Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45 ؉ , CD13 ؉ , collagen 1 ؉ , CD34 ؊ ). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2 ؉/؉ mice than from CCR2 ؊/؊ mice. Transplant of CCR2 ؉/؉ bone marrow into CCR2 ؊/؊ recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2 ؉/؉ lung fibrocytes also migrated to injured airspaces of CCR2 ؊/؊ recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury. Pulmonary fibrosis is characterized by alveolar epithelial cell injury, hyperplasia, inflammatory cell accumulation, fibroblast proliferation, and deposition of extracellular matrix.

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2−/− mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5−/− mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2−/− mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2−/− mice have increased levels of GM-CSF and reduced levels of TNF-α compared with FITC-treated CCR2+/+ mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

We have previously shown that mice that are genetically deficient in the CCR2 gene (CCR2؊/؊ mice) are protected from fluorescein isothiocyanate (FITC)-induced lung fibrosis. Protection from fibrosis correlated with impaired recruitment of fibrocytes (bone marrowderived cells, which share both leukocyte and mesenchymal markers). There are three ligands for CCR2 in the mouse: CCL2, CCL7, and CCL12. CCL2 and CCL12 are both elevated in the lung after FITC injury, but with different kinetics. CCL2 is maximal at Day 1 and absent by Day 7 after FITC. In contrast, CCL12 peaks at Day 3, but remains elevated through Day 21 after FITC. We now demonstrate that while CCR2؊/؊ mice are protected from FITC-induced fibrosis, CCL2؊/؊ mice are not. CCL2؊/؊ mice are able to recruit fibrocytes to FITC-injured airspaces, unlike CCR2؊/؊ mice. Adoptive transfer of CCR2-expressing fibrocytes augments FITC-induced fibrosis in both wild-type and CCR2؊/؊ mice, suggesting that these cells play a pathogenic role in the disease process. Both CCL2 and CCL12 are chemotactic for fibrocytes. However, neutralization of CCL12 in wild-type mice significantly protects from FITC-induced fibrosis, whereas neutralization of CCL2 was less effective. Thus, CCL12 is likely the CCR2 ligand responsible for driving fibroproliferation in the mouse. As murine CCL12 is homologous to human CCL2, we suggest that the pathobiology of murine CCL12 in fibroproliferation may correlate to human CCL2 biology.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

Rationale: Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen. Objectives: To investigate the role of EDA-containing fibronectin in lung fibrogenesis. Methods: Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and a-smooth muscle actin (a-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes. Measurements and Main Results: Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and a-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wildtype controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer a-SMA-expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-b and decreased lung fibroblast responsiveness to active TGF-b in vitro. Conclusions: The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-b activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.Keywords: fibrosis; fibronectin; TGF-b; myofibroblast Fibroproliferative disorders, characterized by the increased production and deposition of extracellular matrix (ECM) proteins in tissues, are not well understood despite major efforts to elucidate pathogenic mechanisms. Recent attention has focused on ECM components and mesenchymal cell phenotypes as being critical to the development of fibrosis (1, 2). The ECM is a highly dynamic complex that varies in composition according to its tissue localization and physiologic circumstances. Collagens are the predominant ECM proteins identified in fibrotic lesions and are the hallmark of fibroproliferative diseases, but fibronectins (FNs) are also present in abnormally large quantities and localize to areas of active fibrogenesis (3, 4).FNs are multifunctional glycoproteins found in the ECM of tissues and plasma. Two main forms of FN exist: plasma FN, a dimeric and soluble form produced by hepatocytes that lacks the EDA and EDB sequences, and cellular FN (cFN), a multimeric form synthesized by mesenchymal, epithelial, and inflammatory cells, which is deposited in ECM fibrils and that contains variable proportions of the extra type III domains A and B (EDA and EDB) (5...