CD14 Subpopulations Research Articles

Chitinase 3-like 1 Produced By Fibrocytes Promotes Myelofibrosis

Introduction Monocyte-derived fibrocytes are spindle-shaped fibroblast-like blood cells derived from a sub-population of CD14+ monocytes. Recent studies have shown that fibrocytes play a key role in the pathogenesis of primary myelofibrosis (MF), and these cells have attracted attention as novel therapeutic targets for MF treatment. Using a Romiplostim (Rom)-induced murine MF model, we have previously reported that fibrocyte differentiation, which is directly induced by activating the thorombopoietin receptor signaling pathway, can potentially trigger the progression of MF (Leukemia 2017; 31: 2709). However, the precise molecular mechanism of fibrocyte activity in MF have not been elucidated. To address this question, we compared the RNA sequencing-based gene expression profiles of monocytes and fibrocytes and detected that fibrocytes exhibited a higher level of Chitinase 3-like 1 (CHI3L1) expression than monocytes. CHI3L1, an 18-glycosyl hydrolase-related molecule, is a member of the enzymatically inactive chitinase-like protein family. These proteins are associated with inflammation, tissue remodeling, and organ fibrosis. Therefore, we hypothesized that CHI3L1 produced by fibrocytes is potentially involved in the progression of MF and could be a therapeutic target. In the present study, we evaluated CHI3L1 using serum samples obtained from patients with myeloproliferative neoplasms (MPN) and a Rom-induced murine MF model. Methods Monocytes and fibrocytes (Day 9 and Day 12) were prepared using peripheral blood samples from healthy volunteers. Following total RNA extraction, RNA qualification, and library preparation, a Hiseq4000 (Illumina) system was used for RNA sequencing. ELISA-based analyses were performed using the supernatant of the blood cells to confirm the presence of the CHI3L1 protein. Furthermore, we determined the serum CHI3L1 levels of MPN patients with or without MF using ELISA-based analyses. Overall, 21 patients without MF and 31 patients with MF (MF-1 to MF-3) were enrolled after obtaining written informed consent. Using the Rom-induced murine MF model, we performed in vivo analyses of Chi3l1. Female C57BL/6J mice (8-week-old) were administered with 1 mg/kg Rom once a week for 2 or 3 weeks, following which serum samples, spleens, and bone marrow samples were examined. To eliminate fibrocytes, clodronate liposomes were administered to mice. Chi3l1-deficient (Chi3l1−/−) mice were administered with Rom in the same manner. Following these treatments, RT-PCR and histopathological analyses of bone marrow were conducted. Non-contact co-culture assays of human fibrocytes and fibroblasts were performed using the human fibroblast cell line HS-5 to evaluate the interaction between CHI3L1 and fibroblasts. HS-5 cells were cultured using the supernatant of fibrocytes with or without the neutralizing antibody for CHI3L1 and mRNA expression of HS-5 cells was examined. Results Comparing the RNA sequencing gene expression profiles of monocytes and fibrocytes revealed a significant increase in CHI3L1 expression associated with fibrocyte differentiation. ELISA-based analyses of human monocyte and fibrocyte supernatants confirmed that the secretion of CHI3l1 protein concurred with the amount of mRNA expression (P < 0.01) (a). We demonstrated that CHI3L1 levels were elevated in in serum samples obtained from MPN patients with MF (N = 31) compared with those obtained from patients without MF (N = 21; P = 0.027) (b). High levels of Chi3l1 mRNA expression were observed in the bone marrow of the Rom-induced murine MF model (P < 0.01), which was abrogated by clodronate treatment. Furthermore, compared with wild-type mice, Chi3l1−/− mice showed fewer reticulin fibers in silver-stained bone marrow sections (c) and significantly decreased Col3A1 and Acta2 mRNA expression in the bone marrow (P < 0.05). In addition, co-culture assays demonstrated that HS-5 cells cultured with the supernatant of fibrocytes showed significantly higher levels of COL1A1 and COL3A1 expression (P < 0.05) than that of monocytes and the addition of CHI3L1 neutralizing antibody abrogated these increases (d). Conclusion CHI3L1 is involved in the interaction between fibrocytes and fibroblasts and this molecule exacerbates the progression of bone marrow fibrosis by promoting the production of the extracellular matrix. Therefore, CHI3L1 could be a potential therapeutic target for MF treatment. Figure Disclosures Makishima: Bristol-Myers Squibb: Research Funding. Komatsu:Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Novartis K.K: Speakers Bureau. Kimura:JSPS KAKENHI: Research Funding.

A novel CD14(high) CD16(high) subset of peritoneal macrophages from cirrhotic patients is associated to an increased response to LPS.

The aim of this study was to characterize monocyte-derived macrophages (M-DM) from blood and ascites of cirrhotic patients comparatively with those obtained from blood of healthy controls. The phenotypic profile based on CD14/CD16 expression was analyzed by flow cytometry. Cells were isolated and stimulated in vitro with LPS and heat killed Candida albicans. Phosphorylation of ERK, c-Jun, p38 MAPK, and PKB/Akt was analyzed by Western blotting. A novel CD14(high)CD16(high) M-DM subpopulation is present in ascites (∼33%). The CD14(++)CD16(+) intermediate subset is increased in the blood of cirrhotic patients (∼from 4% to 11%) and is predominant in ascites (49%), while the classical CD14(++)CD16(-) subpopulation is notably reduced in ascites (18%). Basal hyperactivation of ERK and JNK/c-Jun pathways observed in ascites M-DM correlates with CD14/CD16 high expressing subsets, while PI3K/PKB does it with the CD16 low expressing cells. In vitro LPS treatment highly increases ERK1/2, PKB/Akt and c-Jun phosphorylation, while that of p38 MAPK is decreased in M-DM from ascites compared to control blood M-DM. Stimulation of healthy blood M-DM with LPS and C. albicans induced higher phosphorylation levels of p38 than those from ascites. Regarding cytokines secretion, in vitro activated M-DM from ascites of cirrhotic patients produced significantly higher amounts of IL-6, IL-10 and TNF-α, and lower levels of IL-1β and IL-12 than control blood M-DM. In conclusion, a new subpopulation of CD14(high)CD16(high) peritoneal M-DM has been identified in ascites of cirrhotic patients, which is very sensitive to LPS stimulation.

DNA methylation profiling of the X chromosome reveals an aberrant demethylation on CXCR3 promoter in primary biliary cirrhosis.

BackgroundAlthough the etiology of primary biliary cirrhosis (PBC) remains enigmatic, there are several pieces of data supporting the thesis that a strong genetic predisposition and environmental factors interact to produce a selective loss of tolerance. The striking female predominance of PBC has suggested that this sex predisposition may be secondary to epigenetic alterations on the X chromosome. In the present study, we rigorously defined the X chromosome methylation profile of CD4, CD8, and CD14 cells from 30 PBC patients and 30 controls. Genomic DNA from sorted CD4, CD8, and CD14 subpopulations was isolated, sonicated, and immunoprecipitated for analysis of methylation. All products were hybridized to a custom-tiled four-plex array containing 27,728 CpG islands annotated by UCSC and 22,532 well-characterized RefSeq promoter regions. Furthermore, bisulfite sequencing was then used for validation on a subsequent group of independent samples from PBC patients and controls. Thence, expression levels of selected X-linked genes were evaluated by quantitative real-time PCR with cDNA samples from all subjects.ResultsWe report herein that a total of 20, 15, and 19 distinct gene promoters reflected a significant difference in DNA methylation in CD4+ T, CD8+ T, and CD14+ cells in patients with PBC. Interestingly, there was hypermethylation of FUNDC2 in CD8+ T cells and a striking demethylation of CXCR3 in CD4+ T cells, which inversely correlated with CXCR3 expression levels in CD4+ T cells from early-stage PBC patients.ConclusionsOur data provides a set of genes with epigenetic alteration likely to be indicators of autoimmunity and emphasizes the role of CXCR3 in the natural history of PBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0098-9) contains supplementary material, which is available to authorized users.

The CD14brightCD16+ monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population

Circulating monocytes contain a subpopulation of CD14+CD16+ cells; this subpopulation of cells has been described to be proinflammatory and to have an increased frequency in rheumatoid arthritis (RA). New evidence suggests that this subpopulation can be further subdivided into CD14(dim) CD16+ and CD14(bright) CD16+ cells. The aim of this study was to determine which of the two CD16+ monocyte subpopulations is expanded in patients with RA and to investigate their possible role in disease pathogenesis. The frequencies of monocyte subpopulations in the peripheral blood of healthy donors and patients with RA were determined by flow cytometry. Monocyte subpopulations were sorted and cocultured with CD4+ T cells. Cytokines were determined in the supernatant, and Th17 cell frequencies were measured by flow cytometry. In comparison with the other monocyte subpopulations, CD14(bright) CD16+ cells showed higher HLA-DR and CCR5 expression and responded with higher tumor necrosis factor production to direct cell contact with preactivated T cells. They were observed at increased frequencies in the peripheral blood of patients with RA, while CD14(dim) CD16+ monocyte frequencies were not increased. CD14(bright) CD16+ cells were extremely potent inducers of Th17 cell expansion in vitro. Their frequency in the peripheral blood of patients with RA correlated closely with Th17 cell frequencies determined ex vivo. This study is the first to provide a link between the increased frequency of the CD14(bright) CD16+ monocyte subpopulation in RA and the expansion of Th17 cells, which are likely to have a role in the pathogenesis of autoimmunity.

023 Platelet activation induces phenotypic and functional changes in circulating monocytes as a consequence of monocyte–platelet aggregate formation

<h3>Background and Hypothesis</h3> Circulating levels of monocyte-platelet aggregates (MPAs) increase in response to platelet activation, and have previously been shown to be elevated in patients presenting with acute cardiovascular (CV) events and in healthy subjects who have underlying CV risk factors. MPAs are also believed to be crucial mediators involved in the pathophysiology of atherosclerosis. Since circulating monocytes comprise different sub-populations which can infiltrate and migrate into tissues to differing degrees, we hypothesised that activated platelets may enhance their differentiation towards a pro-atherogenic phenotype. <h3>Methods</h3> MPAs and monocytes in peripheral blood obtained from healthy volunteers were measured and characterised by FACS analysis. Additionally, monocytes were isolated through immunomagnetic negative selection for CD14+/CD16− cells and co-incubated with autologous platelets. Flow cytometry and adhesion assays on pre-activated endothelial cells (treated with TNF-α 10 ng/ml for 3 h) were performed at different time points. <h3>Results</h3> Three monocytic subsets were identified in blood: classical CD14+/CD16−, CD14high/CD16+ and CD14low/CD16+ cells. They displayed different expression patterns of the adhesion molecules CD11b and CD11c, although no difference was found in their ability to form MPAs in vivo. Platelet co-incubation enhanced monocyte phenotypic change toward CD14+/CD16+ double positivity in a time-dependent manner (33.33±7.79% vs 16.76±6.34% at 48h in the presence or absence of platelets respectively; p=0.0001), with CD16 expression being directly correlated to MPA levels (r²=0.746; p=0.005). This phenotypic change was associated with enhanced monocyte adhesion to activated endothelium (11.5±1.7 vs 5.7±1.5 cells per field, when co-incubated or not with platelets respectively; p=0.005). Adhesion assay demonstrated that only cells positive for both CD14 and CD16 could bind to endothelium, while classical CD14+/CD16− monocytes could not (Abstract 023 Figure 1a). In keeping with this, the sub-population of CD14+/CD16+ monocytes exhibited higher levels of both CD11b and CD11c on the cell surface compared to the monocyte sub-population only positive for CD14. PSGL-1 blocking antibody, which abrogates monocyte-platelet interaction, abolished all these effects (Abstract 023 Figure 2). Moreover, induction of cyclooxygenase 2 (COX-2) was observed in monocytes at 18 h of co-incubation with platelets (Abstract 023 Figure 1b), and specific blockade of COX-2 with NS-398 (10 μM) reduced platelet-dependent CD16 upregulation in CD14+ monocytes, without affecting MPA formation in vitro (Abstract 023 Figure 2). <h3>Conclusion</h3> Platelet interaction upregulates CD16 and expression of adhesion molecules on monocytes, in a COX-2-dependent manner, resulting in greater adhesion to the endothelium.

Expansion of CD14+CD16+ peripheral monocytes among patients with aseptic loosening

In this study, we have investigated the relevance of peripheral blood inflammatory CD14(+)CD16(+) monocytes phenotype to patients with aseptic loosening (AL). Immunophenotypes of monocytes were examined among patients with AL (n = 43), patients with mechanical loosening (ML, n = 30), patients with stable implant (SI, n = 16), and patients with osteoarthritis (OA, n = 17) using flow cytometry. Immunological assay was used to measure TNF-alpha and IL-1 beta levels in both sera and culture media of implant wear stimulated CD14(+)CD16(+) and CD14(++)CD16(-) monocytes. Periprosthetic tissues were collected during surgery for histological assessment. The frequency of CD14(+)CD16(+) monocytes showed significant increase in AL patients than in ML, SI, and OA patients. A positive association was found between the subpopulation of CD14(+)CD16(+) monocytes and plasma TNF-alpha and IL-1 beta level in AL patients. Furthermore, a positive correlation existed between the subpopulation of CD14(+)CD16(+) monocytes and the total histopathology score. The results indicate that CD14(+)CD16(+) monocytes represent a sensitive marker for the disease activity of AL, and may serve as an effective prognostic index to identify total joint replacement recipients who are at increased risk for osteolysis and progression of AL.