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.

Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

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.

Bacterial indole-3-lactic acid affects epithelium-macrophage crosstalk to regulate intestinal homeostasis.

Tryptophan and its derivatives perform a variety of biological functions; however, the role and specific mechanism of many tryptophan derivatives in intestinal inflammation remain largely unclear. Here, we identified that an Escherichia coli strain (Ec-TMU) isolated from the feces of tinidazole-treated individuals, and indole-3-lactic acid (ILA) in its supernatant, decreased the susceptibility of mice to dextran sulfate sodium-induced colitis. Ec-TMU and ILA contribute to the relief of colitis by inhibiting the production of epithelial CCL2/7, thereby reducing the accumulation of inflammatory macrophages in vitro and in vivo. Mechanistically, ILA downregulates glycolysis, NF-κB, and HIF signaling pathways via the aryl hydrocarbon receptor, resulting in decreased CCL2/7 production in epithelial cells. Clinical evidence suggests that the fecal ILA level is negatively correlated with the progression indicator of inflammatory bowel diseases. These results demonstrate that ILA has the potential to regulate intestinal homeostasis by modulating epithelium-macrophage interactions.

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

BACKGROUND: The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. METHODS: To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. RESULTS: Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. CONCLUSIONS: Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

Virus-Induced Host Chemokine CCL2 in COVID-19 Pathogenesis: Potential Prognostic Marker and Target of Anti-Inflammatory Strategy.

A wide variety of inflammatory mediators, mainly cytokines and chemokines, are induced during SARS CoV-2 infection. Among these proinflammatory mediators, chemokines tend to play a pivotal role in virus-mediated immunopathology. The C-C chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1) is a potent proinflammatory cytokine and strong chemoattractant of monocytes, macrophages and CD4+ T cells bearing C-C chemokine receptor type-2 (CCR2). Besides controlling immune cell trafficking, CCL2 is also involved in multiple pathophysiological processes including systemic hyperinflammation associated cytokine release syndrome (CRS), organ fibrosis and blood coagulation. These pathological features are commonly manifested in severe and fatal cases of COVID-19. Given the crucial role of CCL2 in COVID-19 pathogenesis, the CCL2:CCR2 axis may constitute a potential therapeutic target to control virus-induced hyperinflammation and multi-organ dysfunction. Herein we describe recent advances on elucidating the role of CCL2 in COVID-19 pathogenesis, prognosis, and a potential target of anti-inflammatory interventions.

Intranasal vaccination with engineered BCG expressing CCL2 induces a stronger immune barrier against Mycobacterium tuberculosis than BCG.

Intradermal Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination is currently the only licensed strategy for preventing tuberculosis (TB). It provides limited protection against pulmonary TB. To enhance the efficacy of BCG, we developed a recombinant BCG expressing exogenous monocyte chemoattractant CC chemokine ligand 2 (CCL2), termed rBCG-CCL2. Co-culturing macrophages with rBCG-CCL2 enhances their abilities in migration, phagocytosis, and effector molecules expression. In the mouse model, intranasal vaccination with rBCG-CCL2 induced greater immune cells infiltration and a more extensive innate immune responses in lung compared to vaccination with parental BCG, as determined by multiparameter flow cytometry, transcriptomic analysis, and pathological assessments. Moreover, rBCG-CCL2 induced a high frequency of activated macrophages and antigen-specific Th1 and Th17 T cells in lungs. The enhanced immune microenvironment responded more effectively to intravenous challenge with Mycobacterium tuberculosis (Mtb) H37Ra, leading to significant reductions in H37Ra burden and pathological damage to the lungs and spleen. Intranasal rBCG-CCL2 vaccinated mice rapidly initiated pro-inflammatory Th1 cytokine release and reduced pathological damage to the lungs and spleen during the early stage of H37Ra challenge. The finding that co-expression of CCL2 synergistically enhances the immune barrier induced by BCG provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against TB.

Tetrahedral DNA loaded siCCR2 restrains M1 macrophage polarization to ameliorate pulmonary fibrosis in chemoradiation-induced murine model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal disease in the absence of demonstrated efficacy for preventing progression. Although macrophage-mediated alveolitis is determined to participate in myofibrotic transition during disease development, the paradigm of continuous macrophage polarization is still under-explored due to lack of proper animal models. Here, by integrating 2.5 U/kg intratracheal Bleomycin administration and 10 Gy thorax irradiation at day 7, we generated a murine model with continuous alveolitis-mediated fibrosis, which mimics most of the clinical features of our involved IPF patients. In combination with data from scRNA-seq of patients and a murine IPF model, a decisive role of CCL2/CCR2 axis in driving M1 macrophage polarization was revealed, and M1 macrophage was further confirmed to boost alveolitis in leading myofibroblast activation. Multiple sticky-end tetrahedral framework nucleic acids conjunct with quadruple ccr2-siRNA (FNA-siCCR2) was synthesized in targeting M1 macrophages. FNA-siCCR2 successfully blocked macrophage accumulation in pulmonary parenchyma of the IPF murine model, thus preventing myofibroblast activation and leading to the disease remitting. Overall, our studies lay the groundwork to develop a novel IPF murine model, reveal M1 macrophages as potential therapeutic targets, and establish new treatment strategy by using FNA-siCCR2, which are highly relevant to clinical scenarios and translational research in the field of IPF.

The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.

Ultrasensitive CCL2 Detection in Urine for Diabetic Nephropathy Diagnosis Using a WS2-Based Plasmonic Biosensor.

The accurate diagnosis of diabetic nephropathy relies on achieving ultrasensitive biosensing for biomarker detection. However, existing biosensors face challenges such as poor sensitivity, complexity, time-consuming procedures, and high assay costs. To address these limitations, we report a WS2-based plasmonic biosensor for the ultrasensitive detection of biomarker candidates in clinical human urine samples associated with diabetic nephropathy. Leveraging plasmonic-based electrochemical impedance microscopy (P-EIM) imaging, we observed a remarkable charge sensitivity in monolayer WS2 single crystals. Our biosensor exhibits an exceptionally low detection limit (0.201 ag/mL) and remarkable selectivity in detecting CC chemokine ligand 2 (CCL2) protein biomarkers, outperforming conventional techniques such as ELISA. This work represents a breakthrough in traditional protein sensors, providing a direction and materials foundation for developing ultrasensitive sensors tailored to clinical applications for biomarker sensing.

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.

CCL2-CCR2 signaling axis in obesity and metabolic diseases.

Obesity and metabolic diseases, such as insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular ailments, represent formidable global health challenges, bearing considerable implications for both morbidity and mortality rates. It has become increasingly evident that chronic, low-grade inflammation plays a pivotal role in the genesis and advancement of these conditions. The involvement of C-C chemokine ligand 2 (CCL2) and its corresponding receptor, C-C chemokine receptor 2 (CCR2), has been extensively documented in numerous inflammatory maladies. Recent evidence indicates that the CCL2/CCR2 pathway extends beyond immune cell recruitment and inflammation, exerting a notable influence on the genesis and progression of metabolic syndrome. The present review seeks to furnish a comprehensive exposition of the CCL2-CCR2 signaling axis within the context of obesity and metabolic disorders, elucidating its molecular mechanisms, functional roles, and therapeutic implications.

Tumor cell-intrinsic MELK enhanced CCL2-dependent immunosuppression to exacerbate hepatocarcinogenesis and confer resistance of HCC to radiotherapy.

BACKGROUND: The outcome of hepatocellular carcinoma (HCC) is limited by its complex molecular characteristics and changeable tumor microenvironment (TME). Here we focused on elucidating the functional consequences of Maternal embryonic leucine zipper kinase (MELK) in the tumorigenesis, progression and metastasis of HCC, and exploring the effect of MELK on immune cell regulation in the TME, meanwhile clarifying the corresponding signaling networks. METHODS: Bioinformatic analysis was used to validate the prognostic value of MELK for HCC. Murine xenograft assays and HCC lung metastasis mouse model confirmed the role of MELK in tumorigenesis and metastasis in HCC. Luciferase assays, RNA sequencing, immunopurification-mass spectrometry (IP-MS) and coimmunoprecipitation (CoIP) were applied to explore the upstream regulators, downstream essential molecules and corresponding mechanisms of MELK in HCC. RESULTS: We confirmed MELK to be a reliable prognostic factor of HCC and identified MELK as an effective candidate in facilitating the tumorigenesis, progression, and metastasis of HCC; the effects of MELK depended on the targeted regulation of the upstream factor miR-505-3p and interaction with STAT3, which induced STAT3 phosphorylation and increased the expression of its target gene CCL2 in HCC. In addition, we confirmed that tumor cell-intrinsic MELK inhibition is beneficial in stimulating M1 macrophage polarization, hindering M2 macrophage polarization and inducing CD8 + T-cell recruitment, which are dependent on the alteration of CCL2 expression. Importantly, MELK inhibition amplified RT-related immune effects, thereby synergizing with RT to exert substantial antitumor effects. OTS167, an inhibitor of MELK, was also proven to effectively impair the growth and progression of HCC and exert a superior antitumor effect in combination with radiotherapy (RT). CONCLUSIONS: Altogether, our findings highlight the functional role of MELK as a promising target in molecular therapy and in the combination of RT therapy to improve antitumor effect for HCC.

Dipeptidyl peptidase 4 deficiency improves survival after focal cerebral ischemia in mice and ameliorates microglia activation and specific inflammatory markers.

Dipeptidyl peptidase 4 (DPP4; CD26) is involved in the regulation of various metabolic, immunological, and neurobiological processes in healthy individuals. Observations based on epidemiological data indicate that DPP4 inhibition by gliptins, typically used in patients with diabetes, may reduce the risk for cerebral ischemia and may also improve related outcomes. However, as DPP4 inhibitor application is neither complete nor specific for suppression of DPP4 enzymatic activity and DPP4 has non-enzymatic functions as well, the variety of consequences is a matter of debate. Therefore, we here used DPP4 knock-out (KO) mice to analyze the specific contribution of DPP4 to cellular, immunological, and functional consequences of experimental focal cerebral ischemia. We observed a significantly higher survival rate of DPP4 KO mice after ischemia, which was accompanied by a lower abundance of the pro-inflammatory chemokine CCL2 and reduced activation of Iba1-positive microglia cells in brain tissue of DPP4 KO mice. In addition, after ischemia for 24 h to 72 h, decreased concentrations of CCL5 and CCL12 in plasma and of CCL17 in brain tissue of DPP4 KO mice were observed when compared to wild type mice. Other aspects analyzed, such as the functional Menzies score, astrocyte activation and chemokine levels in plasma and brain tissue were affected by ischemia but appeared to be unaffected by the DPP4 KO genotype. Taken together, experimental ablation of DPP4 functions in mice improves survival and ameliorates aspects of cellular and molecular inflammation after focal cerebral ischemia.

Hsp90aa1/JUN/Ccl2 regulatory axis mediates migration and differentiation of NSPCs, promoting the onset and progression of early post-ischemic stroke epilepsy.

Early-onset epilepsy following ischemic stroke is a severe neurological condition, the pathogenesis of which remains incompletely understood. Recent studies suggest that Neural stem/progenitor cells (NSPCs) play a crucial role in the disease process, yet the precise molecular mechanisms regulating NSPCs have not been thoroughly investigated. This study utilized single-cell transcriptome sequencing and bioinformatics analysis to identify disease-related genes, which were subsequently validated in both in vitro and in vivo experiments. The findings revealed that Hsp90aa1 (heat shock protein 90 kDa alpha, class A member 1), Jun proto-oncogene (JUN), and CC Motif Ligation 2 (Ccl2) constitute an important regulatory axis influencing the migration and differentiation of NSPCs, potentially impacting the onset and progression of early-onset epilepsy post-ischemic stroke. Additionally, the expression of Hsp90aa1 was found to influence the likelihood of seizure occurrence and the severity of brain ischemia.

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.

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion.

BACKGROUND & AIMS: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown. METHODS: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids. Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing. Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC single-cell RNA sequencing data sets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion. RESULTS: We established 32 genetically engineered esophageal organoids and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN knockout also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via nuclear factor-κB. Moreover, comparative single-cell transcriptomic analyses stratified patients with ESCC and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. CONCLUSIONS: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.

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.