High CCL18 Research Articles

ACE Phenotyping in Human Blood and Tissues: Revelation of ACE Outliers and Sex Differences in ACE Sialylation.

Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by blood ACE levels) is associated with an increased risk of cardiovascular diseases. Elevated blood ACE is also a marker for granulomatous diseases. Decreased blood ACE activity is becoming a new risk factor for Alzheimer's disease. We applied our novel approach-ACE phenotyping-to characterize pairs of tissues (lung, heart, lymph nodes) and serum ACE in 50 patients. ACE phenotyping includes (1) measurement of ACE activity with two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of these substrates (ZPHL/HHL ratio); (3) determination of ACE immunoreactive protein levels using mAbs to ACE; and (4) ACE conformation with a set of mAbs to ACE. The ACE phenotyping approach in screening format with special attention to outliers, combined with analysis of sequencing data, allowed us to identify patient with a unique ACE phenotype related to decreased ability of inhibition of ACE activity by albumin, likely due to competition with high CCL18 in this patient for binding to ACE. We also confirmed recently discovered gender differences in sialylation of some glycosylation sites of ACE. ACE phenotyping is a promising new approach for the identification of ACE phenotype outliers with potential clinical significance, making it useful for screening in a personalized medicine approach.

CCL18 as a Biomarker of Interstitial Lung Disease (ILD) and Progressive Fibrosing ILD in Patients with Idiopathic Inflammatory Myopathies.

To assess CCL18 and OX40L as biomarkers of interstitial lung disease (ILD) and/or progressive fibrosing (PF-) ILD in idiopathic inflammatory myopathies (IIMs). Patients with IIMs seen in our center from July 2020 to March 2021 were consecutively enrolled. ILD was detected by high-resolution CT. CCL18 and OX40L serum levels were measured by validated ELISA assays in 93 patients and 35 controls. At the 2-year follow-up, PF-ILD was evaluated according to the INBUILD criteria. ILD was diagnosed in 50 (53.7%) patients. CCL18 serum levels were higher in IIMs patients vs. controls (232.9 [IQR 134.7-399.07] vs. 48.4 [29.9-147.5], p < 0.0001), with no difference for OX40L. IIMs-ILD patients exhibited higher levels of CCL18 than those without ILD (306.8 [190.8-520.5] vs. 162 [75.4-255.8], p < 0.0001). High CCL18 serum levels were independently associated with IIMs-ILD diagnosis. At follow-up, 22/50 (44%) patients developed a PF-ILD. Patients who developed PF-ILD had higher CCL18 serum levels than non-progressors (511 [307-958.7] vs. 207.1 [149.3-381.7], p < 0.0001). Multivariate logistic regression analysis revealed CCL18 as the only independent predictor of PF-ILD (OR 1.006 [1.002-1.011], p = 0.005). Although in a relatively small sample, our data suggest that CCL18 is a useful biomarker in IIMs-ILD, particularly in the early identification of patients at risk of developing PF-ILD.

Abstract 4705: Profiling of immune cell components and soluble factors in ovarian cancer ascites highlights impaired immune environment

Abstract Background: It’s now increasingly recognized that ovarian cancer (OC) ascites play a significant role in OC progression - behavior of tumor cells is influenced by the nature of their surrounding microenvironment. Thus, characterization of ascites composition is essential to understand how this milieu affects tumor progression and particularly the immunosuppressive pathways that would underlie immune response dysfunctions and, in turn, how biological ascites effects can be influenced by that composition. Methods: Ascites samples were collected from advanced OC patients. Through respective multiplexed approaches of flow cytometry-based marker expression analysis and quantitation of mediators, immune context was profiled by investigating immune cell composition and levels of a plethora of soluble factors, including cytokines/chemokines, and metabolic pathways of some amino acids known to be involved in immunosuppression. Furthermore, ascites fluids were functionally screened for their biological effects on healthy monocytes, either undifferentiated or undergoing M1 polarization. Results: Unlike healthy PBMCs, OC ascites were mostly “enriched” in regulatory/immunosuppressive immune cell subsets including T and myeloid populations. Also, T cells were shown to highly express immune checkpoints such as PD1 in T cells and TIGIT in Tregs. Intriguingly, a high CD4/CD8 ratio was seen. Also, CD163+ tumor-associated macrophages were shown to express CSF1R, CCR8 and CCR2, and to even display a mixed phenotype since also expressing Arg1, CD80, and iNOS. On acellular fractions, most ascites demonstrated elevated CCL18, IL6, LIF, VEGF, and CCL2 levels, and low IL2, IL4, and IL17 levels. Interestingly, unlike healthy plasmas, these ascites appeared to harbor a metabolically-immunosuppressive profile characterized by high glutaminolysis and tryptophan (Trp) degradation in kynurenine (Kyn). Functionally, we demonstrated that not only ascites basically polarized monocytes into M2 macrophages, but even antagonized with their M1 polarization to ultimately tilt to M2 status. Conclusions: Taken together, our data show that ascites fluids most favorable to the M2 phenotype were associated with high LIF, VEGF, IL6, CCL2, and CCL18 levels, and with elevated Kyn to Trp ratios - Kyn levels being strongly higher than in healthy plasmas. Our results thus highlight a peculiar altered environment of OC ascites where a mixture of suppressive cells and signaling factors mediate extracellular cues leading to immune cell activity dysfunction. Altogether, these translational findings highlight OC ascites as a valuable tool to understand the mechanisms of suppression and develop predictive profiles, and to provide new insights for the identification of new targets and development of targeted-therapies. Citation Format: Assia Chaibi, Coriolan Lebreton, Dominique Bodet, Jean-Philippe Guegan, Guillaume Babin, Antoine Italiano, Alban Bessede, Imane Nafia. Profiling of immune cell components and soluble factors in ovarian cancer ascites highlights impaired immune environment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4705.

CCL18 promotes migration and invasion of multiple myeloma cells and is associated with poor prognosis.

CCL18 has recently been implicated in malignancies and is increasingly mentioned as a potential tumoral biomarker and furtherly a molecular target for therapeutic intervention, but its expression and clinical significance in multiple myeloma have not been explored. Serum CCL18 levels were measured by ELISA method in 254 newly diagnosed multiple myeloma (NDMM), 21 monoclonal gammopathy of undetermined significance (MGUS) and 22 healthy adults. The study suggests that the serum CCL18 level in NDMM patients was significantly higher than that in MGUS and healthy adults. High level of CCL18 were associated with advanced ISS and R-ISS stages in MM. Patients with high serum CCL18 displayed a significantly more frequent occurrence of renal impairment and hypercalcemia, while the proportion of achieving complete remission (CR) was lower. More importantly, Cox analysis identified CCL18 and LDH as independent predictors of PFS in MM patients, whereas CCL18, creatinine and LDH were independent predictors of OS. Finally, we show that CCL18 can promote migration and invasion of myeloma cell lines RPMI8226 and MM.1S. CCL18 may play a tumor-promoting role by increasing the migration and invasion abilities of myeloma cells.

CCL18 overexpression predicts a worse prognosis in oral squamous cell carcinoma (OSCC).

Oral squamous cell carcinoma (OSCC) presents severe morbidity and high mortality owing to local recurrence or remote metastasis. Molecular markers, including chemokines, might provide more efficient prognostic information or even therapeutic targets for the treatment of OSCC. Using quantitative RT-qPCR, we found that CCL18 was dramatically overexpressed in 30 OSCC tissues at the mRNA level in comparison with their adjacent non-cancerous oral mucosa tissues and 15 oral mucosa tissues from non-malignant patients. We then analyzed the relationship between CCL18 overexpression and patient clinical characters and outcomes using immunohistochemistry staining (IHC) in 102 paired OSCC cancerous and adjacent non-cancerous tissues; the increase in CCL18 expression was significantly higher in male patients (p=0.047), tumors of the palate and floor of the mouth (p=0.014), patients with positive lymph node metastasis (p=0.007), and patients with poor tumor differentiation (p=0.029). The median overall survival time and time-to-recurrence were 80.6 and 61.4 months in patients with high CCL18 expression, respectively, as against 93.4 and 81.6 months in patients with comparatively lower CCL18 expression, respectively (p=0.033 and 0.012, respectively; log-rank test). Multivariate analyses indicated age, poor differentiation, and CCL18 levels to be independent prognostic factors for predicting both overall and disease-free survival time. Our study suggests that CCL18 is a novel candidate marker for the OSCC malignancy and prognosis, including lymph node metastasis, time-to-recurrence, and disease-free survival time.

High serum CCL18 predicts a poor prognosis in patients with laryngeal squamous cell carcinoma.

CCL18 is a cytokine secreted by M2 type tumor associated macrophages, which frequently over-expressed in diverse human cancers. However, the clinical significance of serum CCL18 in patients with laryngeal squamous cell carcinoma (LSCC) remains unknown. In this study, serum CCL18 was initially quantified by enzyme-linked immunosorbent assay (ELISA) in 146 patients with LSCC, 25 patients with precancerous lesions and 72 healthy volunteers. In addition, the correlations between serum CCL18 and clinicopathological parameters were analyzed. Our data revealed that serum CCL18 was obviously increased in patients with LSCC. Moreover, serum CCL18 level was significantly associated with primary tumor site (Glottic vs Others), T classification (T1+T2 vs T3+T4), clinical stage (I+II vs III+IV) and lymph node metastasis (N0 vs N+). Survival analysis demonstrated that patients with high serum CCL18 displayed a shorter survival time than those in patients with low serum CCL18. Importantly, serum CCL18 level and clinical stage were independent prognostic factors in patients with LSCC. Taken together, serum CCL18 could be used as a promising biomarker in patients with LSCC.

High Level of Chemokine CCL18 Is Associated With Pulmonary Function Deterioration, Lung Fibrosis Progression, and Reduced Survival in Systemic Sclerosis

Markers for early identification of progressive interstitial lung disease (ILD) in systemic sclerosis (SSc) are in demand. Chemokine CCL18, which has been linked to pulmonary inflammation, is an interesting candidate, but data have not been consistent. We aimed to assess CCL18 levels in a large, prospective, unselected SSc cohort with longitudinal, paired data sets on pulmonary function and lung fibrosis. Sera from the Oslo University Hospital SSc cohort (n= 298) and healthy control subjects (n= 100) were analyzed for CCL18 by enzyme immunoassay. High CCL18 (>53ng/mL) was defined using the mean value plus 2 SD in sera obtained from healthy control subjects as the cutoff. High serum CCL18 was identified in 35%(105 of 298). Annual decline in FVC differed significantly between high and low CCL18 subsets (13.3%and 4.7%; P= .016), as did the annual progression rate of lung fibrosis (0.9%[SD, 2.9] and 0.2%[SD, 1.9]). Highest rates of annual FVC decline > 10%(21%) and annual fibrosis progression (1.2%) were seen in patients with high CCL18 and early disease (< 3 years). In multivariate analyses, CCL18 was associated with annual FVC decline > 10%(OR, 1.1; 95%CI, 1.01-1.11) and FVC< 70%at follow-up (OR, 3.1; 95%CI, 1.08-8.83). Survival analyses showed that patients with high CCL18 had reduced 5- and 10-year cumulative survival compared with patients with low CCL18 (85%and 74%, compared with 97%and 89%, respectively; P= .001). The results from this prospective cohort reinforce the notion that high CCL18mayserve as a marker for early identification of progressive ILD in SSc.

CCL18 as an independent favorable prognostic biomarker in patients with colorectal cancer

BackgroundCCL18 has been shown to have an important role in the progression of gastric and breast cancers. However, the prognostic value of CCL18 in colorectal cancer (CRC) remains unknown. Materials and methodsWe used immunohistochemistry to examine the expression of CCL18 in CRC patients. We applied both univariate and multivariate analysis to evaluate the prognostic value of CCL18 on CRC patients' survival. We used double staining to investigate the relationship between CCL18 and macrophages. ResultsA total 371 CRC patient samples were enrolled in immunohistochemical analysis. According to our criteria, 118 samples (31.8%) showed a high CCL18 expression level. Clinicopathologic analysis revealed an association between the expression level of CCL18 and the preoperative carcino embryonic antigen level (P = 0.001), and the preoperative carbohydrate antigen 19-9 level (P = 0.003). Survival analysis and multivariate analysis revealed that CCL18 was an independent favorable prognostic factor in patients with CRC (P = 0.033). Double staining implied that CCL18 was expressed by macrophages. ConclusionsA high CCL18 level might be an independent biomarker for predicting better survival of patients with CRC.

Addition of Rituximab to Chemotherapy Alters the Prognostic Impact of Tumor-Associated Macrophages in Diffuse Large B-Cell Lymphoma

Abstract 2650 Background:Tumor associated macrophages (TAM) have at least two potential roles in promoting tumor growth: suppression of immune responses and potentiation of angiogenesis. In numerous cancer types, including lymphomas, high M2 type TAM content has been associated with worse prognosis. Rarely, high TAM content correlates with better survival. We have recently shown that CD68 positive TAMs in DLBCL contribute to unfavorable survival after high dose chemotherapy. Here we have extended our analyses on M2 type macrophages and questioned how combination of rituximab with chemotherapy influences TAM-associated clinical outcome. Patients and Methods:Expression of CD163 and CCL18, which are primarily expressed in M2 type macrophages, were identified immunohistochemically from samples of 101 de novo DLBCL patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). With a median follow-up of 65 months, (range 16–114 months), 5-year progression free survival (PFS) was 70% and overall survival (OS) 73%. 29 DLBCL patients previously treated with up front high dose chemotherapy served as a control group. Results:Correlation between CD163 and CCL18 positive TAMs was found (rs=0.427, p<0.001). In the Kaplan-Meier analyses the cutoff level of 67% was found to best discriminate between subgroups with different outcomes. Consistent with previous data, chemotherapy-treated patients with high CD163 or CCL18 positive TAM counts displayed a significantly inferior OS and PFS than the low group (Table). In contrast, after rituximab containing regimen, the patients with high CD163 and CCL18 positive TAM content tended to have favorable survival. Among the patients with low counts in both CD163 and CCL18 positive TAMs, PFS and OS were found to be significantly worse in comparison to others.SurvivalMarkerImmunochemotherapy groupControl groupLow (%)High (%)P-valueLow (%)High (%)P-value5-year OSCD16370800.23575330.019CCL1869840.15174330.037CD163+CCL1865850.035100380.0035-year PFSCD16366820.09175400.054CCL1868800.20674390.131CD163+CCL1863830.04075420.013 Conclusions:In contrast to data on chemotherapy treated DLBCL or other lymphoma types, M2 type TAM content is associated with favorable prognosis in DLBCL patients after immunochemotherapy. Disclosures:No relevant conflicts of interest to declare.

Thrombin facilitates invasion of ovarian cancer along peritoneum by inducing monocyte differentiation toward tumor-associated macrophage-like cells.

Peritoneal metastasis is a distinct pathologic characteristic of advanced epithelial ovarian cancer (EOC), which is the most deadly disease of the female reproductive tract. The inflammatory environment of the peritoneum in EOC contains abundant macrophages, activated thrombin, and thrombin-associated receptors. However, little is known about the mechanism by which the thrombin-macrophages interaction contributes to tumor invasion and metastasis. We investigated the phenotype and cytokine/chemokine expression of thrombin-treated peripheral blood monocytes (MOs)/macrophages, it was found that the phenotype of MOs was altered toward a TAM-like macrophage CD163(high)IL-10(high)CCL18(high)IL-8(high) after thrombin stimulation. By Matrigel invasion assay, the conditioned medium of thrombin-stimulated MOs accelerated remarkable invasion of ES-2, SKOV3, and HO-8910, which was similar to invasive cell numbers of ascites stimuli (P < 0.05) and higher than MOs medium alone (P < 0.05). IL-8 was proposed as the major chemoattractant mediating EOC invasion based on MOs mRNA and protein expression profiling. It was observed that anti IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner. Increased transcriptional activation of NF-kappaB p50/p65 was identified in thrombin-treated MOs. This study provided insight the role of thrombin in the regulation of EOC peritoneal invasion via "educating" MOs.

Serum CC-Chemokine Ligand 18 Concentration Predicts Outcome in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. There is great effort to find predictors of outcome. Conclusive data for any serum biomarker are lacking. We have recently documented that serum CCL18 concentrations correlate with the course of pulmonary function data in patients with pulmonary fibrosis of various causes. To test the value of serum CCL18 concentrations in IPF, we included 72 patients in a prospective study. IPF was defined according to the ATS/ERS criteria. Serum CCL18 concentrations were measured by a commercially available ELISA. Patients were followed for 24 months. Pulmonary function tests were performed at least every 6 months. Baseline serum CCL18 concentrations predicted the change in TLC and FVC at the 6-month follow-up. Receiver operating characteristics (ROC) revealed a significant relation between survival and baseline CCL18 concentrations. By ROC analysis, the cutoff value with the highest diagnostic accuracy was defined as 150 ng/ml (sensitivity, 0.83; specificity, 0.77). There was a significantly higher mortality in patients with serum CCL18 concentrations above 150 ng/ml (P < 0.0001). The hazard proportional ratio adjusted for age, sex, and baseline pulmonary function data was 8.0. There was a higher incidence of disease progression in the group with high serum CCL18 concentrations. Our data demonstrate that serum CCL18 concentrations have a predictive value in IPF and may be a useful tool in the clinical management of patients with IPF and in clinical trials.

Enhanced interleukin‐10 production by dendritic cells upon stimulation with Toll‐like receptor 4 agonists in systemic sclerosis that is possibly implicated in CCL18 secretion

Background: It has been suggested that the T‐cell attracting and profibrotic chemokine CCL18 might play a role in the pathogenesis of systemic sclerosis (SSc). However, it is unclear what underlies the higher CCL18 levels in SSc. The aim of our study was to determine whether Toll‐like receptor (TLR)‐mediated stimulation of monocytes and dendritic cells (DCs) contributes to the higher levels of CCL18 in SSc.Methods: CCL18 levels were measured in 40 patients with SSc, primary Raynaud's phenomenon (RP) and healthy controls. The presence of TLR4 agonists in the circulation of SSc patients was investigated using TLR4 transgenic Chinese hamster ovary (CHO) cells. CCL18 and interleukin (IL)‐10 secretion by monocytes/macrophages and monocyte‐derived DCs (moDCs) was measured in the supernatant. The indirect effect of lipopolysaccharide (LPS)‐stimulated moDCs on CCL18 secretion by monocytes/macrophages was investigated using a transwell system.Results: CCL18 levels were significantly elevated in SSc patients compared to patients with RP and healthy controls. SSc sera strongly induced CD25 expression on CHO cells genetically modified to express TLR4 but not on those expressing CD14 only. By contrast, serum from systemic lupus erythematosus (SLE) patients or healthy individuals did not have an effect. Neither monocytes/macrophages nor moDCs from SSc patients secreted higher levels of CCL18 compared to healthy controls. However, moDCs matured with the TLR4 ligand LPS from patients with SSc did secrete significantly higher amounts of IL‐10 compared to those from healthy counterparts, which were IL‐10 dependent.Conclusions: Our results suggest that elevated CCL18 levels in SSc are not caused by an intrinsically enhanced CCL18 secretion by monocytes/macrophages but are, at least partly, orchestrated by an enhanced IL‐10 secretion by TLR4‐stimulated DCs. These observations suggest a role for TLR4 ligands and DCs in the pathogenesis of SSc, a topic that warrants further investigation.

Changes in the Atherogenic Profile in Type 1 Gaucher Disease Patients after Substrate Reduction Therapy

Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid β-glucosidase (GC) leading to accumulation of glucosylceramide within the macrophages of the reticuloendothelial systems. Plasma of GD patients contains low levels of total cholesterol, LDL-cholesterol and HDL-cholesterol. Enzyme replacement therapy (ERT) with imiglucerase and Substrate Reduction Therapy (SRT) with miglustat restores the haemoglobin levels, platelet counts and organomegalies. Previously we have demonstrated that ERT had significant effects on the concentration and metabolism of plasma lipoproteins. However, the effects of SRT on plasma lipid and other atherogenic profiles of GD patients have not been explored. We report the results of long term (36 months) SRT on plasma lipoprotein concentrations in 26 (11 men and 15 women) GD patients. Mean age of patients was 51 (SD ±20.7; range 22–74) years. Ten patients were therapy-naïve to SRT and 16 switched from previous enzyme replacement therapy (ERT). All patients received 100 mg of Miglustat t.i.d with recommendations of a low hydrocarbonate diet in the first weeks. Total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-C), Apolipoproteins (Apo AI, ApoB and Lp(a)), CRP, MPI1b, CCL18 concentrations and chitotriosidase activity were measured before (baseline) and after 12, 24 and 36 months of follow-up. Treatment resulted in a significant increases in HDL-c concentration in naïve patients during the first 24 months (p<0.02) on therapy and it is maintain till 36 months. The same trend was observed in HDL-cholesterol in switch patient but with a less increase that in naïve patients. TC increase slightly in switch (p<0.05) and naïve (p<0.04) patients after 24 and 36 months of treatment respectively. No significant changes in LDL-c during treatment were observed in both switch and naïve patient. In naïve patients, TG, Lp(a) and CRP concentrations, as well as the atherogenic index TC/HDL-c decreased significantly in patients after 24 months therapy and no changes were observed in switch patients. Our results indicate that miglustat treatment have an apparent beneficial effects on plasma lipid, lipoprotein and CRP concentrations resulting in an apparently less atherogenic lipid profile in GD patients. However further studies will be required to determine the impact of miglustat treatment on the risk of coronary heart disease.

Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines, Toll-like receptors and rheumatoid synovial fluid

BackgroundThe T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells.ResultsIn contrast to a large panel of pro-inflammatory stimuli (IL-1β, TNF-α, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-γ), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid.ConclusionIn summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA.