BACKGROUND: Obesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. Weight loss is a recommended intervention to resolve obesity, but the impact of weight loss on the mammary gland microenvironment and in tumors has not been well identified.
METHODS: To examine the effects of weight loss following obesity, mice were fed a high-fat diet for 16 weeks to induce obesity, then switched to a low-fat diet for 6 weeks. We examined changes in immune cells, including fibrocytes, which are myeloid lineage cells that have attributes of both macrophages and myofibroblasts, and collagen deposition within the mammary glands of non-tumor-bearing mice and within the tumors of mice that were transplanted with estrogen receptor alpha positive TC2 tumor cells.
RESULTS: In formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. Within tumors of obese mice, increased myeloid-derived suppressor cells and diminished CD8+ T cells were identified, while the microenvironment of tumors of formerly obese mice were more similar to tumors from lean mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, which are the cells of origin for fibrocytes, and transplanted into mammary glands of lean and obese mice, collagen deposition within the tumors of both lean and obese was significantly greater than when tumor cells were mixed with CD11b+CD34- monocytes or total CD45+ immune cells.
CONCLUSIONS: Overall, these studies demonstrate that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression. Additionally, fibrocytes may contribute to early collagen deposition in mammary tumors of obese mice leading to the growth of desmoplastic tumors.

BACKGROUND: Previous studies have shown that IL-25 levels are increased in patients with asthma with fixed airflow limitation (FAL). However, the mechanism by which IL-25 contributes to airway remodeling and FAL remains unclear. Here, we hypothesized that IL-25 facilitates pro-fibrotic phenotypic changes in bronchial epithelial cells (BECs) and circulating fibrocytes (CFs), orchestrates pathological crosstalk from BECs to CFs, and thereby contributes to airway remodeling and FAL.
METHODS: Fibrocytes from asthmatic patients with FAL and chronic asthma murine models were detected using flow cytometry, multiplex staining and multispectral imaging analysis. The effect of IL-25 on BECs and CFs and on the crosstalk between BECs and CFs was determined using cell culture and co-culture systems.
RESULTS: We found that asthmatic patients with FAL had higher numbers of IL-25 receptor (i.e., IL-17RB)+-CFs, which were negatively correlated with forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC). The number of airway IL-17RB+-fibrocytes was significantly increased in ovalbumin (OVA)- and IL-25-induced asthmatic mice versus the control subjects. BECs stimulated with IL-25 exhibited an epithelial-mesenchymal transition (EMT)-like phenotypic changes. CFs stimulated with IL-25 produced high levels of extracellular matrix (ECM) proteins and connective tissue growth factors (CTGF). These profibrotic effects of IL-25 were partially blocked by the PI3K-AKT inhibitor LY294002. In the cell co-culture system, OVA-challenged BECs facilitated the migration and expression of ECM proteins and CTGF in CFs, which were markedly blocked using an anti-IL-17RB antibody.
CONCLUSIONS: These results suggest that IL-25 may serve as a potential therapeutic target for asthmatic patients with FAL.

Obesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. To examine the impact of weight loss on the mammary microenvironment, mice were fed high-fat diet to induce obesity, then switched to a low-fat diet. In formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, collagen deposition within the tumors was significantly greater compared to when tumor cells were mixed with CD11b+CD34- monocytes, suggesting that fibrocytes contribute to early collagen deposition in mammary tumors of obese mice. Overall, these studies show that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression.

Obesity rates continue to rise, and obese individuals are at higher risk for multiple types of cancer, including breast cancer. Obese mammary fat is a site of chronic, macrophage-driven inflammation, which enhances fibrosis within adipose tissue. Elevated fibrosis within the mammary gland may contribute to risk for obesity-associated breast cancer. To understand how inflammation due to obesity enhanced fibrosis within mammary tissue, we utilized a high-fat diet model of obesity and elimination of CCR2 signaling in mice to identify changes in immune cell populations and their impact on fibrosis. We observed that obesity increased a population of CD11b+ cells with the ability to form myofibroblast-like colonies in vitro. This population of CD11b+ cells is consistent with fibrocytes, which have been identified in wound healing and chronic inflammatory diseases but have not been examined in obesity. In CCR2-null mice, which have limited ability to recruit myeloid lineage cells into obese adipose tissue, we observed reduced mammary fibrosis and diminished fibrocyte colony formation in vitro. Transplantation of myeloid progenitor cells, which are the cells of origin for fibrocytes, into the mammary glands of obese CCR2-null mice resulted in significantly increased myofibroblast formation. Gene expression analyses of the myeloid progenitor cell population from obese mice demonstrated enrichment for genes associated with collagen biosynthesis and extracellular matrix remodeling. Together these results show that obesity enhances recruitment of fibrocytes to promote obesity-induced fibrosis in the mammary gland.

UNASSIGNED: The circulating progenitor cells of fibroblasts (fibrocytes) have been shown to infiltrate the airway smooth muscle compartment of asthma patients; however, the pathological significance of this discovery has yet to be elucidated. This study established a co-culture model of airway smooth muscle cells (ASMCs) and fibrocytes from asthmatic or normal subjects to evaluate innate cytokine production, corticosteroid responses, and signaling in ASMCs.
UNASSIGNED: CD34+ fibrocytes were purified from peripheral blood of asthmatic (Global Initiative for Asthma treatment step 4-5) and normal subjects and cultured for 5∼7 days. In a transwell plate, ASMCs were co-cultured with fibrocytes at a ratio of 2:1, ASMCs were cultured alone (control condition), and fibrocytes were cultured alone for 48 h. Measurements were obtained of interleukin-8 (IL-8), IL-6, IL-17, thymic stromal lymphopoietin, and IL-33 levels in the supernatant and IL-33 levels in the cell lysate of the co-culture. Screening for intracellular signaling in the ASMCs after stimulation was performed using condition medium from the patients\' co-culture (PtCM) or IL-8. mRNA and western blot analysis were used to analyze AKT/mTOR signaling in ASMCs stimulated via treatment with PtCM or IL-8.
UNASSIGNED: Compared with ASMCs cultured alone, IL-8 levels in the supernatant and IL-33 levels in the ASMCs lysate were significantly higher in samples co-cultured from asthmatics, but not in those co-cultured from normal subjects. Corticosteroid-induced suppression of IL-8 production was less pronounced in ASMCs co-cultured with fibrocytes from asthma patients than in ASMCs co-cultured from normal subjects. ASMCs stimulated using PtCM and IL-8 presented elevating activated AKT substrate PRAS40. Treatment with IL-8 and PtCM increased mRNA expression of mTOR and P70S6 kinases in ASMCs. Treatment with IL-8 and PtCM also significantly increased phosphorylation of AKT and mTOR subtract S6 ribosomal protein in ASMCs.
UNASSIGNED: The interaction between ASMCs and fibrocytes from asthmatic patients was shown to increase IL-8 and IL-33 production and promote AKT/mTOR signaling in ASMCs. IL-8 production in the co-culture from asthmatic patients was less affected by corticosteroid than was that in the co-culture from normal subjects. Our results elucidate the novel role of fibrocytes and ASMCs in the pathogenesis of asthma.

Fibrosis is a prominent feature of chronic allograft rejection, caused by an excessive production of matrix proteins, including collagen-1. Several cell types produce collagen-1, including mesenchymal fibroblasts and cells of hematopoietic origin. Here, we sought to determine whether tissue-resident donor-derived cells or allograft-infiltrating recipient-derived cells are responsible for allograft fibrosis, and whether hematopoietic cells contribute to collagen production. A fully MHC-mismatched mouse heterotopic heart transplantation model was used, with transient depletion of CD4+ T cells to prevent acute rejection. Collagen-1 was selectively knocked out in recipients or donors. In addition, collagen-1 was specifically deleted in hematopoietic cells. Tissue-resident macrophages were depleted using anti-CSF1R antibody. Allograft fibrosis and inflammation were quantified 20 days post-transplantation. Selective collagen-1 knock-out in recipients or donors showed that tissue-resident cells from donor hearts, but not infiltrating recipient-derived cells, are responsible for production of collagen-1 in allografts. Cell-type-specific knock-out experiments showed that hematopoietic tissue-resident cells in donor hearts substantially contributed to graft fibrosis. Tissue resident macrophages, however, were not responsible for collagen-production, as their deletion worsened allograft fibrosis. Donor-derived cells including those of hematopoietic origin determine allograft fibrosis, making them attractive targets for organ preconditioning to improve long-term transplantation outcomes.

Every organ develops fibrosis that compromises functions in response to infections, injuries, or diseases. The cornea is a relatively simple, avascular organ that offers an exceptional model to better understand the pathophysiology of the fibrosis response. Injury and defective regeneration of the epithelial basement membrane (EBM) or the endothelial Descemet\'s basement membrane (DBM) triggers the development of myofibroblasts from resident corneal fibroblasts and bone marrow-derived blood borne fibrocytes due to the increased entry of TGF beta-1/-2 into the stroma from the epithelium and tears or residual corneal endothelium and aqueous humor. The myofibroblasts, and disordered extracellular matrix these cells produce, persist until the source of injury is removed, the EBM and/or DBM are regenerated, or replaced surgically, resulting in decreased stromal TGF beta requisite for myofibroblast survival. A similar BM injury-related pathophysiology can underly the development of fibrosis in other organs such as skin and lung. The normal liver does not contain traditional BMs but develops sinusoidal endothelial BMs in many fibrotic diseases and models. However, normal hepatic stellate cells produce collagen type IV and perlecan that can modulate TGF beta localization and cognate receptor binding in the space of Dissé. BM-related fibrosis is deserving of more investigation in all organs.

Fibrocytes are hematopoietic-derived cells that directly contribute to tissue fibrosis by producing collagen following injury, during disease, and with aging. The lack of a fibrocyte-specific marker has led to the use of multiple strategies for identifying these cells in vivo. This review will detail how past studies were performed, report their findings, and discuss their strengths and limitations. The motivation is to identify opportunities for further investigation and promote the adoption of best practices during future study design.

Abdominal aortic aneurysm (AAA) is potentially life-threatening in aging population due to the risk of aortic rupture and a lack of optimal treatment. The roles of different vascular and immune cells in AAA formation and pathogenesis remain to be future characterized. Single-cell RNA sequencing was performed on an angiotensin (Ang) II-induced mouse model of AAA. Macrophages, B cells, T cells, fibroblasts, smooth muscle cells and endothelial cells were identified through bioinformatic analyses. The discovery of multiple subtypes of macrophages, such as the re-polarization of Trem2 + Acp5 + osteoclast-like and M2-like macrophages toward the M1 type macrophages, indicates the heterogenous nature of macrophages during AAA development. More interestingly, we defined CD45+COL1+ fibrocytes, which was further validated by flow cytometry and immunostaining in mouse and human AAA tissues. We then reconstituted these fibrocytes into mice with Ang II-induced AAA and found the recruitment of these fibrocytes in mouse AAA. More importantly, the fibrocyte treatment exhibited a protective effect against AAA development, perhaps through modulating extracellular matrix production and thus enhancing aortic stability. Our study reveals the heterogeneity of macrophages and the involvement of a novel cell type, fibrocyte, in AAA. Fibrocyte may represent a potential cell therapy target for AAA.

OBJECTIVE: Graves\' disease (GD) is an autoimmune disorder affecting primarily the thyroid gland. The most common extrathyroidal manifestation of GD is known as Graves\' orbitopathy (GO). Bone marrow-derived fibrocytes represent a subset of monocytes in peripheral blood mononuclear cells (PBMCs), infiltrate the orbital tissues, and contribute to the pathogenesis of GO. Hence objectives of the study included whether the concentration of fibrocytes in peripheral blood was higher in GO, whether TSHR m RNA expression and TSHR surface expression in peripheral blood were higher in GO in comparison to Graves\' Disease (GD) and Control subjects.
METHODS: The percentage of circulating fibrocytes (FC) along with TSHR on its cell surface (CD 34+, CD 45+, CXCR4+, Collagen 1+, TSHR+) were assessed by flow cytometry of 50 patients with GD and GO and 15 healthy donors (Control). TSHR mRNA expression was measured by q RT PCR.
RESULTS: The concentration of circulating fibrocytes was significantly higher in GO compared to GD and control [GO 17% vs GD 3% vs control 0.7% (p < 0.05)]. Moreover, these fibrocytes express a significantly higher level of TSHR in GO. This was corroborated by the measure of TSH mRNA; in GD it was 2.3-fold higher and in GO it was 3.9 fold higher than in control, in GO this transcript level was 1.7fold higher than GD (p < 0.05). TSHR+ fibrocytes were significantly positively correlated with CAS (p = 0.004) and negatively correlated with age (p = 0.01) and duration of disease (p = 0.01) in GO.
CONCLUSIONS: This study sheds further light on the pathogenesis of GO.