B7H3 expression and significance in idiopathic pulmonary fibrosis.

The clinical significance of B7H3 (CD276) and its cleavage product soluble B7H3 (sB7H3) in idiopathic pulmonary fibrosis (IPF) is unknown. Mounting evidence suggests the potential utility of peripheral blood myeloid cell enumeration to predict disease outcome and indicate active lung disease. Here we hypothesized that sB7H3 is involved in regulation of circulating myeloid cells in pulmonary fibrosis. In support of this possibility, both plasma sB7H3 and B7H3+ cells were elevated in IPF patient blood samples, which correlated negatively with lung function. To analyze its function, the effects of sB7H3 on naïve or bleomycin-treated mice were examined. The results revealed that sB7H3 injection induced an influx of myeloid-derived suppressor cells (MDSCs) and Ccl2 expression in lung tissue of naïve mice, accompanied by enhanced overall inflammation. Additionally, sB7H3 caused accumulation of MDSCs in bone marrow with increased expression of inflammatory cytokines. Notably, in vitro assays revealed chemotaxis of MDSCs to sB7H3, which was dependent on TLT-2 (TREML2), a putative receptor for sB7H3. Thus, increased circulating sB7H3 and/or B7H3+ cells in IPF patient blood samples correlated with lung function decline and potential immunosuppressive status. The correlation of sB7H3 with deterioration of lung function might be due to its ability to enhance inflammation and recruitment of MDSCs into the lung and their expansion in the bone marrow, and thus potentially contribute to IPF exacerbation. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Noncanonical Wnt Signaling Promotes Myofibroblast Differentiation in Pulmonary Fibrosis.

The Wnt/ß-catenin pathway initiates a signaling cascade that is critical in cell differentiation and the normal development of multiple organ systems. The reactivation of this pathway has been documented in experimental and human idiopathic pulmonary fibrosis, wherein Wnt/ß-catenin activation has been implicated in epithelial-cell repair. Furthermore, the canonical ligand Wnt3a is known to induce myofibroblast differentiation; however, the role of noncanonical Wnt ligands remains unclear. This study showed significantly higher levels of Wnt11 expression in cells from both patients with idiopathic pulmonary fibrosis and bleomycin-treated mice, as well as in TGFß-treated mouse lung fibroblasts. Moreover, Wnt11 induced myofibroblast differentiation as manifested by increased α-SMA (ACTA2) expression, which was similar to that induced by canonical Wnt3a/ß-catenin signaling. Further investigation revealed that Wnt11 induction of α-SMA was associated with the activation of JNK (c-Jun N-terminal kinase)/c-Jun signaling and was inhibited by a JNK inhibitor. The potential importance of this signaling pathway was supported by in vivo evidence showing significantly increased levels of Wnt11 and activated JNK in the lungs of mice with bleomycin-induced pulmonary fibrosis. Interestingly, fibroblasts did not express canonical Wnt3a, but treatment of these cells with exogenous Wnt3a induced endogenous Wnt11 and Wnt5a, resulting in repression of the Wnt3a/ß-catenin target gene Axin2. These findings suggested that the noncanonical Wnt induction of myofibroblast differentiation mediated by the JNK/c-Jun pathway might play a significant role in pulmonary fibrosis, in addition to or in synergy with canonical Wnt3a/ß-catenin signaling. Moreover, Wnt3a activation of noncanonical Wnt signaling might trigger a switch from canonical to noncanonical Wnt signaling to induce myofibroblast differentiation.

Telomerase reverse transcriptase ameliorates lung fibrosis by protecting alveolar epithelial cells against senescence.

Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.

Inhibition of the stem cell factor 248 isoform attenuates the development of pulmonary remodeling disease.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.

Role of B7H3/IL-33 Signaling in Pulmonary Fibrosis-induced Profibrogenic Alterations in Bone Marrow.

Rationale: The impact of lung insult on the bone marrow (BM) and subsequent disease is unknown.Objectives: To study alterations in the BM in response to lung injury/fibrosis and examine their impact on subsequent lung insult.Methods: BM cells from control or bleomycin-treated donor mice were transplanted into naive mice, which were subsequently evaluated for bleomycin-induced pulmonary fibrosis. In addition, the effect of prior bleomycin treatment on subsequent fibrosis was examined in wild-type and B7H3-knockout mice. Samples from patients with idiopathic pulmonary fibrosis were analyzed for potential clinical relevance of the findings.Measurements and Main Results: Recipient mice transplanted with BM from bleomycin-pretreated donors showed significant exacerbation of subsequent fibrosis with increased B7H3+ cell numbers and a T-helper cell type 2-skewed phenotype. Pretreatment with a minimally fibrogenic/nonfibrogenic dose of bleomycin also caused exacerbation, but not in B7H3-deficient mice. Exacerbation was not observed if the mice received naive BM cell transplant after the initial bleomycin pretreatment. Soluble B7H3 stimulated BM Ly6Chi monocytic cell expansion in vitro and caused similar expansion in the lung in vivo. Notably, soluble B7H3 was elevated in plasma of patients with idiopathic pulmonary fibrosis and in BAL fluid in those with acute exacerbation. Finally, ST2 deficiency diminished the bleomycin-induced B7H3 and IL-13 upregulation, suggesting a role for type 2 innate lymphoid cells.Conclusions: Pulmonary fibrosis caused significant alterations in BM with expansion and activation of monocytic cells, which enhanced fibrosis when transplanted to naive recipients with potential mediation by a novel role for B7H3 in the pathophysiology of pulmonary fibrosis in both mice and humans.

An ST2-dependent role of bone marrow-derived group 2 innate lymphoid cells in pulmonary fibrosis.

Recent evidence supports that bone marrow (BM)-derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)-expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM-derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin-induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2-sufficient control mice exhibited activation of lung IL-33/ST2 signaling, ILC2 recruitment, IL-13 induction, and fibrosis, these responses were significantly diminished in ST2-deficient-BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild-type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2+ BM cells. ST2-deficient BM cells were unresponsive to IL-33-induced ILC2 maturation. Finally, lineage-negative wild-type, but not ST2-deficient BM cells from bleomycin-treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFß expression in the BM cells. Taken together, these findings suggested that the BM-derived ILC2s were recruited to fibrotic lung through the IL-33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bone Marrow CD11c+ Cell-Derived Amphiregulin Promotes Pulmonary Fibrosis.

Amphiregulin (AREG), an epidermal growth factor receptor ligand, is implicated in tissue repair and fibrosis, but its cellular source and role in regeneration versus fibrosis remain unclear. In this study, we hypothesize that AREG induced in bone marrow-derived CD11c(+) cells is essential for pulmonary fibrosis. Thus, the objectives were to evaluate the importance and role of AREG in pulmonary fibrosis, identify the cellular source of AREG induction, and analyze its regulation of fibroblast function and activation. The results showed that lung AREG expression was significantly induced in bleomycin-induced pulmonary fibrosis. AREG deficiency in knockout mice significantly diminished pulmonary fibrosis. Analysis of AREG expression in major lung cell types revealed induction in fibrotic lungs predominantly occurred in CD11c(+) cells. Moreover, depletion of bone marrow-derived CD11c(+) cells suppressed both induction of lung AREG expression and pulmonary fibrosis. Conversely, adoptive transfer of bone marrow-derived CD11c(+) cells from bleomycin-treated donor mice exacerbated pulmonary fibrosis, but not if the donor cells were made AREG deficient prior to transfer. CD11c(+) cell-conditioned media or coculture stimulated fibroblast proliferation, activation, and myofibroblast differentiation in an AREG-dependent manner. Furthermore, recombinant AREG induced telomerase reverse transcriptase, which appeared to be essential for the proliferative effect. Finally, AREG significantly enhanced fibroblast motility, which was associated with increased expression of α6 integrin. These findings suggested that induced AREG specifically in recruited bone marrow-derived CD11c(+) cells promoted bleomycin-induced pulmonary fibrosis by activation of fibroblast telomerase reverse transcriptase-dependent proliferation, motility, and indirectly, myofibroblast differentiation.

Mesenchymal deficiency of Notch1 attenuates bleomycin-induced pulmonary fibrosis.

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I-expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.

FIZZ1-induced myofibroblast transdifferentiation from adipocytes and its potential role in dermal fibrosis and lipoatrophy.

Subcutaneous lipoatrophy characteristically accompanies dermal fibrosis with de novo emergence of myofibroblasts such as in systemic sclerosis or scleroderma. Recently dermal adipocytes were shown to have the capacity to differentiate to myofibroblasts in an animal model. Transforming growth factor ß can induce this phenomenon in vitro; however its in vivo significance is unclear. Because found in inflammatory zone 1 (FIZZ1) is an inducer of myofibroblast differentiation but an inhibitor of adipocyte differentiation, we investigated its potential role in adipocyte transdifferentiation to myofibroblast in dermal fibrosis. FIZZ1 caused significant and rapid suppression of the expression of fatty acid binding protein 4 and peroxisome proliferator-activated receptor-γ in adipocytes, consistent with dedifferentiation with loss of lipid and Oil Red O staining. The suppression was accompanied subsequently with stimulation of α-smooth muscle actin and type I collagen expression, indicative of myofibroblast differentiation. In vivo FIZZ1 expression was significantly elevated in the murine bleomycin-induced dermal fibrosis model, which was associated with significant reduction in adipocyte marker gene expression and subcutaneous lipoatrophy. Finally, FIZZ1 knockout mice exhibited significantly reduced bleomycin-induced dermal fibrosis with greater preservation of the subcutaneous fat than wild-type mice. These findings suggested that the FIZZ1 induction of adipocyte transdifferentiation to myofibroblast might be a key pathogenic mechanism for the accumulation of myofibroblasts in dermal fibrosis.

Notch in fibrosis and as a target of anti-fibrotic therapy.

The Notch pathway represents a highly conserved signaling network with essential roles in regulation of key cellular processes and functions, many of which are critical for development. Accumulating evidence indicates that it is also essential for fibrosis and thus the pathogenesis of chronic fibroproliferative diseases in diverse organs and tissues. Different effects of Notch activation are observed depending on cellular and tissue context as well as in both physiologic and pathologic states. Close interactions of Notch signaling pathway with other signaling pathways have been identified. In this review, current knowledge on the role of the Notch signaling with special focus on fibrosis and its potential as a therapeutic target is summarized.

Reemergence of hedgehog mediates epithelial-mesenchymal crosstalk in pulmonary fibrosis.

Hedgehog signaling plays important roles in cell development and differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce myofibroblast differentiation was analyzed in isolated human lung fibroblasts, and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary fibrosis. The results showed that SHH could induce myofibroblast differentiation in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis revealed that a Gli1 binding consensus in the α-SMA gene promoter was important for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was significantly induced in bleomycin-injured lung along with Gli1. The induction of SHH was only noted in epithelial cells, and its expression was undetectable in lung fibroblasts or macrophages. transforming growth factor (TGF)-ß induced SHH significantly in cultured alveolar epithelial cells, whereas SHH induced TGF-ß in lung fibroblasts. Pulmonary fibrosis and α-smooth muscle actin (α-SMA) expression were significantly reduced in mice that were Smo deficient only in type I collagen-expressing cells. Thus, the reemergence of SHH in epithelial cells could result in induction of myofibroblast differentiation in a Smo-dependent manner and subsequent Gli1 activation of the α-SMA promoter.

Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.

Regulation of myofibroblast differentiation by poly(ADP-ribose) polymerase 1.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with an expression plasmid activated expression of the α-SMA gene (Acta2), a marker of myofibroblast differentiation in lung fibroblasts. Suppression of PARP-1 activity or gene expression with PARP-1 inhibitors or siRNA, respectively, had the opposite effect on these cells. PARP-1-deficient cells also had reduced α-SMA gene expression. DNA pyrosequencing identified hypermethylated regions of the α-SMA gene in PARP-1-deficient cells, relative to wild-type cells. Interestingly, and of potential relevance to human idiopathic pulmonary fibrosis, PARP activity in lung fibroblasts isolated from idiopathic pulmonary fibrosis patients was significantly higher than that in cells isolated from control subjects. Furthermore, PARP-1-deficient mice exhibited reduced pulmonary fibrosis in response to bleomycin-induced lung injury, relative to wild-type controls. These results suggest that PARylation is important for myofibroblast differentiation and the pathogenesis of pulmonary fibrosis.

Essential role of stem cell factor-c-Kit signalling pathway in bleomycin-induced pulmonary fibrosis.

Stem cell factor (SCF) and its receptor c-Kit have been implicated in tissue remodelling and fibrosis. Alveolar fibroblasts from patients with diffuse interstitial fibrosis secrete more SCF. However, its precise role remains unclear. In this study the potential role of the SCF-c-Kit axis in pulmonary fibrosis was examined. Fibrosis was induced by intratracheal instillation of bleomycin (BLM), which caused increased SCF levels in plasma, bronchoalveolar lavage fluid (BALF) and lung tissue, as well as increased expression by lung fibroblasts. These changes were accompanied by increased numbers of bone marrow-derived c-Kit(+) cells in the lung, with corresponding depletion in bone marrow. Both recombinant SCF and lung extracts from BLM-treated animals induced bone-marrow cell migration, which was blocked by c-Kit inhibitor. The migrated cells promoted myofibroblast differentiation when co-cultured with fibroblasts, suggesting a paracrine pathogenic role. Interestingly, lung fibroblast cultures contained a subpopulation of cells that expressed functionally active c-Kit, which were significantly greater and more responsive to SCF induction when isolated from fibrotic lungs, including those from patients with idiopathic pulmonary fibrosis (IPF). This c-Kit(+) subpopulation was αSMA-negative and expressed lower levels of collagen I but significantly higher levels of TGFß than c-Kit-negative cells. SCF deficiency achieved by intratracheal treatment with neutralizing anti-SCF antibody or by use of Kitl(Sl)/Kitl(Sl-d) mutant mice in vivo resulted in significant reduction in pulmonary fibrosis. Taken together, the SCF-c-Kit pathway was activated in BLM-injured lung and might play a direct role in pulmonary fibrosis by the recruitment of bone marrow progenitor cells capable of promoting lung myofibroblast differentiation.

Lung bone marrow-derived hematopoietic progenitor cells enhance pulmonary fibrosis.

RATIONALE: Bone marrow (BM)-derived cells have been implicated in pulmonary fibrosis. However, their precise role in pathogenesis is incompletely understood. OBJECTIVES: To elucidate roles of BM-derived cells in bleomycin-induced pulmonary fibrosis, and clarify their potential relationship to lung hematopoietic progenitor cells (LHPCs). METHODS: GFP BM-chimera mice treated with or without bleomycin were used to assess the BM-derived cells. MEASUREMENTS AND MAIN RESULTS: GFP(+) cells in the chimera lung were found to be comprised of two distinct phenotypes: GFP(hi) and GFP(low) cells. The GFP(hi), but not GFP(low), population was significantly increased after bleomycin treatment. Flow-cytometric analysis and quantitative real-time polymerase chain reaction revealed that GFP(hi) cells exhibited phenotypic characteristics of CD11c(+) dendritic cells and macrophages. GFP(hi) cell conditioned media were chemotactic for fibroblasts obtained from fibrotic but not normal lung in vitro. Moreover, adoptive transfer of GFP(hi) cells exacerbated fibrosis in recipient mice, similar to that seen on adoptive transfer of BM-derived CD11c(+) cells from donor bleomycin-treated mice. Next, we evaluated the potential of LHPCs as the source of GFP(hi) cells. Isolation of LHPCs by flow sorting revealed enrichment in cKit(+)/Sca1(-)/Lin(-) cells, most of which were GFP(+) indicating their BM origin. The number of LHPCs increased rapidly after bleomycin treatment. Furthermore, stem cell factor induced LHPC proliferation, whereas granulocyte-macrophage-colony stimulating factor induced differentiation to GFP(hi) cells. CONCLUSIONS: BM-derived LHPCs with a novel phenotype could differentiate into GFP(hi) cells, which enhanced pulmonary fibrosis. Targeting this mobilized LHPCs might represent a novel therapeutic approach in chronic fibrotic lung diseases.

Detection of myeloid-derived suppressor cells by flow cytometry.

Recently discovered heterogeneous myeloid-derived suppressor cells (MDSCs) are some of the most discussed immunosuppressive cells in contemporary immunology, especially in the tumor microenvironment, and are defined primarily by their T cell immunosuppressive function. The importance of these cells extend to other chronic pathological conditions as well, including chronic infection, inflammation, and tissue remodeling. In many of these conditions, their accumulation/expansion correlates with disease progression, poor prognosis, and reduced survival, which highlights the potential of how these cells may be used in a clinical setting as both prognostic factor and therapeutic target. In healthy individuals, these cells are usually not present in the circulation. Therefore, monitoring this cell population is of potential clinical significance, and utility in basic research. However, these cells have a complex phenotype without one single marker of sufficient specificity for their identification. Flow cytometry is a powerful tool allowing multi-parameter analysis of heterogeneous cell populations, which makes it ideally suitable for the complex phenotypic analysis essential for identification and enumeration of circulating MDSCs. This approach has the potential to provide a novel clinically useful tool for assessment of prognosis and treatment outcomes. The protocol in this chapter describes a flow cytometric analysis to identify and quantify MDSCs from human or mouse whole blood leukocytes and peripheral blood mononuclear cells, as well as a single cell suspension from solid tissue, by using multicolor fluorescence-conjugated antibodies against their surface markers.

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.

Myofibroblasts.

PURPOSE OF REVIEW: Interest in the myofibroblast as a key player in propagation of chronic progressive fibrosis continues to elicit many publications, with focus on its cellular origins and the mechanisms underpinning their differentiation and/or transition. The objective of the review is to highlight this recent progress. RECENT FINDINGS: The epithelial origin of the myofibroblast in fibrosis has been challenged by recent studies, with the pericyte suggested as a possible precursor instead. Additional signaling pathways, including Notch, Wnt, and hedgehog, are implicated in myofibroblast differentiation. The importance of NADPH oxidase 4 was highlighted recently to suggest a potential link between cellular/oxidative stress and the genesis of the myofibroblast. Recent observations on the importance of lysophosphatidic acid in fibrosis suggest that this may be due, in part, to its ability to regulate myofibroblast differentiation. Finally, there is increasing evidence for the role of epigenetic mechanisms in regulating myofibroblast differentiation, including DNA methylation and miRNA regulation of gene expression. SUMMARY: These recent discoveries open up a whole new array of potential targets for novel antifibrotic therapies. This is of special importance given the current bleak outlook for chronic progressive fibrotic diseases, such as scleroderma, due to lack of effective therapies.

Mesenchymal-specific deletion of C/EBPß suppresses pulmonary fibrosis.

The CCAAT/enhancer-binding protein ß (C/EBPß) regulates a variety of factors and cellular responses associated with pulmonary fibrosis. To distinguish its role in the mesenchyme from that in other compartments, the effects of mesenchymal-specific deletion of C/EBPß on pulmonary fibrosis was examined. Crossing of mice with the floxed C/EBPß gene with α2(I) collagen enhancer-CreER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of C/EBPß in collagen I-expressing ("mesenchymal") cells only on treatment with tamoxifen (C/EBPß CKO). When treated with an endotracheal bleomycin injection, C/EBPß CKO mice showed significant attenuation of pulmonary fibrosis relative to control C/EBPß-intact mice. C/EBPß CKO mice also had reduced myofibroblasts in the lung. However, no significant differences in inflammatory/immune cell influx were noted in the mutant mice relative to the control mice. DNA microarray and real-time PCR analyses identified a series of myofibroblast differentiation regulators as novel target genes of C/EBPß. Interestingly, C/EBPß deficiency caused a marked induction of matrix metalloproteinase 12 expression, suggesting its potential role as a repressor, which could account for the noted reduction in fibrosis in the C/EBPß-deficient mice. Thus, these findings indicate an essential role for C/EBPß in the mesenchymal compartment in pulmonary fibrosis that is independent of its effects on inflammation or immune cell infiltration.