C-C Motif Chemokine Ligand 2 Research Articles

Tumour-derived exosomes promote the induction of monocytic myeloid-derived suppressor cells from peripheral blood mononuclear cells by delivering miR-106a-5p and miR-146a-5p in multiple myeloma.

The shift of the tumour immune microenvironment to a suppressive state promotes not only the development and progression of the disease in multiple myeloma (MM) but also the development of resistance to immunotherapy. We previously demonstrated that myeloma cells can induce monocytic myeloid-derived suppressor cells (M-MDSCs) from healthy peripheral blood mononuclear cells (PBMCs) via the concomitant secretion of CC motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF), but an unknown mediator also promotes M-MDSC induction. This study demonstrates that miR-106a-5p and miR-146a-5p delivered by tumour-derived exosomes (TEXs) from myeloma cells play essential roles in M-MDSC induction in MM. MiR-106a-5p and miR-146a-5p upregulate various immunosuppressive/inflammatory molecules in PBMCs, such as IDO1, CD38, programmed death-ligand 1, CCL5 or MYD88, which are involved in interferon (IFN)-α response, IFN-γ response, inflammatory response, tumour necrosis factor-α signalling and Interleukin-6-JAK-STAT3 signalling. These molecular features mirror the increases in myeloid cellular compartments of PBMCs when co-cultured with myeloma cells. MiR-106a-5p and miR-146a-5p have a compensatory relationship, and these two miRNAs collaborate with CCL5 and MIF to promote M-MDSC induction. Collectively, novel therapeutic candidates may be involved in TEX-mediated sequential cellular and molecular events underlying M-MDSC induction, potentially improving the efficacy of immunotherapy.

Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

Abstract P1012: Wnt Signaling Is Required For Myxomatous Valve Disease In A Mouse Model Of Marfan Syndrome

Introduction: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) includes valve leaflet thickening and increased immune cell infiltration. However, the underlying mechanisms by which immune cells are recruited in MFS valves remain undefined. Hypothesis: Inhibition of Wnt signaling prevents immune cell recruitment and MVD progression in MFS valves. Methods: Mice with the mutation of Fibrillin 1 ( Fbn1 C1039G/+ ) recapitulate histopathological features of MFS and Wnt signaling activity was detected in TCF/Lef-lacZ reporter mice. The expression level of C-C motif chemokine ligand 2 (CCL2) was detected in Fbn1 C1039G/+ mice by qPCR and RNAscope. Inhibition of Wnt signaling was achieved by conditional induction of the soluble Wnt inhibitor Dkk1 in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1 C1039G/+ mice. Dietary doxycycline was administered for one month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for immune cells were performed. Results: Wnt signaling as detected by TCF/Lef-lacZ reporters and CCL2 expression were upregulated early in mitral valve disease progression, prior to immune cell infiltration in Fbn1 C1039G/+ mice. Inhibition of Wnt signaling by Dkk1 induction at 1-month of age prevented the induction of myxomatous mitral valve disease, indicated by reduced valve leaflet thickness with decreased CD45+ leukocytes and CCR2+ immune cells in Fbn1 C1039G/+ mice. However, later Wnt inhibition starting at 2 months when inflammation and myxomatous changes are occurring did not prevent the progression of myxomatous mitral valve disease. Conclusions: Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation, but is not responsible for valve disease progression once it has been initiated.

Influence of food emulsifiers on cellular function and inflammation, a preliminary study.

Emulsifiers are extensively used as food additives and their consumption is increasing in Western countries. However, so far only few studies examined their potential effects on intestinal cellular functions and gut inflammation. The aim of this preliminary analysis was to study the emulsifiers and their concentrations capable of causing cellular damage compared to extra virgin olive oil (EVOO). We tested two commonly used emulsifiers (EMI, EMII) and EVOO on Caco-2 cells, derived from a colon carcinoma and widely used as a model of the intestinal inflammation. The diphenyltetrazolium bromide test MTT and clonogenic assay were used to study the effect of emulsifiers on cell viability. Cell migration was determined by the wound-healing assay. The inflammation was studied by measuring the levels of interleukin 6 (IL-6) and monocyte chemoattractant protein-1/C-C motif chemokine ligand 2 (CCL2), multifunctional cytokines with a major role in the acute-phase response. Furthermore, we analyzed the effect of conditioned media of Caco-2 cells treated with EMs on macrophages activation. In conclusion, our preliminary data provide evidence that EMs increase the proliferation and migration rate of Caco-2 cells. Moreover, Caco-2 cells treated with EMs enhance the IL-6 and CCL2 release and activated macrophages, supporting their role as proinflammatory molecules.

Immune-checkpoint proteins, cytokines, and microbiome impact on patients with cervical insufficiency and preterm birth.

Microenvironmental factors, including microbe-induced inflammation and immune-checkpoint proteins that modulate immune cells have been associated with both cervical insufficiency and preterm delivery. These factors are incompletely understood. This study aimed to explore and compare interactions among microbiome and inflammatory factors, such as cytokines and immune-checkpoint proteins, in patients with cervical insufficiency and preterm birth. In particular, factors related to predicting preterm birth were identified and the performance of the combination of these factors was evaluated. A total of 220 swab samples from 110 pregnant women, prospectively recruited at the High-Risk Maternal Neonatal Intensive Care Center, were collected between February 2020 and March 2021. This study included 63 patients with cervical insufficiency receiving cerclage and 47 control participants. Endo- and exocervical swabs and fluids were collected simultaneously. Shotgun metagenomic sequencing for the microbiome and the measurement of 34 immune-checkpoint proteins and inflammatory cytokines were performed. First, we demonstrated that immune-checkpoint proteins, the key immune-regulatory molecules, could be measured in endocervical and exocervical samples. Secondly, we identified significantly different microenvironments in cervical insufficiency and preterm birth, with precise cervical locations, to provide information about practically useful cervical locations in clinical settings. Finally, the presence of Moraxella osloensis (odds ratio = 14.785; P = 0.037) and chemokine CC motif ligand 2 levels higher than 73 pg/mL (odds ratio = 40.049; P = 0.005) in endocervical samples were associated with preterm birth. Combining M. osloensis and chemokine CC motif ligand 2 yielded excellent performance for predicting preterm birth (area under the receiver operating characteristic curve = 0.846, 95% confidence interval = 0.733-0.925). Multiple relationships between microbiomes, immune-checkpoint proteins, and inflammatory cytokines in the cervical microenvironment were identified. We focus on these factors to aid in the comprehensive understanding and therapeutic modulation of local microbial and immunologic compositions for the management of cervical insufficiency and preterm birth.

Exploration of the Perturbation of PKIG in Lung Squamous Cell Carcinoma and the Role in Tumor Microenvironment Based on Bioinformatics Method

Lung cancer is the leading cause of cancer-related death worldwide, and patients have limited survival benefits from traditional treatments such as surgery, radiotherapy and chemotherapy. As a new treatment for lung cancer, immunotherapy has significantly prolonged the overall survival (OS) of patients. However, only some patients can benefit from it. We need to explore immunotherapy biomarkers more deeply to screen for advantages. The original data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database, and the immunological and prognostic genes of lung squamous cell carcinoma (LUSC) were screened using R software and TIMER database. The expression of target genes was studied in TCGA and GEO databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and correlation analysis with tumor immune characteristics were performed by R software and TISIDB database. We screened out a gene related to immunity and prognosis, cAMP dependent protein kinase inhibitor γ (PKIG), which is significantly differentially expressed in LUSC and normal tissues, and has important reference value for the diagnosis and prognosis assessment of LUSC. PKIG differential genes are mainly concentrated in the regulation of humoral immune response and other processes. The expression of PKIG was positively correlated with the infiltration level of regulatory T cells (Tregs) (r=0.340, P<0.001). In addition, the expression level of PKIG was positively correlated with the expression of chemokines/chemokine receptors such as chemokine C-C motif ligand 2 (CCL2) (r=0.503, P<0.001), CXC chemokine ligand 12 (CXCL12) (r=0.386, P<0.001) and CXC-chemokine receptor 4 (CXCR4) (r=0.492, P<0.001), and immunoinhibitors such as programmed cell death protein 1 (PDCD1) (r=0.359, P<0.001), cytotoxic T-lymphocyte associated antigen 4 (CTLA4) (r=0.375, P<0.001) and T cell immunoglobulin and ITIM domains (TIGIT) (r=0.305, P<0.001) in LUSC. The immunological and prognostic gene PKIG in lung squamous cell carcinoma was screened through bioinformatics analysis. PKIG is highly correlated with LUSC prognosis and immune microenvironment, and is expected to be a potential biomolecular marker for LUSC immunotherapy.

Establishment of a Novel Anti-Human CCR6 Monoclonal Antibody C6Mab-19 with the High Binding Affinity in Flow Cytometry.

CC chemokine receptor 6 (CCR6) is a member of the G-protein-coupled receptor family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19 (mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), human liver carcinoma (HepG2), and human differentiated hepatoma (HuH-7) cells in flow cytometry. The dissociation constant (KD) of C6Mab-19 was determined as 3.0 × 10-10 M for CHO/hCCR6, 6.9 × 10-10 M for HepG2, and 1.8 × 10-10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitable for detecting hCCR6-expressing cells and tissues and could be useful for pathological analysis and diagnosis.

CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis.

Lymphangiogenesis in tumors provides an auxiliary route for cancer cell invasion to drainage lymph nodes, facilitating the development of lymphatic metastasis (LM). However, the mechanisms governing tumor lymphangiogenesis and lymphatic permeability in gastric cancer (GC) remain largely unknown. Here, the unprecedented role and mechanism of cysteine-rich intestinal protein-1 (CRIP1) in mediating the development of GC LM is uncovered. A series of assays are performed to identify downstream targets of CRIP1, and rescue experiments are performed to confirm the effects of this regulatory axis on LM. CRIP1 overexpression facilitates LM in GC by promoting lymphangiogenesis and lymphatic vessel permeability. CRIP1 promotes phosphorylation of cAMP responsive element binding protein 1(CREB1), which then mediates vascular endothelial growth factor C (VEGFC) expression necessary for CRIP1-induced lymphangiogenesis and transcriptionally promotes C-C motif chemokine ligand 5 (CCL5) expression. CCL5 recruits macrophages to promote tumor necrosis factor alpha (TNF-α) secretion, eventually enhancing lymphatic permeability. The study highlights CRIP1 regulates the tumor microenvironment to promote lymphangiogenesis and LM in GC. Considering the current limited understanding of LM development in GC, these pathways provide potential targets for future therapeutics.

CCL2 promotes osteogenesis by facilitating macrophage migration during acute inflammation.

Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1day) but not sustained (7days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

Absolute monocyte count has a diagnostic role in distinguishing tumor marker-negative TGCT from benign testicular tumor via CCL2 regulation.

Clinically, for testicular tumor patients with negative tumor markers, how to distinguish the malignant from the benign is a difficult problem. This study aimed to assess the clinical significance of the absolute monocyte count (AMC) in differential diagnosis of testicular germ cell tumor with stage S0 (TGCTS0) and benign testicular tumor. In this retrospective single-center study, a total of 90 patients newly diagnosed with benign testicular tumor or TGCTS0 were reviewed. All patients received surgical intervention as the primary treatment method. AMC and other clinicopathological parameters were analyzed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic power of investigated parameters, and to determine the optimal cutoff values. Kaplan-Meier curve analysis was used to study the survival of patients with TGCTS0. qRT-PCR and immunohistochemistry (IHC) were performed to examine the expression of C-C motif chemokine ligand 2 (CCL2) mRNA and protein respectively. Differential gene expression and functional enrichment analysis were performed using Gene Expression Omnibus and the Cancer Genome Atlas databases. The mean preoperative AMC in patients with TGCTS0 was significantly higher than that in patients with benign testicular tumor (P = .020). AMC > 0.485*10^9/L was identified to be associated with the presence of TGCTS0 (hazard ratio [HR] = 3.074, P = .026), and patients with higher AMC level had worse progression free survival (PFS) (P = .047). Furthermore, AMC combined with lactate dehydrogenase (LDH) achieved a better diagnostic efficacy for TGCTS0 (area under curve [AUC] = 0.695). Tumor-associated macrophages (TAMs) signature gene CCL2 was highly expressed in TGCT compared with normal testicular tissue. Functional enrichment analysis showed that CCL2 is closely involved in the Extracellular Matrix Organization pathway and positively correlated with the expression of various matrix metalloproteinases (MMPs). Elevated AMC may serve as a predictor of higher risk of TGCTS0, and CCL2 mediated TAMs infiltration and MMPs secretion is essential for the tumorigenesis of TGCT.

The response of human macrophages to 3D printed titanium antibacterial implants does not affect the osteogenic differentiation of hMSCs.

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.

1641-P: C-C Motif Chemokine Ligand 2 Promotes Myogenesis Recovery, Muscle Strength, and Muscle Performance In Vivo and Has Clinical Relevance for Muscle Performance in Human

Muscle mass decreases with aging, while the C-C motif chemokine ligand 2 (CCL2) increases with aging. In our previous study, CCL2 positively affects in vitro myogenesis. In this context, CCL2 can be considered a potential aging-promoting factor. To further confirm the relationship between muscle and CCL2, we sought to establish the role of CCL2 in vivo and in humans. It was divided into Dimethyl Sulfoxide (DMSO) control, dexamethasone (Dexa) intraperitoneal treatment group, and Dexa with the CCL2 treatment group. As a result, grip power and exercise endurance were reduced in the Dexa treatment group compared to the control group but recovered with the CCL2 treatment. In muscle mass, there was no significant difference in mass between groups. We examined the cross-sectional area distribution (CSA) with the tibialis anterior muscle. The Dexa treatment group was distributed between CSA 1000-3000 µm² and was left-shifted. However, the Dexa and CCL2 group was similar or slightly higher than the DMSO group; it was right-shifted and distributed in the 2000-5000 µm². In humans, the median value was 303.8 pg/ml in the no sarcopenia group and 321.7 pg/ml in the sarcopenia group. Subsequently, the CCL2 level was checked by dividing the groups into whether there was sarcopenia, low mass, low strength, or low speed. The CCL2 level was statistically significantly higher in the group showing low speed. In conclusion, CCL2 promotes myogenesis recovery, muscle strength, and muscle performance in vivo and has clinical relevance for muscle performance in humans. Disclosure E.Hong: None. E.Ha: None. J.Lee: None. Y.Choi: None. K.Lee: None. J.Won: None. M.Kwak: None. Funding Ministry of Education, Republic of Korea (2020R1I1A3073230)

475-P: Intermittent Hypoxia Increased the Expression of Intercellular Adhesion Molecule-1 and Endothelial Cell–Specific Molecule-1 in Vascular Endothelial Cells via the Downregulation of MicroRNA-181a1

Sleep apnea syndrome (SAS), which is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), is a risk factor for vascular dysfunction, as well as hypertension, and insulin resistance/Type 2 diabetes. We have reported correlations between IH and insulin resistance/Type 2 diabetes, and hypertension. However, the question of why vascular dysfunction is induced by IH has remained unanswered. Here, we investigated the effects of IH on the expression of endothelial inflammatory transcripts such as intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8, C-C motif chemokine ligand 5 (CCL5), endothelial nitric oxide synthase (NOS3), vascular cell adhesion molecule-1 (VCAM-1), endothelial cell specific molecule 1 (ESM1), and endothelin-1 (EDN-1) using an in vitro IH system. Human and mouse vascular endothelial cells (HUEhT-1 and UV2) were exposed to IH or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of ICAM-1 and ESM1 in both HUEhT-1 (P=0.0436 and P=0.0145, respectively) and UV2 cells (P&amp;lt;0.0001 and P=0.0422, respectively). ELISA showed that ICAM-1 and ESM1 were significantly increased (P=0.0105 and P&amp;lt;0.0001, respectively) in the UV2 cell medium by IH. Reporter assays revealed that the promoter activities of ICAM-1 and ESM1 were not increased by IH. The microRNA (miR)-181a1 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells (P=0.0374). The IH-induced upregulation of ICAM-1 and ESM1 was abolished by the introduction of miR-181a1 mimic, indicating that IH stress increased levels of ICAM-1 and ESM1 via the inhibition of miR-181a1-mediated mRNA degradation, leading SAS patients to develop atherosclerosis. Disclosure S.Takasawa: None. M.Makino: None. A.Yamauchi: None. S.Sakuramoto-tsuchida: None. Y.Takeda: None. T.Uchiyama: None. R.Shobatake: None.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

#2974 SERUM CHEMOKINE CC-MOTIF LIGAND 17 IS A PREDICTIVE MARKER OF SEVERE COVID-19 IN HAEMODIALYSIS PATIENTS

Abstract Background and Aims Maintenance haemodailaysis (HD) patients are at higher risk for severe coronavirus disease 2019 (COVID-19). Because of a limited number of facilities that can provide inpatient treatment for COVID-19 and HD, it is important to identify HD patients who are at high risk for severe COVID-19. For mild to moderate COVID-19 patients, chemokine CC-motif ligand 17 (CCL17) was reported to be a predictive marker for severe COVID-19; however, the validity of CCL17 among HD patients is unknown. Method This retrospective observational study enrolled 107 HD patients with mild or moderate COVID-19 at hospitalisation (mean age 70.1 ± 15.1 years; 71.0% male). Receiver operating characteristic and logistic regression analyses were used to examine the predictive validity of indices for severe COVID-19 which is defined as partial pressure of oxygen/fraction of inspired oxygen (P/F) ratio &amp;lt;300 or SpO2 &amp;lt;94%. Multi-variate logistic regression models were used to investigate the association of CCL17 level with the development of severe COVID-19. Furthermore, to evaluate the degree of improvement in predicting performance in the pre-existing model with the addition of CCL17 compared to the pre-existing model, we calculated net reclassification improvement (NRI) and the integrated discrimination improvement (IDI). Results During hospitalisation, 32 patients developed severe COVID-19. Serum CCL17 collected at admission exhibited a higher area under the curve value (0.818) compared with that of other indicators including LDH and C-reactive protein for the prediction of severe COVID-19. The optimal cut-off value for CCL17 was 150.5 pg/ml. A multi-variate logistic analysis revealed that CCL17 (above 150.5 pg/ml) was significantly associated with severe COVID-19 (Odds ratio, 0.063; 95% Confidence interval [CI], 0.017–0.227; P &amp;lt; 0.001) even after adjustment for covariates. The addition of the CCL17 to a model consisting of vaccination status, albumin, blood urea nitrogen, C-reacting protein and lactate dehydrogenase, that are variables incorporated in multivariate logistic model, significantly improved classification performance for severe COVID-19 using the NRI (1.16, 95% CI: 0.82–1.50, P &amp;lt; 0.001) and IDI (0.18, 95% CI: 0.09–0.26, P &amp;lt; 0.001). Conclusion CCL17 levels in HD patients with mild or moderate COVID-19 predict risk of developing severe COVID-19.

EPEN-04. DEVELOPMENT OF B7-H3-TARGETED CAR T CELL THERAPY FOR EPENDYMOMA

Abstract Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T cells against EPNs. We found that B7-H3 is highly expressed on pediatric brain tumor patient-derived orthotopic xenografts from EPN patients, making it a promising target for CAR-T cells. The project’s goal is to develop a safe, effective B7-H3 targeted CAR T cell immunotherapy for EPN. We generated second generation human B7-H3-CAR T cells and examined their anti-EPN cytolytic activity, expansion, and cytokine production. We found that B7-H3-CAR T cells had potent in vitro antitumor activity against all EPN cell lines tested (1425, ST1, CPITT, ST2, EPI, L6SJ). In repeated-stimulation assays, CAR T cells expanded 7-10 times, and up to one million-fold when co-cultured with EPN cells. Interestingly, while the EPN cells were found to secrete high levels of C-C motif chemokine ligand 2 (CCL2) which is consistent with published data, our Multiplex analysis of co-culture supernatants shows that CCL2 production increased up to 10-fold when EPN cells were co-cultured with CAR T cells. Finally, we demonstrate that B7-H3-CAR T cells had antitumor activity in a supratentorial-EPN xenograft mouse model (1425 cell line) without apparent toxicities, although 4 out of 12 tumors re-lapsed. Recurrent tumor analysis is underway. Together, our data shows establishment of a clinically relevant model to study resistance to CAR T cell therapy in EPN, and preliminary mechanistic insight into CAR T cell persistence and modulation through CCL2 signaling. Given that little is known about the function of CCL2 and its receptor, C-C chemokine receptor type 2 (CCR2) in EPNs or CAR T cells, future work will focus on defining these biological mechanisms, enabling us to develop improvements to B7-H3-CAR T cells for EPN.

Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone.

FFAR2 antagonizes TLR2- and TLR3-induced lung cancer progression via the inhibition of AMPK-TAK1 signaling axis for the activation of NF-κB

BackgroundFree fatty acid receptors (FFARs) and toll-like receptors (TLRs) recognize microbial metabolites and conserved microbial products, respectively, and are functionally implicated in inflammation and cancer. However, whether the crosstalk between FFARs and TLRs affects lung cancer progression has never been addressed.MethodsWe analyzed the association between FFARs and TLRs using The Cancer Genome Atlas (TCGA) lung cancer data and our cohort of non-small cell lung cancer (NSCLC) patient data (n = 42), and gene set enrichment analysis (GSEA) was performed. For the functional analysis, we generated FFAR2-knockout (FFAR2KO) A549 and FFAR2KO H1299 human lung cancer cells and performed biochemical mechanistic studies and cancer progression assays, including migration, invasion, and colony-formation assays, in response to TLR stimulation.ResultsThe clinical TCGA data showed a significant down-regulation of FFAR2, but not FFAR1, FFAR3, and FFAR4, in lung cancer, and a negative correlation with TLR2 and TLR3. Notably, GSEA showed significant enrichment in gene sets related to the cancer module, the innate signaling pathway, and the cytokine-chemokine signaling pathway in FFAR2DownTLR2UpTLR3Up lung tumor tissues (LTTs) vs. FFAR2upTLR2DownTLR3Down LTTs. Functionally, treatment with propionate (an agonist of FFAR2) significantly inhibited human A549 or H1299 lung cancer migration, invasion, and colony formation induced by TLR2 or TLR3 through the attenuation of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB. Moreover, FFAR2KO A549 and FFAR2KO H1299 human lung cancer cells showed marked increases in cell migration, invasion, and colony formation in response to TLR2 or TLR3 stimulation, accompanied by elevations in NF-κB activation, cAMP levels, and the production of C-C motif chemokine ligand (CCL)2, interleukin (IL)-6, and matrix metalloproteinase (MMP) 2 cytokines.ConclusionOur results suggest that FFAR2 signaling antagonized TLR2- and TLR3-induced lung cancer progression via the suppression of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB, and its agonist might be a potential therapeutic agent for the treatment of lung cancer.

CCL20 inhibition for treating inflammation in ankylosing spondylitis.

Th17 cells are known to play a significant role in AS. C-C motif chemokine ligand 20 (CCL20) binds to C-C chemokine receptor 6 (CCR6) on Th17 cells, promoting their migration to inflammation sites. The aim of this research is to examine the effectiveness of CCL20 inhibition in treating inflammation in AS. Mononuclear cells from peripheral blood (PBMC) and SF (SFMC) were collected from healthy individuals and AS. Flow cytometry was used to analyse cells producing inflammatory cytokines. CCL20 levels were determined using ELISA. The impact of CCL20 on Th17 cell migration was verified using a Trans-well migration assay. The in vivo efficacy of CCL20 inhibition was evaluated using an SKG mouse model. The presence of Th17 cells and CCL20 expressing cells was higher in SFMCs from AS patients compared with their PBMCs. The CCL20 level in AS SF was significantly higher than in OA patients. The percentage of Th17 cells in PBMCs from AS patients increased when exposed to CCL20, whereas the percentage of Th17 cells in SFMCs from AS patients decreased when treated with CCL20 inhibitor. The migration of Th17 cells was found to be influenced by CCL20, and this effect was counteracted by the CCL20 inhibitor. In the SKG mouse model, the use of CCL20 inhibitor significantly reduced joint inflammation. This research validates the critical role of CCL20 in AS and suggests that targeting CCL20 inhibition could serve as a novel therapeutic approach for AS treatment.

ASCT2-mediated glutamine uptake of epithelial cells facilitates CCL5-induced T cell infiltration via ROS-STAT3 pathway in oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disease characterized by T cell infiltration at lesion sites. T cell migration is greatly facilitated by chemokines produced by epithelial cells. Studies have noted the potential role of glutamine uptake in OLP and other inflammatory diseases. Here, we investigated the effect of altered glutamine uptake of epithelial cells on T cell infiltration and its underlying mechanisms in OLP. Immunohistochemistry was used to identify the expressions of glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2) and C-C motif chemokine ligand 5 (CCL5) in oral tissues of OLP and healthy controls. Human gingival epithelial cells (HGECs) were treated with glutamine deprivation and ASCT2 inhibiter GPNA respectively to detect the expressions of CCL5 and its related signaling molecules. Additionally, we had determined the impact of epithelial cell-derived CCL5 on T-cell migration using a co-culture system in vitro. ASCT2 and CCL5 expressions in OLP were significantly higher than healthy controls and positively correlated with the density of inflammatory infiltrations. Glutamine supplement significantly increased CCL5 production in HGECs, which was effectively inhibited by GPNA. Besides, glutamine could inhibit reactive oxygen species (ROS) production to activate the signal transducer and activator of transcription 3 (STAT3) causing higher expression level of CCL5 in HGECs. Simultaneously, T cell migration could be blocked by anti-CCL5 neutralizing antibody and STAT3 inhibitor stattic in the co-culture system. The upregulated ASCT2-mediated glutamine uptake in epithelial cells promotes CCL5 production via ROS-STAT3 signaling, which boosts the T-cell infiltration in OLP lesion.