Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b+ macrophage subpopulations and aggravates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.

Serum proteomic profiling of rheumatoid arthritis-interstitial lung disease with a comparison to idiopathic pulmonary fibrosis.

Although interstitial lung disease (ILD) causes significant morbidity and mortality in rheumatoid arthritis (RA), it is difficult to predict the development or progression of ILD, emphasising the need for improved discovery through minimally invasive diagnostic tests. Aptamer-based proteomic profiling was used to assess 1321 proteins from 159 patients with rheumatoid arthritis with interstitial lung disease (RA-ILD), RA without ILD, idiopathic pulmonary fibrosis and healthy controls. Differential expression and gene set enrichment analyses revealed molecular signatures that are strongly associated with the presence and severity of RA-ILD and provided insight into unexplored pathways of disease. These warrant further study as non-invasive diagnostic tools and future therapeutic targets.

Type I interferon regulates proteolysis by macrophages to prevent immunopathology following viral infection.

The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28-2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.

Characterization of the COPD alveolar niche using single-cell RNA sequencing.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

Mesenchymal stromal cell-derived syndecan-2 regulates the immune response during sepsis to foster bacterial clearance and resolution of inflammation.

Sepsis is a life-threatening process related to a dysregulated host response to an underlying infection, which results in organ dysfunction and poor outcomes. Therapeutic strategies using mesenchymal stromal cells (MSCs) are under investigation for sepsis, with efforts to improve cellular utility. Syndecan (SDC) proteins are transmembrane proteoglycans involved with cellular signaling events including tissue repair and modulating inflammation. Bone marrow-derived human MSCs express syndecan-2 (SDC2) at a level higher than other SDC family members; thus, we explored SDC2 in MSC function. Administration of human MSCs silenced for SDC2 in experimental sepsis resulted in decreased bacterial clearance, and increased tissue injury and mortality compared with wild-type MSCs. These findings were associated with a loss of resolution of inflammation in the peritoneal cavity, and higher levels of proinflammatory mediators in organs. MSCs silenced for SDC2 had a decreased ability to promote phagocytosis of apoptotic neutrophils by macrophages in the peritoneum, and also a diminished capability to convert macrophages from a proinflammatory to a proresolution phenotype via cellular or paracrine actions. Extracellular vesicles are a paracrine effector of MSCs that may contribute to resolution of inflammation, and their production was dramatically reduced in SDC2-silenced human MSCs. Collectively, these data demonstrate the importance of SDC2 for cellular and paracrine function of human MSCs during sepsis.

FK506-Binding Protein 13 Expression Is Upregulated in Interstitial Lung Disease and Correlated with Clinical Severity. A Potentially Protective Role.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. We sought to investigate the role of FKBP13 (13-kD FK506-binding protein), an endoplasmic reticulum-resident molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy specimens from 24 patients with idiopathic pulmonary fibrosis and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of idiopathic pulmonary fibrosis lung tissues and correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsy specimens of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21), and resolution (Day 50) phases. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity of interstitial lung diseases and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation, and fibrosis.

IL-33 Mediates Lung Inflammation by the IL-6-Type Cytokine Oncostatin M.

The interleukin-1 family member IL-33 participates in both innate and adaptive T helper-2 immune cell responses in models of lung disease. The IL-6-type cytokine Oncostatin M (OSM) elevates lung inflammation, Th2-skewed cytokines, alternatively activated (M2) macrophages, and eosinophils in C57Bl/6 mice in vivo. Since OSM induces IL-33 expression, we here test the IL-33 function in OSM-mediated lung inflammation using IL-33-/- mice. Adenoviral OSM (AdOSM) markedly induced IL-33 mRNA and protein levels in wild-type animals while IL-33 was undetectable in IL-33-/- animals. AdOSM treatment showed recruitment of neutrophils, eosinophils, and elevated inflammatory chemokines (KC, eotaxin-1, MIP1a, and MIP1b), Th2 cytokines (IL-4/IL-5), and arginase-1 (M2 macrophage marker) whereas these responses were markedly diminished in IL-33-/- mice. AdOSM-induced IL-33 was unaffected by IL-6-/- deficiency. AdOSM also induced IL-33R+ ILC2 cells in the lung, while IL-6 (AdIL-6) overexpression did not. Flow-sorted ILC2 responded in vitro to IL-33 (but not OSM or IL-6 stimulation). Matrix remodelling genes col3A1, MMP-13, and TIMP-1 were also decreased in IL-33-/- mice. In vitro, IL-33 upregulated expression of OSM in the RAW264.7 macrophage cell line and in bone marrow-derived macrophages. Taken together, IL-33 is a critical mediator of OSM-driven, Th2-skewed, and M2-like responses in mouse lung inflammation and contributes in part through activation of ILC2 cells.

Targeted inhibition of PI3 kinase/mTOR specifically in fibrotic lung fibroblasts suppresses pulmonary fibrosis in experimental models.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low-molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.

Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis.

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor ß solely on activated myeloid cells, we have created a folate-targeted TLR7 agonist (FA-TLR7-54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis-inducing cytokine production. We demonstrate that FA-TLR7-54 reprograms M2-like fibrosis-inducing macrophages into fibrosis-suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7-54 is lethal at fibrosis-suppressing doses, FA-TLR7-54 halts fibrosis without evidence of toxicity. Taken together, FA-TLR7-54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose-limiting systemic toxicities.

The importance of interventional timing in the bleomycin model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown aetiology, which makes drug development challenging. Single administration of bleomycin directly to the lungs of mice is a widely used experimental model for studying pulmonary fibrogenesis and evaluating the effect of therapeutic antifibrotic strategies. The model works by inducing an early inflammatory phase, which transitions into fibrosis after 5-7 days. This initial inflammation makes therapeutic timing crucial. To accurately assess antifibrotic efficacy, the intervention should inhibit fibrosis without impacting early inflammation.Studies published between 2008 and 2019 using the bleomycin model to investigate pulmonary fibrosis were retrieved from PubMed, and study characteristics were analysed. Intervention-based studies were classified as either preventative (starting <7 days after bleomycin installation) or therapeutic (>7 days). In addition, studies were cross-referenced with current major clinical trials to assess the availability of preclinical rationale.A total of 976 publications were evaluated. 726 investigated potential therapies, of which 443 (61.0%) were solely preventative, 166 (22.9%) were solely therapeutic and 105 (14.5%) were both. Of the 443 preventative studies, only 70 (15.8%) characterised inflammation during the model's early inflammatory phase. In the reported 145 IPF clinical trials investigating 93 compounds/combinations, only 25 (26.9%) interventions had any preclinical data on bleomycin available on PubMed.Since 2008, we observed a shift (from <5% to 37.4%) in the number of studies evaluating drugs in the therapeutic setting in the bleomycin model. While this shift is encouraging, further characterisation of early inflammation and appropriate preclinical therapeutic testing are still needed. This will facilitate fruitful drug development in IPF, and more therapeutic strategies for patients with this devastating disease.

Palmitic Acid-Rich High-Fat Diet Exacerbates Experimental Pulmonary Fibrosis by Modulating Endoplasmic Reticulum Stress.

The impact of lipotoxicity on the development of lung fibrosis is unclear. Saturated fatty acids, such as palmitic acid (PA), activate endoplasmic reticulum (ER) stress, a cellular stress response associated with the development of idiopathic pulmonary fibrosis (IPF). We tested the hypothesis that PA increases susceptibility to lung epithelial cell death and experimental fibrosis by modulating ER stress. Total liquid chromatography and mass spectrometry were used to measure fatty acid content in IPF lungs. Wild-type mice were fed a high-fat diet (HFD) rich in PA or a standard diet and subjected to bleomycin-induced lung injury. Lung fibrosis was determined by hydroxyproline content. Mouse lung epithelial cells were treated with PA. ER stress and cell death were assessed by Western blotting, TUNEL staining, and cell viability assays. IPF lungs had a higher level of PA compared with controls. Bleomycin-exposed mice fed an HFD had significantly increased pulmonary fibrosis associated with increased cell death and ER stress compared with those fed a standard diet. PA increased apoptosis and activation of the unfolded protein response in lung epithelial cells. This was attenuated by genetic deletion and chemical inhibition of CD36, a fatty acid transporter. In conclusion, consumption of an HFD rich in saturated fat increases susceptibility to lung fibrosis and ER stress, and PA mediates lung epithelial cell death and ER stress via CD36. These findings demonstrate that lipotoxicity may have a significant impact on the development of lung injury and fibrosis by enhancing pro-death ER stress pathways.

Cell surface expression of 78-kDa glucose-regulated protein (GRP78) mediates diabetic nephropathy.

The 78-kDa glucose-regulated protein (GRP78) is a well-established endoplasmic reticulum (ER)-resident chaperone that maintains protein homeostasis and regulates the unfolded protein response. Under conditions of ER stress, GRP78 is also expressed at the cell surface and implicated in tumorigenesis, immunity, and cellular signaling events. The role of cell surface-associated GRP78 (csGRP78) in the pathogenesis of diabetic nephropathy has not yet been defined. Here we explored the role of csGRP78 in regulating high glucose (HG)-induced profibrotic AKT Ser/Thr kinase (AKT) signaling and up-regulation of extracellular matrix proteins. Using primary kidney mesangial cells, we show that HG treatment, but not the osmotic control mannitol, induces csGRP78 expression through an ER stress-dependent mechanism. We found that csGRP78, known to be located on the outer membrane leaflet, interacts with the transmembrane protein integrin ß1 and activates focal adhesion kinase and downstream PI3K/AKT signaling. Localization of GRP78 at the cell surface and its interaction with integrin ß1 were also required for extracellular matrix protein synthesis in response to HG. Surprisingly, both the N and C termini of csGRP78 were necessary for this profibrotic response. Increased localization of GRP78 at the plasma membrane was also found in the glomerular mesangial area of type 1 diabetic mice in two different models (streptozotocin-induced and Akita). In freshly isolated glomeruli from Akita mice, csGRP78 co-localized with the mesangial cell surface marker α8-integrin. In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy.

Mechanical stress-induced mast cell degranulation activates TGF-ß1 signalling pathway in pulmonary fibrosis.

BACKGROUND: The role of mast cells accumulating in idiopathic pulmonary fibrosis (IPF) lungs is unknown. OBJECTIVES: We investigated the effect of fibrotic extracellular matrix (ECM) on mast cells in experimental and human pulmonary fibrosis. RESULTS: In IPF lungs, mast cell numbers were increased and correlated with disease severity (control vs 60%90% vs 60%90% vs FVC<60%, mean difference=-268.6, 95% CI of difference -441.0 to -96.17, p=0.0007). Plasma tryptase levels were increased in IPF and negatively correlated with FVC (control vs FVC<60%, mean difference=-17.12, 95% CI of difference -30.02 to -4.22, p=0.006: correlation curves R=-0.045, p=0.025). In a transforming growth factor (TGF)-ß1-induced pulmonary fibrosis model, chymase-positive and tryptase-positive mast cells accumulated in fibrotic lung. Lung tissue was decellularised and reseeded with bone marrow or peritoneum-derived mast cells; cells on fibrotic ECM released more TGF-ß1 compared with normal ECM (active TGF-ß1: bone marrow-derived mast cell (BMMC)-DL vs BMMC-TGF-ß1 p=0.0005, peritoneal mast cell (PMC)-DL vs PMC-TGF-ß1 p=0.0003, total TGF-ß1: BMMC-DL vs BMMC-TGF-ß1 p=0.013, PMC-DL vs PMC-TGF-ß1 p=0.001). Mechanical stretch of lungs caused mast cell degranulation; mast cell stabilisers inhibited degranulation (histamine: cont vs doxantrazole p=0.004, ß-hexosaminidase: cont vs doxantrazole, mean difference=1.007, 95% CI of difference 0.2700 to 1.744, p=0.007) and TGF-ß1 activation (pSmad2/Smad2: cont vs dox p=0.006). Cromoglycate attenuated pulmonary fibrosis in rats (collagen: phosphate-buffered saline (PBS) vs cromoglycate p=0.036, fibrotic area: PBS vs cromoglycate p=0.031). CONCLUSION: This study suggests that mast cells may contribute to the progression of pulmonary fibrosis.

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL-6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL-6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1-XBP1 expansion pathway, IL-4/IL-13 and IL-4/IL-13/IL-6-mediated alternative programming of murine bone marrow-derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase-1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1-XBP1 arm was achieved using STF-083010 and was verified by RT-PCR. The addition of IL-6 to the conventional alternative programming cocktail IL-4/IL-13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response-mediated induction of molecular chaperones. IRE1-XBP1 inhibition substantially reduced the IL-6-mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL-6 enhances ER expansion and the profibrotic capacity of IL-4/IL-13-mediated activation of macrophages. Therapeutic strategies targeting IL-6 or the IRE1-XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.

Separate roles of IL-6 and oncostatin M in mouse macrophage polarization in vitro and in vivo.

Arginase-1 (Arg-1)-expressing M2-like macrophages are associated with Th2-skewed immune responses, allergic airway pathology, ectopic B16 melanoma cancer growth in murine models, and can be induced by Oncostatin M (OSM) transient overexpression in vivo. Here, we compare OSM to the gp130-cytokine IL-6 in mediating macrophage polarization, and find that IL-6 overexpression alone (Ad vector, AdIL-6) did not induce Arg-1 protein in mouse lungs at day 7, nor ectopic melanoma tumor growth at day 14, in contrast to overexpression of OSM (AdOSM). AdOSM elevated levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid, whereas AdIL-6 did not. Bone marrow-derived macrophages respond with Arg-1 enzymatic activity to M2 stimuli (IL-4/IL-13), which was further elevated in combination with IL-6 stimulation; however, OSM or LIF had no detectable activity in vitro. Arg-1 mRNA expression induced by AdOSM was attenuated in IL-6-/- and STAT6-/- mice, suggesting requirements for both IL-6 and IL-4/IL-13 signaling in vivo. Ectopic B16 tumor burden was also reduced in IL-6-/- mice. Thus, OSM induces Arg-1+ macrophage accumulation indirectly through elevation of Th2 cytokines and IL-6 in vivo, whereas IL-6 acts directly on macrophages but requires a Th2 microenvironment, demonstrating distinct roles for OSM and IL-6 in M2 macrophage polarization.

Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced lung fibrosis.

Although recent evidence indicates that gp130 cytokines, Oncostatin M (OSM) and IL-6 are involved in alternative programming of macrophages, their role in lung fibrogenesis is poorly understood. Here, we investigated the effect of transient adenoviral overexpression of OSM or IL-6 in mice during bleomycin-induced lung fibrosis. Lung fibrosis and M2-like macrophage accumulation were assessed by immunohistochemistry, western blotting, gene expression and flow cytometry. Ex-vivo isolated alveolar and bone marrow-derived macrophages were examined for M2-like programming and signalling. Airway physiology measurements at day 21 demonstrated that overexpression of OSM or IL-6 exacerbated bleomycin-induced lung elastance, consistent with histopathological assessment of extracellular matrix and myofibroblast accumulation. Flow cytometry analysis at day 7 showed increased numbers of M2-like macrophages in lungs of mice exposed to bleomycin and OSM or IL-6. These macrophages expressed the IL-6Rα, but were deficient for OSMRß, suggesting that IL-6, but not OSM, may directly induce alternative macrophage activation. In conclusion, the gp130 cytokines IL-6 and OSM contribute to the accumulation of profibrotic macrophages and enhancement of bleomycin-induced lung fibrosis. This study suggests that therapeutic strategies targeting these cytokines or their receptors may be beneficial to prevent the accumulation of M2-like macrophages and the progression of fibrotic lung disease.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Disruption of Calcium Signaling in Fibroblasts and Attenuation of Bleomycin-Induced Fibrosis by Nifedipine.

Fibrotic lung disease afflicts millions of people; the central problem is progressive lung destruction and remodeling. We have shown that external growth factors regulate fibroblast function not only through canonical signaling pathways but also through propagation of periodic oscillations in Ca(2+). In this study, we characterized the pharmacological sensitivity of the Ca(2+)oscillations and determined whether a blocker of those oscillations can prevent the progression of fibrosis in vivo. We found Ca(2+) oscillations evoked by exogenously applied transforming growth factor ß in normal human fibroblasts were substantially reduced by 1 µM nifedipine or 1 µM verapamil (both L-type blockers), by 2.7 µM mibefradil (a mixed L-/T-type blocker), by 40 µM NiCl2 (selective at this concentration against T-type current), by 30 mM KCl (which partially depolarizes the membrane and thereby fully inactivates T-type current but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and T-type currents). In our in vivo study in mice, nifedipine prevented bleomycin-induced fibrotic changes (increased lung stiffness, overexpression of smooth muscle actin, increased extracellular matrix deposition, and increased soluble collagen and hydroxyproline content). Nifedipine had little or no effect on lung inflammation, suggesting its protective effect on lung fibrosis was not due to an antiinflammatory effect but rather was due to altering the profibrotic response to bleomycin. Collectively, these data show that nifedipine disrupts Ca(2+) oscillations in fibroblasts and prevents the impairment of lung function in the bleomycin model of pulmonary fibrosis. Our results provide compelling proof-of-principle that interfering with Ca(2+) signaling may be beneficial against pulmonary fibrosis.

Protein misfolding and endoplasmic reticulum stress in chronic lung disease.

The pathogenesis of chronic lung disorders is poorly understood but is often thought to arise because of repeated injuries derived from exposure to exogenous or endogenous stress factors. Protein-misfolding events have been observed in a variety of genetic and nongenetic chronic lung disorders and may contribute to both the initiation and the progression of lung disease through endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Evidence indicates that exposure to common lung irritants such as cigarette smoke, environmental pollutants, and infectious viral or bacterial agents can induce ER stress and protein misfolding. Although the UPR is thought to be a molecular mechanism involved in the repair and restoration of protein homeostasis or "proteostasis," prolonged activation of the UPR may lead to compromised cellular functions, cellular transformation, or cell death. Here, we review literature that associates protein-misfolding events with ER stress and UPR activation and discuss how this basic molecular repair mechanism may contribute to the initiation and progression of various genetic and nongenetic chronic lung diseases.