Human Fibrocytes Research Articles

Targeting IL-11 to reduce fibrocyte circulation and lung accumulation in animal models of pulmonary hypertension-associated lung fibrosis.

IL-11 is a member of the IL-6 family of cytokine initially considered as haematopoietic and cytoprotective factor. Recent evidence indicates that IL-11 promotes lung fibrosis and pulmonary hypertension in animal models and is elevated in lung tissue of patients with pulmonary fibrosis and pulmonary hypertension. Fibrocytes are bone marrow-derived circulating cells that participate in lung fibrosis and pulmonary hypertension, but the role of IL-11 on fibrocytes is unknown. We investigated the role of IL-11 system on fibrocyte activation in different in vitro and in vivo models of lung fibrosis associated with pulmonary hypertension. Human fibrocytes were isolated from peripheral blood of six healthy donors. Recombinant human (rh)-IL-11 and soluble rh-IL-11 receptor, α subunit (IL-11Rα) were used to stimulated fibrocytes in vitro to measure:- cell migration in a chemotactic migration chamber, fibrocyte to endothelial cell adhesion in a microscope-flow chamber and fibrocyte to myofibroblast transition. Mouse lung fibrosis and pulmonary hypertension was induced using either IL-11 (s.c.) or bleomycin (intra-tracheal), while in the rat monocrotaline (intra-tracheal) was used. In vivo siRNA-IL-11 was administered to suppress IL-11 in vivo. RhIL-11 and soluble rhIL-11Rα promote fibrocyte migration, endothelial cell adhesion and myofibroblast transition. Subcutaneous (s.c.) IL-11 infusion elevates blood, bronchoalveolar and lung tissue fibrocytes. SiRNA-IL-11 transfection in bleomycin and monocrotaline animal models reduces blood and lung tissue fibrocytes and reduces serum CXCL12 and CXCL12/CXCR4 lung expression. Targeting IL-11 reduces fibrocyte circulation and lung accumulation in animal models of pulmonary hypertension-associated lung fibrosis.

Increased Expression of Galectin-3 in Skin Fibrosis: Evidence from In Vitro and In Vivo Studies.

Skin fibrosis is a hallmark of a wide array of dermatological diseases which can greatly impact the patients' quality of life. Galectin-3 (GAL-3) has emerged as a central regulator of tissue fibrosis, playing an important pro-fibrotic role in numerous organs. Various studies are highlighting its importance as a skin fibrotic diseases biomarker; however, there is a need for further studies that clarify its role. This paper aims to ascertain whether the expression of GAL-3 is increased in relevant in vitro and in vivo models of skin fibrosis. We studied the role of GAL-3 in vitro using normal human dermal fibroblasts (NHDF) and fibrocytes. In addition, we used a skin fibrosis murine model (BALB/c mice) and human biopsies of healthy or keloid tissue. GAL-3 expression was analyzed using real time PCR, Western blot and immunostaining techniques. We report a significantly increased expression of GAL-3 in NHDF and fibrocytes cell cultures following stimulation with transforming growth factor β1 (TGFβ1). In vivo, GAL-3 expression was increased in a murine model of systemic sclerosis and in human keloid biopsies. In sum, this study underlines the involvement of GAL-3 in skin fibrosis using several models of the disease and highlights its role as a relevant target.

Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system

Fibrocytes are bone marrow–derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.

NOD/scid IL‐2Rγnull mice reconstituted with peripheral blood mononuclear cells from patients with Crohn's disease reflect the human pathological phenotype

IntroductionCrohn's disease (CD) is characterized by pronounced intestinal fibrosis and severe mucosal damage and conventional animal models are limited to reflect these pathological manifestations. The aim of this study was to examine whether the combination of patient immune‐profiling and preclinical studies in a mouse model based on NOD/scid IL‐2Rγnull (NSG) reconstituted with peripheral blood mononuclear cells (PBMC) from CD patients has the capacity to harmonize ex vivo human and in vivo animal studies.MethodsImmunological profiles of CD (n = 24) and ulcerative colitis (UC) patients (n = 47) were established by flow cytometry of subgroups of immune cells and subjected to hierarchical cluster and estimation graphics analyses. Pathological phenotypes of NSG mice, which were reconstituted with PBMC from CD, UC, and non‐IBD donors (NSG‐CD, NSG‐UC, and NSG‐non‐IBD) were compared. Readouts were the clinical, colon, and histological scores; subtypes of immune cells from spleen and colon; and levels of inflammatory markers, such as c‐reactive protein (CRP), monocyte chemotactic protein (MCP)‐3, transforming growth factor‐beta (TGFß), and hepatocyte growth factor (HGF). Fibrocytes were identified by immunohistochemistry in colonic sections.ResultsCD patients were significantly clustered in a group characterized by increased levels of TH1, TH2 cells, and decreased levels of CD14+ CD163+ monocytes (p = .003). In contrast to NSG‐UC mice, NSG‐CD mice exhibited an immune‐remodeling phenotype characterized by enhanced collagen deposition, elevated levels of CD14+ CD163+ monocytes, HGF, and TGFß. This phenotype was further corroborated by the presence of human fibrocytes as components of fibrotic areas.ConclusionThe NSG‐CD model partially reflects the human disease and allows for studying the development of fibrosis.

Blockade of PD-1/PD-L1 Pathway Enhances the Antigen-Presenting Capacity of Fibrocytes.

Fibrocytes, a distinct population of collagen-producing, monocyte-derived cells, are involved in wound healing as well as fibrotic diseases. Recently, fibrocytes have been revealed to play a role in the tumor microenvironment, particularly under antiangiogenic therapy. In addition, combination cancer immunotherapy with immune checkpoint inhibitor and antiangiogenic agents have been developed for various cancers in the clinical setting, although the immunological background is not clear. In the current study, we aimed to determine the function of fibrocytes in tumor immunity induced by immune checkpoint inhibitor therapy. Human and murine fibrocytes were generated from PBMCs and lungs, respectively. The expression of costimulatory and inhibitory molecules on fibrocytes was examined by flow cytometry. The stimulation of CD8+ T cells by fibrocytes was examined in MLRs with a 3H-thymidine incorporation assay. Fibrocytes expressed CD80low and CD86high as a costimulatory molecule, and expressed PD-L1high, but not PD-L2, as a coinhibitory molecule. Without any stimulation, fibrocytes strongly enhanced the proliferation of CD8+ T cells in mice and humans. Treatment with anti-CD86 and -CD54 Abs inhibited the growth of CD8+ T cells induced by fibrocytes. Anti-PD-L1 Ab further enhanced the proliferation of CD8+ T cells, even in the OVA-specific MLR with OT-1Rag-/- mice. Importantly, fibrocytes derived from PBMCs of patients with lung adenocarcinoma or murine MC38 tumors augmented the proliferation of CD8+ T cells with PD-L1 blockade. These results suggest that fibrocytes infiltrating tumor sites may play a role in the antitumor immunity mediated by CD8+ T cells when the activity is further enhanced by PD-L1/PD-1 blockade.

Expression of extracellular matrix components and cytokine receptors in human fibrocytes during rheumatoid arthritis

ABSTRACT Purpose: Fibroblast-like synoviocytes (FLS) represent one of the principal effectors of joint damage in rheumatoid arthritis (RA). Recent discovery of the circulating fibrocyte, a potential precursor of FLS, has raised issues regarding the characterization of fibrocytes with respect to their morphology and their biological role. In this study, we evaluated the morphology of fibrocytes in vitro and their ability to produce different extracellular matrix (ECM) components in comparison with two populations of RA FLS: synovial fluid FLS (fd-FLS) and intimal lining FLS (td-FLS). We also studied the expression of ECM regulators and a set of cytokine receptors involved in the pathogenesis of RA. Materials and Methods: Fibrocytes were cultured from peripheral blood of patients with RA. FLS were cultured from synovial fluids and tissues. ECM proteins (collagen I (col I) and fibronectin), Matrix metalloproteinases (MMP) (MMP3, and MMP9), ECM regulators (β catenin, TCF4, and c-fos), and cytokine receptors (CXCR1, CXCR2, CXCR3, IL1RI, IL1RII, and IL6Rα) were analyzed using qRT-PCR and/or western blot. Results: Our results demonstrated that fibronectin and MMP3 levels were higher in FLS compared to fibrocytes. Although MMP9 was expressed in the three cell types, its level was greater in fibrocytes than in td/fd FLS. The three cell types expressed CXCR3, IL1RI, IL1RII, and IL6Rα, while the expression of CXCR1 and CXCR2 was restricted to fibrocytes. Conclusion: Our results demonstrated that fibrocytes express ECM molecules and cytokines receptors. The observed differences between fibrocytes and FLS may be due to their distinct functions or differentiation state during RA. Abbreviations: RA: Rheumatoid ArthritisFLS: fibroblast-like synoviocytestd: tissue derivedfd: fluid derivedSF: Synovial FluidWnt: WinglessMMP: Matrix MetalloproteinaseCIA: murine collagen induced arthritisECM: Extracellular matrixcol I: Collagen ITCF/LEF: T-cell factor/lymphoid enhancer-binding factorAP1: Activator Protein 1

Slit2 May Underlie Divergent Induction by Thyrotropin of IL-23 and IL-12 in Human Fibrocytes.

IL-23 and IL-12, two structurally related heterodimeric cytokines sharing a common subunit, divergently promote Th cell development and expansion. Both cytokines have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves disease. In TAO, CD34+ fibrocytes, putatively derived from bone marrow, can be identified in the orbit. There they masquerade as CD34+ orbital fibroblasts (OF) (CD34+ OF) and cohabitate with CD34- OF in a mixed fibroblast population (GD-OF). Slit2, a neural axon repellent, is expressed and released by CD34- OF and dampens the inflammatory phenotype of fibrocytes and CD34+ OF. In this study we report that thyrotropin (TSH) and the pathogenic, GD-specific monoclonal autoantibody, M22, robustly induce IL-23 in human fibrocytes; however, IL-12 expression is essentially undetectable in these cells under basal conditions or following TSH-stimulation. In contrast, IL-12 is considerably more inducible in GD-OF, cells failing to express IL-23. This divergent expression and induction of cytokines appears to result from cell type-specific regulation of both gene transcription and mRNA stabilities. It appears that the JNK pathway activity divergently attenuates IL-23p19 expression while enhancing that of IL-12p35. The shift from IL-23p19 expression in fibrocytes to that of IL-12p35 in their derivative CD34+ OF results from the actions of Slit2. Thus, Slit2 might represent a molecular determinant of balance between IL-23 and IL-12 expression, potentially governing immune responses in TAO.

Functional analysis of human fibrocytes derived from monocytes reveals their profibrotic phenotype through paracrine effects.

Fibrocytes, which are bone marrow-derived collagen-producing cells, were reported to play a role in the pathogenesis of pulmonary fibrosis. However, their function in pulmonary fibrosis is unclear. We analyzed their function compared with that of monocytes and localization in fibrotic tissues in patients with idiopathic pulmonary fibrosis (IPF). We compared the gene expression profile of monocyte-derived fibrocytes with that of monocytes by microarray analysis. Proliferation and differentiation into myofibroblasts were examined by 3H-thymidine incorporation assay and Western blotting. We measured the level of growth factors in the culture supernatant of fibrocytes by ELISA. The localization of fibrocytes in lung tissues of patients with IPF was determined by immunofluorescence staining. Compared with monocytes, fibrocytes had higher expression of extracellular matrix- and growth factor-encoding genes, including PDGF-B, FGF-2 and VEGF-B. Although fibrocytes did not proliferate in response to PDGF, co-culture of fibrocytes stimulated the growth of lung fibroblasts through the production of PDGF-BB. In the lung of IPF patients, CD45+Collagen-I+FSP-1+ cells, which have a similar phenotype to fibrocytes, were detected and co-stained with anti-PDGF antibody. This study suggested that fibrocytes function in pulmonary fibrosis partly by producing PDGF in the lungs of IPF patients. J. Med. Invest. 67 : 102-112, February, 2020.

The role of fibrocytes in myelofibrosis and fibrocyte regulation as a novel treatment approach

Recently, monocyte-derived fibroblast-like cells, called fibrocytes, garnered attention as involved in the novel pathogenesis of various fibrotic diseases. They also play a role in the induction of myelofibrosis (MF). Neoplastic fibrocytes are overrepresented in the bone marrow of patients with primary MF, and the suppression of fibrocyte differentiation by serum amyloid P has been shown to remarkably improve MF. Further, thrombopoietin (TPO) or a TPO receptor agonist directly induces fibrocyte differentiation, and fibrocyte elimination reversed the MF phenotype in a murine model. Human fibrocytes highly express signaling lymphocytic activation molecule-F7 (SLAMF7) compared with macrophages. Myeloproliferative neoplasm (MPN) patients harboring JAK2V617F with MF had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. Furthermore, the JAK2V617F allele burden and the tendency to differentiate into fibrocytes of SLAMF7high monocytes was significantly higher than that of JAK2V617Flow monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used to treat multiple myeloma. Elo independently inhibited the differentiation of fibrocytes derived not only from healthy controls but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. Thus, Elo could be a therapeutic agent for MPN patients harboring JAK2V617F with MF.

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.

Increased SLAMF7high monocytes in myelofibrosis patients harboring JAK2V617F provide a therapeutic target of elotuzumab

AbstractMonocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.

Essential Oil Composition and Antiproliferative Potential of the Aerial Parts of Majorana syriaca (L. Rafi): An Aromatic Essential Oil Bearing Ethnomedicinal Herb from Jordan

This study aimed to analyze the chemical composition of essential oils of dried aerial parts of five different specimens of Majorana syriaca (L. Rafi) grown in Jordan obtained by hydrodistillation using GC-FID and GC/MS. Also, in vitro antiproliferative activity of plant extracts against MCF-7, Caco-2 and Panc-1 cancer cell lines in addition to normal fibrocytes cells was assessed using MTT assay. Thirty three components were identified in the oil hydrodistilled from the tested specimens of M. syriaca amounting for an average of 94.0 % of oil content. Oxygenated monoterpenes predominated the volatile fractions with carvacrol (0.6-88.2 %) and/or thymol (0.9-70.3 %) as the principal constituents, p-cymene (2.1-14.5 %) and γ-terpinene (2.6-15.4 %) as major constituents. M. syriaca essential oils exhibited excellent in vitro antiproliferative activity against all tested cells. While water extracts were ineffective; M. syriaca ethanol extract showed significant activity only against MCF-7 (IC50 value= 6.96 μg/mL). Moreover, all extracts were safe on normal human fibrocytes. Based on the obtained results, ethanol extract and EO of M. syriaca may be advocated as candidates for breast, colorectal and pancreatic cancers management.

PS1435 SLAMF7 HIGH CD16 NEGATIVE MONOCYTES INCREASE IN PERIPHERAL BLOOD OF PATIENTS WITH MYELOFIBROSIS IN CORRELATION WITH JAK2V617F MUTATION

Background:Monocyte‐derived fibrocytes are vital in the pathogenesis of primary myelofibrosis (MF) (J Exp Med 2016;213:1723). We established the direct induction of fibrocyte differentiation using TPO receptor activation in a mouse model (Leukemia 2017;31:2709). Moreover, we demonstrated that elotuzumab (Elo; anti‐SLAMF7 antibody) inhibited human fibrocyte differentiation in vitro and relieved MF in the mouse model, determining Elo as a potential therapeutic agent (manuscript in preparation). In the peripheral blood (PB) of patients with MF, SLAMF7high CD16− monocytes were significantly increased than that of healthy controls (HCs). Thus, we hypothesize that SLAMF7high CD16− monocytes reflected fibrocyte activation and were the target of Elo.Aims:Through this cross‐sectional study, we aimed to identify suitable candidates for a clinical trial of Elo by evaluating SLAMF7high CD16− monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with or without MF, and non‐MPN patients with MF.Methods:We enrolled 6 HCs, 12 non‐MPN patients with MF, and 44 patients with MPN (18 without and 26 with MF) and collected their blood samples. All patients with MPN underwent a bone marrow biopsy in 2016–2018 and were diagnosed per the 2016 WHO classification and diagnostic criteria. Non‐MPN patients underwent a bone marrow biopsy in 2015–2018, and MF was established over MF‐1 per the European Consensus Criteria. We evaluated the SLAMF7high CD16– monocyte percentage in PB monocytes of HCs and patients using flow cytometry. All PB samples were tested for JAK2V617F, CALR, or MPL genetic mutations, and the allele burden of the JAK2V617F mutation was measured in PB samples and cultured fibrocytes. Furthermore, we collected SLAMF7high/low CD16– monocytes separately of 2 patients harboring the JAK2V617F mutation and assessed the JAK2V617F allele burden of that fraction and the ratio of fibrocytes in culture.Results:The SLAMF7high CD16− monocyte percentage significantly increased in MPN and non‐MPN patients compared with HCs and MPN patients without MF (P < 0.001 and P < 0.01, respectively). JAK2V617F MPN patients with MF exhibited a significantly higher SLAMF7high CD16− monocyte percentage than those without MF (P < 0.001). We noted a similar trend in patients not harboring the JAK2V617F mutation (P = 0.05). The ROC analysis revealed that >25% of SLAMF7high CD16− monocytes in PB estimates MF in patients with MPN harboring the JAK2V617F mutation (sensitivity: 82.4%, specificity: 87.5%). Moreover, the JAK2V617F allele burden of fibrocytes markedly correlated with the SLAMF7high CD16− monocyte percentage in the PB (P = 0.0003). In CD16– monocytes of 2 patients, the JAK2V617F allele burden of the SLAMF7high fraction was high (87.96% and 94.75%) compared with the SLAMF7low fraction (49.09% and 45.64%, respectively). Furthermore, the culture assay of isolated SLAMF7high/low monocytes revealed that the frequency of fibrocytes derived from SLAMF7high monocytes markedly increased compared with SLAMF7low monocytes.Summary/Conclusion:SLAMF7high CD16– monocytes are neoplastic cells with homozygous JAK2V617F, and the levels closely correlate with MF and a neoplastic clone of fibrocytes harboring the JAK2V617F mutation; this fraction indicates fibrocyte activation and denotes a noninvasive marker of MF progression and a potential target for Elo treatment.

SLAMF7high CD16negative Monocytes Increase in Peripheral Blood of Patients with Myelofibrosis in Correlation with JAK2V617F Mutation

IntroductionMyelofibrosis (MF) occurrence can be attributed to various pathogenic mechanisms. A recent study showed that the neoplastic clone of fibrocytes (spindle shaped, fibroblast-like blood cells derived from monocyte lineage) was essential in primary MF pathogenesis; moreover, serum amyloid P (PRM-151), which suppresses fibrocyte differentiation, markedly improved survival and MF in a murine xenograft model (J Exp Med 2016; 213: 1723). Using a romiplostim-induced murine MF model, direct induction of fibrocyte differentiation using TPO receptor activation, leading to MF progression, was demonstrated (Leukemia 2017; 31: 2709).Using DNA microarray analysis, we previously showed that compared with macrophages, human fibrocytes highly express SLAMF7 (J Immunol 2015; 195: 4341). Elotuzumab (Elo) is an anti-SLAMF7 antibody recently developed for treating multiple myeloma. We showed that Elo inhibits human fibrocyte differentiation in vitro and relieves MF using the mouse model in vivo, identifying Elo as a potential therapeutic agent (manuscript in preparation). Additionally, monocytes with high SLAMF7 expression and negative CD16 expression were significantly elevated in the peripheral blood (PB) of MF patients compared with that of healthy controls (HCs). We hypothesized that this monocyte subset reflects fibrocyte activation and is the target of Elo.For the clinical study of Elo, we evaluated SLAMF7high CD16− monocyte percentage in PB of HCs, myeloproliferative neoplasm (MPN) patients with or without MF, and non-MPN patients with MF in a cross-sectional manner.MethodsSix HCs, 12 non-MPN patients with MF, and 44 MPN patients (18 without and 26 with MF) were enrolled; their blood samples were collected. All MPN patients underwent bone marrow biopsy in 2016-2018 and were diagnosed by the 2016 WHO classification and diagnostic criteria. Non-MPN patients underwent bone marrow biopsy in 2015-2018, and MF was confirmed over MF-1 according to the European Consensus Criteria.SLAMF7high CD16- monocyte percentage in PB monocytes of HCs and patients was calculated using flow cytometry. Further, all patients were tested for JAK2V617F, CALR, or MPL genetic mutations. The allele burden of JAK2V617F was measured. Moreover, fibrocytes were differentiated from PB mononuclear cells of patients with genetic mutation and were verified for genetic mutation in their fibrocytes.ResultsPatients were classified in three groups (MPN without MF, MPN with MF, and non-MPN with MF). The median percentage of SLAMF7high CD16− monocytes in PB of HCs, MPN patients without MF, MPN patients with MF, and non-MPN patients with MF were 1.66%, 2.48%, 27.4%, and 19.8%, respectively. Compared with HCs and MPN patients without MF, SLAMF7high CD16− monocyte percentage of MPN and non-MPN patients with MF significantly increased (p < 0.05 and p < 0.01, respectively) (Figure A).MPN patients with MF harboring JAK2V617F (n = 17) had a significantly higher SLAMF7high CD16− monocyte percentage than those without MF harboring JAK2V617F (n = 8) (median: 43.70% vs. 7.00%, p < 0.001). While a similar trend was observed in patients not harboring JAK2V617F (median: 1.15% vs. 3.26%, p = 0.05), the contrast between patients with and without MF patients was significant for those with JAK2V617F (Figure B). More than 25% of SLAMF7high CD16− monocytes in PB indicates MF in MPN patients harboring JAK2V617F (sensitivity: 82.4%, specificity: 87.5%).In addition, the JAK2V617F allele burden of fibrocytes was strongly correlated with the SLAMF7high CD16− monocyte percentage in PB (p = 0.0003) (Figure C). In all patients, fibrocytes and PB harbored the same genetic mutation.ConclusionThe percentage of SLAMF7high CD16− monocytes in PB elevated in MF patients regardless of MPN, whereas it was not elevated in MPN patients without MF. In MPN patients, those with MF harboring JAK2V617F presented a high SLAMF7high CD16-monocyte percentage in PB, positively correlating with the JAK2V617F allele burden of fibrocytes. In conclusion, SLAMF7high CD16- monocyte levels are closely related with MF and neoplastic clones of fibrocytes harboring JAK2V617F, indicating fibrocyte activation and representing a non-invasive marker of MF progression and a potential target for Elo treatment. [Display omitted] DisclosuresUsuki:Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Celgene Corporation: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sanofi K.K.: Research Funding; Shire Japan: Research Funding; SymBio Pharmaceuticals Limited.: Research Funding; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; Novartis: Speakers Bureau; Janssen Pharmaceutical K.K: Research Funding; Pfizer Japan: Research Funding, Speakers Bureau; GlaxoSmithKline K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku: Speakers Bureau; Mochida Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau.

Novel differences in gene expression and functional capabilities of myofibroblast populations in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial pneumonia, is characterized by excessive fibroproliferation. Key effector cells in IPF are myofibroblasts that are recruited from three potential sources: resident fibroblasts, fibrocytes, and epithelial cells. We hypothesized that IPF myofibroblasts from different sources display unique gene expression differences and distinct functional characteristics. Primary human pulmonary fibroblasts (normal and IPF), fibrocytes, and epithelial cells were activated using the profibrotic factors TGF-β and TNF-α. The resulting myofibroblasts were characterized using cell proliferation, soluble collagen, and contractility assays, ELISA, and human fibrosis PCR arrays. Genes of significance in human whole lung were validated by immunohistochemistry on human lung sections. Fibroblast-derived myofibroblasts exhibited the greatest increase in expression of profibrotic genes and genes involved in extracellular matrix remodeling and signal transduction. Functional studies demonstrated that myofibroblasts derived from fibrocytes expressed mostly soluble collagen and chemokine (C-C) motif ligand (CCL) 18 but were the least proliferative of the myofibroblast progeny. Activated IPF fibroblasts displayed the highest levels of contractility and CCL2 production. This study identified novel differences in gene expression and functional characteristics of different myofibroblast populations. Further investigation into the myofibroblast phenotype may lead to potential therapeutic targets in future IPF research.

Bone marrow-derived fibrocytes promote stem cell-like properties of lung cancer cells

Cancer stem cells (CSCs) represent a minor population that have clonal tumor initiation and self-renewal capacity and are responsible for tumor initiation, metastasis, and therapeutic resistance. CSCs reside in niches, which are composed of diverse types of stromal cells and extracellular matrix components. These stromal cells regulate CSC-like properties by providing secreted factors or by physical contact. Fibrocytes are differentiated from bone marrow-derived CD14+ monocytes and have features of both macrophages and fibroblasts. Accumulating evidence has suggested that stromal fibrocytes might promote cancer progression. However, the role of fibrocytes in the CSC niches has not been revealed. We herein report that human fibrocytes enhanced the CSC-like properties of lung cancer cells through secreted factors, including osteopontin, CC-chemokine ligand 18, and plasminogen activator inhibitor-1. The PIK3K/AKT pathway was critical for fibrocytes to mediate the CSC-like functions of lung cancer cells. In human lung cancer specimens, the number of tumor-infiltrated fibrocytes was correlated with high expression of CSC-associated protein in cancer cells. These results suggest that fibrocytes may be a novel cell population that regulates the CSC-like properties of lung cancer cells in the CSC niches.

Intersection of Chemokine and TSH Receptor Pathways in Human Fibrocytes: Emergence of CXCL-12/CXCR4 Cross Talk Potentially Relevant to Thyroid-Associated Ophthalmopathy.

Fibrocytes are monocyte progenitor cells that have been implicated in normal and pathological tissue remodeling. Among the prominent chemokine receptors expressed by these cells is CXC motif receptor 4 (CXCR4), which, with its cognate ligand CXCL motif ligand 12 (CXCL-12), directs fibrocytes to sites of fibrosis. Fibrocytes have been implicated in the pathogenesis of thyroid-associated ophthalmopathy, the ocular manifestation of Graves' disease (GD), by virtue of their unique accumulation as CD34+ orbital fibroblasts (OFs). Fibrocytes also express high levels of functional TSH receptor (TSHR). Here, we determined CXCL-12 and CXCR4 expression in fibrocytes and GD-OF and whether that pathway interacts with TSHR. CXCL-12 is highly expressed in GD-OF, whereas CXCR4 levels are dramatically higher in fibrocytes. Levels of these proteins are differentially regulated by TSH in a cell type-specific manner. Further, CXCL-12 enhances the induction by TSH of IL-6 in fibrocytes but attenuates this induction in GD-OF. In contrast, in pure CD34+ OF, the interplay between TSH and CXCL-12 reverts to that observed in fibrocytes. Our results indicate that CXCL-12 enhances TSH actions in fibrocytes but inhibits them in GD-OF, a dichotomy imposed by factors emanating from CD34- OF. They also suggest a potentially important modulatory role for CD34- OF in determining the factors that influence pathological TSHR signaling in the TAO orbit.

Peritoneal Dialysis Fluid and Some of Its Components Potentiate Fibrocyte Differentiation.

Long-term peritoneal dialysis (PD) often results in the development of peritoneal fibrosis. In many other fibrosing diseases, monocytes enter the fibrotic lesion and differentiate into fibroblast-like cells called fibrocytes. We find that peritoneal tissue from short-term PD patients contains few fibrocytes, while fibrocytes are readily observed in the peritoneal membrane of long-term PD patients. The PD fluid Dianeal (Baxter Healthcare Corporation, Deerfield, IL, USA) contains dextrose, a number of electrolytes including sodium chloride, and sodium lactate. We find that PD fluid potentiates human fibrocyte differentiation in vitro and implicates sodium lactate in this potentiation. The plasma protein serum amyloid P (SAP) inhibits fibrocyte differentiation. Peritoneal dialysis fluid and sodium chloride decrease the ability of human SAP to inhibit human fibrocyte differentiation in vitro Together, these results suggest that PD fluid contributes to the development of peritoneal fibrosis by potentiating fibrocyte differentiation.

TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation

In healing wounds and fibrotic lesions, fibroblasts and monocyte-derived fibroblast-like cells called fibrocytes help to form scar tissue. Although fibrocytes promote collagen production by fibroblasts, little is known about signaling from fibroblasts to fibrocytes. In this report, we show that fibroblasts stimulated with the fibrocyte-secreted inflammatory signal tumor necrosis factor-α secrete the small leucine-rich proteoglycan lumican, and that lumican, but not the related proteoglycan decorin, promotes human fibrocyte differentiation. Lumican competes with the serum fibrocyte differentiation inhibitor serum amyloid P, but dominates over the fibroblast-secreted fibrocyte inhibitor Slit2. Lumican acts directly on monocytes, and unlike other factors that affect fibrocyte differentiation, lumican has no detectable effect on macrophage differentiation or polarization. α2β1, αMβ2, and αXβ2 integrins are needed for lumican-induced fibrocyte differentiation. In lung tissue from pulmonary fibrosis patients with relatively normal lung function, lumican is present at low levels throughout the tissue, whereas patients with advanced disease have pronounced lumican expression in the fibrotic lesions. These data may explain why fibrocytes are increased in fibrotic tissues, suggest that the levels of lumican in tissues may have a significant effect on the decision of monocytes to differentiate into fibrocytes, and indicate that modulating lumican signaling may be useful as a therapeutic for fibrosis.

Pentraxin-3 Is a TSH-Inducible Protein in Human Fibrocytes and Orbital Fibroblasts.

CD34(+) fibrocytes are bone marrow-derived monocyte progenitor cells that traffic to sites of tissue injury and repair. They putatively infiltrate the orbit in thyroid-associated ophthalmopathy where they appear to transition into CD34(+) orbital fibroblasts (OFs) that interact with residential CD34(-) fibroblasts. A unique phenotypic attribute of fibrocytes and CD34(+) OFs is their expression of the functional thyrotropin receptor (TSHR) and other "thyroid-specific" proteins. When activated through TSHR, fibrocytes express a number of cytokines and other inflammatory genes. Here we sought to determine whether pentraxin-3 (PTX-3), an acute-phase protein involved in inflammation and autoimmunity, might be induced by TSH in fibrocytes and OFs. These cells were collected from patients with Graves disease and healthy individuals. PTX-3 mRNA levels were determined by real-time PCR, protein was determined by ELISA and Western blot, and PTX-3 gene promoter activity was assessed with reporter assays. PTX-3 expression was induced by TSH in both cell types, regardless of the health status of the donor and was a consequence of increased steady-state PTX-3 mRNA levels. M22, a TSHR-activating monoclonal antibody, also induced PTX-3. The induction could be attenuated by dexamethasone and by IGF-I receptor-blocking antibodies, teprotumumab and 1H7. TSH effects were mediated through phosphatidylinositol 3-kinase/AKT, mammalian target of rapamycin/p70(s6k), Janus tyrosine kinase 2 pathways, and enhanced PTX-3 mRNA stability. These findings indicate that PTX-3 is a TSH target gene, the expression of which can be induced in fibrocytes and OFs. They suggest that PTX-3 might represent a previously unidentified nexus between the thyroid axis and the mechanisms involved in tissue remodeling.