Fibrocyte CXCR4 regulation as a therapeutic target in pulmonary fibrosis

Abstract

Fibrotic interstitial lung diseases are characterized by progressive decline in lung function and premature death from respiratory failure. Fibrocytes are circulating bone marrow-derived progenitor cells that traffic to the lungs and contribute to fibrosis and may represent novel therapeutic targets in these diseases. We have previously found the recruitment of fibrocytes to the lung to be dependent on the chemokine ligand CXCL12. Given that the expression of the CXCL12 receptor, CXCR4, can be modulated pharmacologically in other cell types, we tested the hypotheses that the regulation of CXCR4 expression on fibrocytes mediates their influx to the lung in the context of pulmonary fibrosis and that pharmacologic inhibition of this process results in attenuated disease severity. CXCR4 was the predominant chemokine receptor on human fibrocytes, and its expression on fibrocytes was enhanced by hypoxia and by growth factors including platelet-derived growth factor. Both hypoxia-induced and growth factor-induced CXCR4 expressions were attenuated by specific inhibition of PI3-kinase and mTOR. Finally, in the mouse model of bleomycin-induced pulmonary fibrosis, treatment with the mTOR inhibitor rapamycin resulted in reduced numbers of CXCR4-expressing fibrocytes in the peripheral blood and lung as well as reduced lung collagen deposition. Taken together, these experiments support the notion that pharmacologic inhibition of the CXCR4/CXCL12 biological axis is achievable in human fibrocytes and reduces the magnitude of pulmonary fibrosis in an animal model. This approach may hold promise in human fibrotic lung diseases.

Introduction

Fibrotic interstitial lung diseases are illnesses characterized by tissue remodeling, fibroproliferation, and deposition of extra-cellular matrix in the lung parenchyma. Some of these illnesses occur in response to known insults – such as inorganic dusts, inhaled antigens, or autoantigens – whereas others are of unknown cause. The prototypical fibrotic lung disease is idiopathic pulmonary fibrosis (IPF), a progressive disorder that culminates in premature death from respiratory failure and in which no treatment intervention has been effective to date. There is thus an urgent clinical need for new therapeutic approaches these diseases.

A fundamental question in the pathogenesis of fibrotic interstitial lung diseases is the cellular precursors of the lung fibroblasts and myofibroblasts. In this context, we have previously examined the contribution of a population of bone marrow-derived circulating progenitor cells, the fibrocytes (Phillips et al., 2004). Specifically, we found fibrocytes to accumulate in the lungs of mice with bleomycin-induced pulmonary fibrosis in associated with deposition of collagen and that human fibrocytes given to SCID mice challenged with bleomycin specifically trafficked to the lungs (Phillips et al., 2004). Consistent with this, we also found an expanded pool of fibrocytes in the peripheral blood of patients with fibrotic interstitial lung diseases (Mehrad et al., 2007) findings that have been subsequently reproduced by another group (Andersson-Sjoland et al., 2008). We have recently found that the expanded pool of circulating fibrocytes in patients with IPF is an independent predictor of mortality (Moeller et al., 2009), lending further support to the relevance of these cells in the pathogenesis of pulmonary fibrosis.

Identification of the chemokine ligand–receptor pair that mediates the mobilization of human fibrocytes from the bone marrow to the lung in the context of pulmonary fibrosis has the potential to open new avenues for therapy for these illnesses, but the issue remains unsettled in the literature. This is, at least in part, due to differences between human and mouse fibrocyte chemokine receptor repertoire: human fibrocytes express CXCR4, CCR3, CCR5 and CCR7 (Phillips et al., 2004, Quan et al., 2004) whereas mouse fibrocytes express CXCR4, CCR2 and CCR7 (Phillips et al., 2004, Abe et al., 2001, Moore et al., 2005, Quan et al., 2004). CXCR4 is a critical chemokine receptor in haematopoietic and non-haematopoietic stem cell homing in both mice and humans, and the differential expression of CXCL12 in tissue creates a gradient essential for traffic of CXCR4-expressing cells (Murdoch, 2000). Consistent with their progenitor cell phenotype, human fibrocytes express CXCR4 and migrate in response to CXCL12 in vitro (Phillips et al., 2004). In the context of the mouse model, there was a marked induction in lung CXCL12 after bleomycin challenge and the neutralization of this ligand resulted in reduced accumulation of fibrocytes in the lungs and attenuated lung fibrosis (Phillips et al., 2004). In human interstitial lung disease, we found elevated plasma and lung levels of CXCL12 protein associated with ∼90% CXCR4 expression in circulating fibrocytes (Mehrad et al., 2007, Phillips et al., 2004). Other groups have shown mouse fibrocytes to traffic to tissue via the CCR2/CCL12 axis in the FITC-induced model of pulmonary fibrosis (Moore et al., 2005, Moore et al., 2006), via the CCL3–CCR5 axis in bleomycin-induced pulmonary fibrosis (Ishida et al., 2007), and via CCR7 in models of renal fibrosis and wound healing (Abe et al., 2001, Sakai et al., 2006).

Prior work has demonstrated the expression of CXCR4 on fibrocytes in the context of mouse and human pulmonary fibrosis and implicated CXCL12-mediated traffic of fibrocytes to the lungs in the pathogenesis of pulmonary fibrosis (Phillips et al., 2004, Mehrad et al., 2007, Andersson-Sjoland et al., 2008). The purpose of the current work was to provide proof-of-principle that this mechanism can be manipulated pharmacologically as a therapeutic strategy in fibrotic lung disease. We therefore tested the hypotheses that the regulation of CXCR4 expression on fibrocytes mediates their influx to the lung in the context of pulmonary fibrosis and that pharmacologic inhibition of this process results in attenuated disease severity.