the cause-and-effect relationship between the two events remains unclear. We investigated specifically whether attenuating AR activity in prostate luminal cells induces inflammation. Disrupting luminal cell AR signaling in mouse models promotes cytokine production cell-autonomously, impairs epithelial barrier function, and induces immune cell infiltration, which further augments local production of cytokines and chemokines including Il-1 and Ccl2. This inflammatory microenvironment promotes AR-independent prostatic epithelial proliferation, which can be abolished by ablating IL-1 signaling or depleting its major cellular source, the macrophages. This study demonstrates that disrupting luminal AR signaling promotes prostate inflammation, which may serve as a mechanism for resistance to androgen-targeted therapy for prostate-related diseases.