Tianju Liu

Bone marrow–derived progenitor cells in pulmonary fibrosis

The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP(+) cells to appear in active fibrotic lesions, while only a few GFP(+) cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP(+) cells in chimera mice and revealed a significant increase in GFP(+) cells that also express type I collagen. GFP(+) lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not alpha-smooth muscle actin. Treatment of isolated GFP(+) fibroblasts with TGF-beta failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell-derived factor-1 alpha and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells.

Epigenetic regulation of the alternatively activated macrophage phenotype.

Alternatively activated (M2) macrophages play critical roles in diverse chronic diseases, including parasite infections, cancer, and allergic responses. However, little is known about the acquisition and maintenance of their phenotype. We report that M2-macrophage marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 (H3K4) and histone H3 lysine-27 (H3K27) methylation; and the latter methylation marks are removed by the H3K27 demethylase Jumonji domain containing 3 (Jmjd3). We found that continuous interleukin-4 (IL-4) treatment leads to decreased H3K27 methylation, at the promoter of M2 marker genes, and a concomitant increase in Jmjd3 expression. Furthermore, we demonstrate that IL-4-dependent Jmjd3 expression is mediated by STAT6, a major transcription factor of IL-4-mediated signaling. After IL-4 stimulation, activated STAT6 is increased and binds to consensus sites at the Jmjd3 promoter. Increased Jmjd3 contributes to the decrease of H3K27 dimethylation and trimethylation (H3K27me2/3) marks as well as the transcriptional activation of specific M2 marker genes. The decrease in H3K27me2/3 and increase in Jmjd3 recruitment were confirmed by in vivo studies using a Schistosoma mansoni egg-challenged mouse model, a well-studied system known to support an M2 phenotype. Collectively, these data indicate that chromatin remodeling is mechanistically important in the acquisition of the M2-macrophage phenotype.

Dual Roles of IL-4 in Lung Injury and Fibrosis

Increased lung IL-4 expression in pulmonary fibrosis suggests a potential pathogenetic role for this cytokine. To dissect this role, bleomycin-induced pulmonary inflammation and fibrosis were analyzed and compared in wild type (IL-4+/+) vs IL-4-deficient (IL-4−/−) mice. Lethal pulmonary injury after bleomycin treatment was higher in IL-4−/− vs IL-4+/+ mice. By administration of anti-CD3 Abs, we demonstrated that this early response was linked to the marked T lymphocyte lung infiltration and to the overproduction of the proinflammatory mediators such as TNF-α, IFN-γ, and NO in IL-4−/− mice. In contrast to this early anti-inflammatory/immunosuppressive role, during later stages of fibrosis, IL-4 played a profibrotic role since IL-4−/− mice developed significantly less pulmonary fibrosis relative to IL-4+/+ mice. However, IL-4 failed to directly stimulate proliferation, α-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice. Upon appropriate stimulation with other known fibrogenic cytokines, fibroblasts from IL-4−/− mice were relatively deficient in the studied parameters in comparison to fibroblasts isolated from IL-4+/+ mice. Taken together, these data suggest dual effects of IL-4 in this model of lung fibrosis: 1) limiting early recruitment of T lymphocytes, and 2) stimulation of fibrosis chronically.

FIZZ1 Stimulation of Myofibroblast Differentiation

Bleomycin-induced pulmonary fibrosis is characterized by inflammation, emergence of myofibroblasts, and deposition of extracellular matrix. In an attempt to identify genes that may be involved in fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the lung gene expression profiles in this model in the rat. Cluster analysis showed 628 genes were more than or equal to twofold up- or down-regulated, many of which were known to be involved in fibrosis. However, the most dramatic increase was observed with FIZZ1 (found in inflammatory zone; also known as RELM-alpha or resistin-like molecule-alpha), which was induced 17-fold to approximately 25-fold at the peak of expression. In situ hybridization analysis revealed FIZZ1 expression to localize primarily to alveolar and airway epithelium, which was confirmed in vitro by analysis of isolated type II alveolar epithelial cells. However FIZZ1 expression was not detected in isolated lung fibroblasts. Co-culture of FIZZ1-expressing type II cells with fibroblasts stimulated alpha-smooth muscle actin and type I collagen expression independent of transforming growth factor-beta. Transfection of a FIZZ1-expressing plasmid into fibroblasts or treatment with glutathione S-transferase-FIZZ1 fusion protein stimulated alpha-smooth muscle actin and collagen I production. These results suggest a novel role for FIZZ1 in myofibroblast differentiation in pulmonary fibrosis.

Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule α, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.

Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis

Leukocyte infiltration is characteristic of lung injury and fibrosis, and its role during tissue repair and fibrosis is incompletely understood. We found that overexpression of IL-5 in transgenic mice (IL-5TG) or by adenoviral gene transfer increased bleomycin (blm)-induced lung injury, fibrosis, and eosinophilia. Surprisingly, blm-treated IL-5-deficient (IL-5−/−) mice also developed pronounced pulmonary fibrosis but characterized by marked T lymphocyte infiltration and absence of eosinophilia. In both murine strains however, induction of lung TGF-β expression was evident. Purified lung eosinophils from blm-treated IL-5TG mice stimulated α-smooth muscle actin and collagen expression in mouse lung fibroblasts, without affecting proliferation. Furthermore instillation of purified eosinophils into murine lungs resulted in extension of blm-induced lung fibrosis, thus confirming a role for eosinophils. However, lung T lymphocytes from blm-treated IL-5−/− mice were able to stimulate fibroblast proliferation but not α-smooth muscle actin or collagen expression. Blocking T cell influx by anti-CD3 Abs abrogated lung fibrosis, thus also implicating T lymphocytes as a key participant in fibrosis. Pulmonary fibrosis in IL-5TG mice was preferentially associated with type 2 cytokines (IL-4 and IL-13), whereas fibrotic lesions in IL-5−/− animals were accompanied by proinflammatory cytokine (TNF-α, IL-1β, and IFN-γ) expression. We suggest that eosinophils and T cells contribute distinctly to the development of blm-induced lung fibrosis potentially via their production of different cytokine components, which ultimately induce TGF-β expression that is intimately involved with the fibrosis.

Notch1 signaling in FIZZ1 induction of myofibroblast differentiation.

Notch1 is an evolutionarily conserved receptor that regulates cell fate, including such events as differentiation, proliferation, and apoptosis. Myofibroblast differentiation is a key feature of lung fibrosis. Found in inflammatory zone 1 (FIZZ1) has direct fibrogenic properties because of its ability to induce myofibroblast differentiation. However, the downstream signaling pathway that mediates FIZZ1 induction of myofibroblast differentiation remains unknown. The objective of this study was to investigate the involvement of Notch signaling in FIZZ1 induction of lung myofibroblast differentiation and thus explore the potential role of Notch1 in pulmonary fibrosis. The results showed that FIZZ1 increased the expression levels of activated intracellular domain of Notch1 (NIC), its ligand Jagged1, and its target gene Hes1, which were associated with elevated alpha-smooth muscle actin expression levels. Fibroblast alpha-smooth muscle actin expression is induced by the overexpression of NIC but is suppressed by the inhibition of NIC. Moreover, lung fibroblasts that were isolated from mice lacking the GDP-4-keto-6-deoxymannose3,5-epimerase-4-reductase enzyme (FX knockout) exhibited significantly reduced responsiveness to FIZZ1, which was reversed by fucose supplementation. In the absence of exogenous fucose, these FX-deficient cells exhibited defective fucosylation, which is required for Notch signaling. These knockout mice also showed impaired lung fibrosis. These findings suggest that Notch1 signaling in response to FIZZ1 may play a significant role in myofibroblast differentiation during lung fibrosis.

Telomerase activity is required for bleomycin-induced pulmonary fibrosis in mice

In addition to its well-known expression in the germline and in cells of certain cancers, telomerase activity is induced in lung fibrosis, although its role in this process is unknown. To identify the pathogenetic importance of telomerase in lung fibrosis, we examined the effects of telomerase reverse transcriptase (TERT) deficiency in a murine model of pulmonary injury. TERT-deficient mice showed significantly reduced lung fibrosis following bleomycin (BLM) insult. This was accompanied by a significant reduction in expression of lung alpha-SMA, a marker of myofibroblast differentiation. Furthermore, lung fibroblasts isolated from BLM-treated TERT-deficient mice showed significantly decreased proliferation and increased apoptosis rates compared with cells isolated from control mice. Transplantation of WT BM into TERT-deficient mice restored BLM-induced lung telomerase activity and fibrosis to WT levels. Conversely, transplantation of BM from TERT-deficient mice into WT recipients resulted in reduced telomerase activity and fibrosis. These findings suggest that induction of telomerase in injured lungs may be caused by BM-derived cells, which appear to play an important role in pulmonary fibrosis. Moreover, TERT induction is associated with increased survival of lung fibroblasts, which favors the development of fibrosis instead of injury resolution.

Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis.

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11(-/-)) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-beta1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis.

Telomerase and telomere length in pulmonary fibrosis.

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.