Xueyan Peng

Circulating Monocytes from Systemic Sclerosis Patients with Interstitial Lung Disease Show an Enhanced Profibrotic Phenotype

Profibrotic cells derived from circulating CD14+ monocytes include fibrocytes and alternatively activated macrophages. These cells are associated with interstitial lung disease (ILD) and are implicated in the pathogenesis of systemic sclerosis (SSc); however, the simultaneous presence of profibrotic cells and their associated mediators in the circulation of these patients has not been defined. We hypothesized that monocytes from patients with SSc-related ILD (SSc-ILD) would show profibrotic characteristics when compared with normal controls. We recruited patients with SSc-ILD (n=12) and normal controls (n=27) and quantified circulating collagen-producing cells by flow cytometry for CD45 and pro-collagen I. The in vitro activation potential of CD14+ monocytes in response to lipopolysaccharide was assessed using flow cytometry for CD163, and by ELISA for CCL18 and IL-10 secretion. Profibrotic mediators in plasma were quantified using Luminex-based assays. The concentration of circulating collagen-producing cells was increased in the SSc-ILD patients when compared with controls. These cells were composed of both CD34+ fibrocytes and a population of CD34+CD14+ cells. Cultured CD14+ monocytes from SSc-ILD patients revealed a profibrotic phenotype characterized by expression of CD163 and by enhanced secretion of CCL18 and IL-10 in response to proinflammatory activation. Plasma levels of IL-10, MCP-1, IL-1RA, and TNF levels were significantly elevated in the plasma of the SSc-ILD cohort. Subgroup analysis of the normal controls revealed that unlike the subjects ≤35 years, subjects ≥60 years old showed higher levels of circulating CD34+CD14+ cells, collagen-producing CD14+ monocytes, CD163+ monocytes, IL-4, IL-10, IL-13, MCP-1, and CCL18. These data indicate that the blood of patients with SSc-ILD and of healthy aged controls is enriched for fibrocytes, profibrotic monocytes, and fibrosis-associated mediators. Investigations defining the factors responsible for this peripheral blood profile may provide new insight into SSc-ILD as well as the pathophysiology of aging.

Simvastatin Inhibits IL-17 Secretion by Targeting Multiple IL-17-Regulatory Cytokines and by Inhibiting the Expression of IL-17 Transcription Factor RORC in CD4+ Lymphocytes

Statins, extensively used as cholesterol-lowering agents, have recently been identified as immunomodulatory agents. This study investigated the statins’ mechanisms that target the autoimmune response in humans, and evaluated their therapeutic potential in multiple sclerosis. Our results demonstrated statin-mediated increases in suppressor of cytokine secretion (SOCS) 3 and suppressor of cytokine secretion 7, which negatively regulate the STAT/JAK signal transduction pathway and IL-6 and IL-23 gene expression in monocytes. Simvastatin also induced IFN-γ, IL-4, and IL-27 production in monocytes, which together inhibited IL-17 transcription and secretion in CD4+ T cells. IL-17-producing CD4+ cells, referred to as Th17 cells, have recently been found to play a central role in the development of autoimmune diseases. Furthermore, simvastatin directly inhibited the expression of retinoic acid-related orphan nuclear hormone receptor C, a transcription factor that controls IL-17 production in CD4+ T cells. This effect was reversed by mevalonic acid, a downstream metabolite of 3-hydroxy-3-methylglutaryl CoA reductase, confirming that simvastatin’s specific effect is through the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase. These results provide evidence for the novel immunomodulatory mechanisms of statins, which selectively target the regulation of cytokine transcription involved in the development of the human autoimmune response. Based on the described immunomodulatory mechanisms, good safety profile and oral bioavailability, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.

Role of semaphorin 7a signaling in transforming growth factor β1-induced lung fibrosis and scleroderma-related interstitial lung disease.

OBJECTIVE Semaphorin 7a regulates transforming growth factor β1 (TGFβ1)-induced fibrosis. This study was undertaken to test the hypothesis that semaphorin 7a exerts its profibrotic effects in part by promoting the tissue accumulation of CD45+ fibrocytes. METHODS A murine model of pulmonary fibrosis in which an inducible, bioactive form of the human TGFβ1 gene is overexpressed in the lung was used. Fibrosis and fibrocytes were evaluated in TGFβ1-transgenic mice in which the semaphorin 7a locus had been disrupted. The effect of replacement or deletion of semaphorin 7a on bone marrow-derived cells was ascertained using bone marrow transplantation. The role of the semaphorin 7a receptor β1 integrin was assessed using neutralizing antibodies. The applicability of these findings to TGFβ1-driven fibrosis in humans was examined in patients with scleroderma-related interstitial lung disease (ILD). RESULTS The appearance of fibrocytes in the lungs of TGFβ1-transgenic mice required semaphorin 7a. Replacement of semaphorin 7a on bone marrow-derived cells restored lung fibrosis and fibrocytes. Immunoneutralization of β1 integrin reduced pulmonary fibrocytes and fibrosis. Peripheral blood mononuclear cells (PBMCs) from patients with scleroderma-related ILD showed increased levels of messenger RNA for semaphorin 7a and its receptors, with semaphorin 7a located on collagen-producing fibrocytes and CD19+ lymphocytes. Peripheral blood fibrocyte outgrowth was enhanced in these patients. Stimulation of normal human PBMCs with recombinant semaphorin 7a enhanced fibrocyte differentiation; these effects were attenuated by β1 integrin neutralization. CONCLUSION Our findings indicate that interventions that reduce semaphorin 7a expression or prevent the semaphorin 7a-β1 integrin interaction may ameliorate TGFβ1-driven or fibrocyte-associated autoimmune fibroses.

Semaphorin 7a+ Regulatory T Cells Are Associated with Progressive Idiopathic Pulmonary Fibrosis and Are Implicated in Transforming Growth Factor-β1–induced Pulmonary Fibrosis

RATIONALE Lymphocytes are increasingly associated with idiopathic pulmonary fibrosis (IPF). Semaphorin 7a (Sema 7a) participates in lymphocyte activation. OBJECTIVES To define the relationship between Sema 7a and lymphocytes in IPF. METHODS We characterized the significance of Sema 7a+ lymphocytes in humans with IPF and in a mouse model of lung fibrosis caused by lung-targeted, transgenic overexpression of TGF-β1. We determined the site of Sema 7a expression in human and murine lungs and circulation and used adoptive transfer approaches to define the relevance of lymphocytes coexpressing Sema7a and the markers CD19, CD4, or CD4+CD25+FoxP3+ in TGF-β1-induced murine lung fibrosis. MEASUREMENTS AND MAIN RESULTS Subjects with IPF show expression of Sema 7a on lung CD4+ cells and circulating CD4+ or CD19+ cells. Sema 7a expression is increased on CD4+ cells and CD4+CD25+FoxP3+ regulatory T cells, but not CD19+ cells, in subjects with progressive IPF. Sema 7a is expressed on lymphocytes expressing CD4 but not CD19 in the lungs and spleen of TGF-β1-transgenic mice. Sema 7a expressing bone marrow-derived cells induce lung fibrosis and alter the production of T-cell mediators, including IFN-γ, IL-4, IL-17A, and IL-10. These effects require CD4 but not CD19. In comparison to Sema 7a-CD4+CD25+FoxP3+ cells, Sema7a+CD4+CD25+FoxP3+ cells exhibit reduced expression of regulatory genes such as IL-10, and adoptive transfer of these cells induces fibrosis and remodeling in the TGF-β1-exposed murine lung. CONCLUSIONS Sema 7a+CD4+CD25+FoxP3+ regulatory T cells are associated with disease progression in subjects with IPF and induce fibrosis in the TGF-β1-exposed murine lung.

Chitinase 3–Like 1 Suppresses Injury and Promotes Fibroproliferative Responses in Mammalian Lung Fibrosis

Chitinase 3–like 1 protects against lung injury but has a profibrotic role during the repair phase. Two-Face Chitinase Idiopathic pulmonary fibrosis is a devastating—currently incurable—disease where scars develop deep in the lung, making it hard to breathe. Now, Zhou et al. report a breath of fresh air for IPF. They found that the protein chitinase 3–like 1 (CHI3L1) is elevated in IPF patients, and that high levels of CHI3L1 are associated with disease progression. However, things were not as simple as they seemed. CHI3L1 actually played a protective, anti-apoptotic role against lung injury, but contributed to fibrosis during the repair phase, in part through inducing myofibroblast transformation. This insight into the two sides of CHI3L1 provides mechanistic understanding of the pathogenesis of IPF, which is necessary to the development of successful therapeutics. Epithelial injury, alternative macrophage accumulation, and fibroproliferation coexist in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Chitinase 3–like 1 (CHI3L1) is a prototypic chitinase-like protein that has been retained over species and evolutionary time. However, the regulation of CHI3L1 in IPF and its ability to regulate injury and/or fibroproliferative repair have not been fully defined. We demonstrated that CHI3L1 levels were elevated in patients with IPF. High levels of CHI3L1 are associated with progression—as defined by lung transplantation or death—and with scavenger receptor–expressing circulating monocytes in an ambulatory IPF population. In preterminal acute exacerbations of IPF, CHI3L1 levels were reduced and associated with increased levels of apoptosis. We also demonstrated that in bleomycin-treated mice, CHI3L1 expression was acutely and transiently decreased during the injury phase and returned toward and eventually exceeded baseline levels during the fibrotic phase. In this model, CHI3L1 played a protective role in injury by ameliorating inflammation and cell death, and a profibrotic role in the repair phase by augmenting alternative macrophage activation, fibroblast proliferation, and matrix deposition. Using three-dimensional culture system of a human fibroblast cell line, we found that CHI3L1 is sufficient to induce low grade myofibroblast transformation. In combination, these studies demonstrate that CHI3L1 is stimulated in IPF, where it represents an attempt to diminish injury and induce repair. They also demonstrate that high levels of CHI3L1 are associated with disease progression in ambulatory patients and that a failure of the CHI3L1 antiapoptotic response might contribute to preterminal disease exacerbations.

Chitinase 1 is a biomarker for and therapeutic target in scleroderma-associated interstitial lung disease that augments TGF-β1 signaling.

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1−/− mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-β1 to augment fibroblast TGF-β receptors 1 and 2 expression and TGF-β–induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-β1 effects by increasing receptor expression and canonical and noncanonical TGF-β1 signaling.

Immunomodulatory effects of 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors, potential therapy for relapsing remitting multiple sclerosis

This study characterized immunomodulatory targets of statins in humans and their potential for treatment of relapsing remitting multiple sclerosis (RR MS). Statins inhibited the proliferative response of mononuclear cells. Simvastatin, the statin with the strongest antiproliferative effect, inhibited IFN-gamma-induced expression of MHC class II DR on monocytes and decreased their antigen presenting capacity. As for T lymphocytes, it inhibited their activation and expression of the Th1 lineage differentiation markers. Simvastatin inhibited IFN-gamma, TNF-alpha, and IL-2 secretion, as well as the expression of T-bet, a transcription factor that regulates Th1 cell differentiation.

Local apoptosis promotes collagen production by monocyte-derived cells in transforming growth factor β1-induced lung fibrosis

BackgroundCollagen-containing leukocytes (CD45+Col-I+) accumulate in diseased and fibrotic tissues. However, the precise identity of these cells and whether injury is required for their recruitment remain unknown. Using a murine model of pulmonary fibrosis in which an inducible, bioactive form of the human transforming growth factor (TGF)-β1 gene is targeted to the lung, we characterized the cell surface phenotype of collagen-containing CD45+ cells in the lung and tested the hypothesis that apoptotic cell death responses are essential to the accumulation of CD45+Col-I+ cells.ResultsOur studies demonstrate that CD45+Col-I+ cells appearing in the TGF-β1-exposed murine lung express markers of the monocyte lineage. Inhibition of apoptosis via pharmacological caspase blockade led to a significant reduction in CD45+Col-I+ cells, which appear to accumulate independently of alternatively activated macrophages. There are also increased levels of apoptosis and greater numbers of CD45+Col-I+ in the lung tissue of patients with two distinct forms of fibrotic lung disease, idiopathic pulmonary fibrosis and connective tissue disease-related interstitial lung disease, when compared to lung from healthy normal controls. These findings are accompanied by an increase in collagen production in cultured monocytes obtained from subjects with fibrotic lung disease. Treatment of these cultured cells with the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD/fmk) reduces both apoptosis and collagen production in all subjects.ConclusionsInterventions that prevent collagen production by monocytes via modulation of caspase activation and of apoptosis may be ameliorative in monocyte-associated, TGF-β1-driven processes such as pulmonary fibrosis.

Role of Chitinase 3–like-1 and Semaphorin 7a in Pulmonary Melanoma Metastasis

The prototypic chitinase-like protein Chi3l1 is induced in cancers and portends a poor prognosis, but whether it contributes to cancer progression is unknown. To address this gap in knowledge, we investigated the production of Chi3l1 in melanoma lung metastases. We found that Chi3l1 was induced during pulmonary melanoma metastasis and that this induction was regulated by the semaphorin Sema7a, interacting in stimulatory or inhibitory ways with its β1 integrin or Plexin C1 receptors, respectively. In mouse strains with genetic deletions of Chi3l1 or Sema7a, there was a significant reduction in pulmonary metastasis. Notably, antiserum raised against Chi3l1 or Sema7a phenocopied the reduction produced by genetic deletions. Melanoma lung metastasis was also decreased in the absence of IL13Rα2, a recently identified receptor for Chi3l1, consistent with a key role for Chi3l1 in melanoma spread. We confirmed roles for Sema7a and Chi3l1 in pulmonary metastasis of EMT6 breast cancer cells. Taken together, our studies establish a novel pathway through which Sem7a and its receptors regulate Chi3l1, revealing a host axis involving IL13Rα2 that plays a critical role in generating a pulmonary microenvironment that is critical to license metastasis.

Fibroblast engraftment in the decellularized mouse lung occurs via a β1-integrin-dependent, FAK-dependent pathway that is mediated by ERK and opposed by AKT

Creation of bioartificial organs has been enhanced by the development of strategies involving decellularized mammalian lung. Because fibroblasts critically support lung function through a number of mechanisms, study of these cells in the context of the decellularized lung has the potential to improve the structure and function of tissue-engineered lungs. We characterized the engraftment and survival of a mouse fibroblast cell line in decellularized rat lung slices and found a time-dependent increase in cell numbers assessed by hematoxylin and eosin staining, cell proliferation assessed by Ki67 staining, and minimal cell death assessed by TUNEL staining. We developed a repopulation index to allow quantification of cell survival that accounts for variation in cell density throughout the seeded scaffold. We then applied this method to the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a β1-integrin-neutralizing antibody significantly reduced the repopulation index after 24 h of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation scores while treatment with AKT inhibitor increased initial scores. Rho-associated kinase inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occur in a β1-integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT.