Bleomycin-induced Lung Injury Model Research Articles

Human Muse cells isolated from preterm- and term-umbilical cord delivered therapeutic effects in rat bleomycin-induced lung injury model without immunosuppressant

BackgroundBleomycin (BLM)-induced lung injury is characterized by mixed histopathologic changes with inflammation and fibrosis, such as observed in human patients with bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. Although no curative therapies for these lung diseases exist, stem cell therapy has emerged as a potential therapeutic option. Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent- and macrophage-like stem cells distributed in various adult and fetal tissues as stage-specific embryonic antigen-3-positive cells. They selectively home to damaged tissue by sensing sphingosine-1-phosphate and replace the damaged/apoptotic cells by in vivo differentiation. Clinical trials for some human diseases suggest the safety and therapeutic efficacy of intravenously injected human leukocyte antigen-mismatched allogenic Muse cells from adult bone marrow (BM) without immunosuppressant. Here, we evaluated the therapeutic effects of human Muse cells from preterm and term umbilical cord (UC), and adult BM in a rat BLM-induced lung injury model.MethodsRats were endotracheally administered BLM to induce lung injury on day 0. On day 3, human preterm UC-Muse, term UC-Muse, or adult BM-Muse cells were administered intravenously without immunosuppressants, and rats were subjected to histopathologic analysis on day 21. Body weight, serum surfactant protein D (SP-D) levels, and oxygen saturation (SpO2) were monitored. Histopathologic lung injury scoring by the Ashcroft and modified American Thoracic Society document scales, quantitative characterization of engrafted Muse cells, RNA sequencing analysis, and in vitro migration assay of infused Muse cells were performed.ResultsRats administered preterm- and term-UC-Muse cells exhibited a significantly better recovery based on weight loss, serum SP-D levels, SpO2, and histopathologic lung injury scores, and a significantly higher rate of both Muse cell homing to the lung and alveolar marker expression (podoplanin and prosurfactant protein-C) than rats administered BM-Muse cells. Rats receiving preterm-UC-Muse cells showed statistically superior results to those receiving term-UC-Muse cells in many of the measures. These findings are thought to be due to higher expression of genes related to cell migration, lung differentiation, and cell adhesion.ConclusionPreterm UC-Muse cells deliver more efficient therapeutic effects than term UC- and BM-Muse cells for treating BLM-induced lung injury in a rat model.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Bleomycin-induced modulations of PARP 1 activity, NAD+ and PARG content in rat lung nuclei

Bleomycin-induced lung pathology in rodents is a well recognized animal model widely used for evaluation of new therapeutic approaches in treatment of lung inflammation and fibrotic diseases. It is documented that poly(ADP-ribose)polymerase 1 activity has a significant role in development of inflammatory processes in the heart, liver and brain. Herein, we used biochemical and immunochemical methods for estimation of poly(ADP-ribose)polymerase 1 (PARP 1) activity, NAD+ and poly(ADP-ribose)glycohydrolase (PARG) protein content in rat lung nuclei during the inflammatory phase in a bleomycin-induced one-hit rat model. To evaluate the influence of bleomycin – induced alterations in DNA structure on regulation of poly(ADP-ribose)polymerase 1 activation pathways, we isolated DNA from nuclei of lung tissues in the phase of acute lung inflammation induced by bleomycin, and DNA melting profiles were investigated. In the present study we investigated whether naturally occurring water-soluble polyphenol tannic acid with widely accepted anti-fibrotic and anti-inflammatory effects can influence poly(ADP-ribose)polymerase 1 activity, NAD+ and poly(ADP-ribose)glycohydrolase protein content in nuclear fraction isolated from rat lung tissues in a bleomycin-induced acute lung injury model. It was demonstrated that NAD+ level and poly(ADP-ribose)glycohydrolase protein content decreased in rat lung nuclei during the inflammatory phase in the bleomycin-induced acute lung injury model. Treatment of rats with tannic acid enhanced the effects displayed by bleomycin in lung nuclei, thus indicating synergistic interaction with the drug in the field covering PARP 1 activity, poly(ADP-ribose)glycohydrolase (PARG) protein and NAD+ content in lung nuclei. We observed PARP 1 inhibition in nuclei of lung tissue during the inflammatory phase in the bleomycin-induced acute lung injury rat model, which could be coupled with the drop of NAD+ level in nuclei. In the present study we highlighted that bleomycin (BLM) can induce DNA destabilization in lung nuclei. It was proposed that bleomycin-induced modulations in DNA structure could hamper PARP 1 binding with DNA and down-regulate the enzyme activating pathway in lung nuclei. The role of poly(ADP-ribose)glycohydrolase depletion in lung nuclei and sequential accumulation of poly(ADP-ribose)polymers in lung cells, which triggers their destruction and tissues damage, was proposed. It is suggested that in the light of synergistic interaction between bleomycin and tannic acid (TA) the anti-inflammatory role of tannic acid should be repurposed.

Thrombocyte-derived Dickkopf1 promotes macrophage polarization in the Bleomycin-induced lung injury model.

Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating inflammation and tissue repair processes. While an immunomodulatory role of Wnt antagonist Dickkopf1 (DKK1) has been implicated, the role of Wnt antagonist DKK1 in regulating macrophage polarization in inflammation and the tissue repair process remains elusive. Here we found that DKK1 induces gene expression profiles to promote inflammation and tissue repair in macrophages. Importantly, DKK1 induced various genes, including inflammation and tissue repair, via JNK (c-jun N-terminal kinase) in macrophages. Furthermore, DKK1 potentiated IL-13-mediated macrophage polarization and activation. The co-inhibition of JNK and STAT6 markedly decreased gene expressions relevant to inflammation and fibrosis by DKK1 and IL-13. Interestingly, thrombocyte-specific deletion of DKK1 in mice reduced collagen deposition and decreased Arg1, CD206, HIF1α, and IL1β protein expressions in monocyte-derived alveolar macrophages in the acute sterile bleomycin (BLM)-induced lung injury model. These data suggested that thrombocytes communicate with macrophages via DKK1 to orchestrate inflammation and repair in this model. Taken together, our study demonstrates DKK1's role as an important regulatory ligand for macrophage polarization in the injury-induced inflammation and repair process in the lung.

The Regenerative Power of Stem Cells: Treating Bleomycin-Induced Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with no known cure, characterized by the formation of scar tissue in the lungs, leading to respiratory failure. Although the exact cause of IPF remains unclear, the condition is thought to result from a combination of genetic and environmental factors. One of the most widely used animal models to study IPF is the bleomycin-induced lung injury model in mice. In this model, the administration of the chemotherapeutic agent bleomycin causes pulmonary inflammation and fibrosis, which closely mimics the pathological features of human IPF. Numerous recent investigations have explored the functions of various categories of stem cells in the healing process of lung injury induced by bleomycin in mice, documenting the beneficial effects and challenges of this approach. Differentiation of stem cells into various cell types and their ability to modulate tissue microenvironment is an emerging aspect of the regenerative therapies. This review article aims to provide a comprehensive overview of the role of stem cells in repairing bleomycin-induced lung injury. It delves into the mechanisms through which various types of stem cells, including mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and lung resident stem cells, exert their therapeutic effects in this specific model. We have also discussed the unique set of intermediate markers and signaling factors that can influence the proliferation and differentiation of alveolar epithelial cells both during lung repair and homeostasis. Finally, we highlight the challenges and opportunities associated with translating stem cell therapy to the clinic for IPF patients. The novelty and implications of this review extend beyond the understanding of the potential of stem cells in treating IPF to the broader field of regenerative medicine. We believe that the review paves the way for further advancements in stem cell therapies, offering hope for patients suffering from this debilitating and currently incurable disease.

Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

Mechanical ventilation (MV) used in patients with acute lung injury (ALI) induces lung inflammation and causes fibroblast proliferation and excessive collagen deposition-a process termed epithelial-mesenchymal transition (EMT). Phosphoinositide 3-kinase-γ (PI3K-γ) is crucial in modulating EMT during the reparative phase of ALI; however, the mechanisms regulating the interactions among MV, EMT, and PI3K-γ remain unclear. We hypothesized that MV with or without bleomycin treatment would increase EMT through the PI3K-γ pathway. C57BL/6 mice, either wild-type or PI3K-γ-deficient, were exposed to 6 or 30 mL/kg MV for 5 h after receiving 5 mg/kg AS605240 intraperitoneally 5 days after bleomycin administration. We found that, after bleomycin exposure in wild-type mice, high-tidal-volume MV induced substantial increases in inflammatory cytokine production, oxidative loads, Masson's trichrome staining level, positive staining of α-smooth muscle actin, PI3K-γ expression, and bronchial epithelial apoptosis (p < 0.05). Decreased respiratory function, antioxidants, and staining of the epithelial marker Zonula occludens-1 were also observed (p < 0.05). MV-augmented bleomycin-induced pulmonary fibrogenesis and epithelial apoptosis were attenuated in PI3K-γ-deficient mice, and we found pharmacological inhibition of PI3K-γ activity through AS605240 (p < 0.05). Our data suggest that MV augmented EMT after bleomycin-induced ALI, partially through the PI3K-γ pathway. Therapy targeting PI3K-γ may ameliorate MV-associated EMT.

Engineered Fibroblast Extracellular Vesicles Attenuate Pulmonary Inflammation and Fibrosis in Bleomycin-Induced Lung Injury.

Pulmonary fibrosis is a progressive disease for which no curative treatment exists. We have previously engineered dermal fibroblasts to produce extracellular vesicles with tissue reparative properties dubbed activated specialized tissue effector extracellular vesicles (ASTEX). Here, we investigate the therapeutic utility of ASTEX in vitro and in a mouse model of bleomycin-induced lung injury. RNA sequencing demonstrates that ASTEX are enriched in micro-RNAs (miRs) cargo compared with EVs from untransduced dermal fibroblast EVs (DF-EVs). Treating primary macrophages with ASTEX reduced interleukin (IL)6 expression and increased IL10 expression compared with DF-EV-exposed macrophages. Furthermore, exposure of human lung fibroblasts or vascular endothelial cells to ASTEX reduced expression of smooth muscle actin, a hallmark of myofibroblast differentiation (respectively). In vivo, intratracheal administration of ASTEX in naïve healthy mice demonstrated a favorable safety profile with no changes in body weight, lung weight to body weight, fibrotic burden, or histological score 3 weeks postexposure. In an acute phase (short-term) bleomycin model of lung injury, ASTEX reduced lung weight to body weight, IL6 expression, and circulating monocytes. In a long-term setting, ASTEX improved survival and reduced fibrotic content in lung tissue. These results suggest potential immunomodulatory and antifibrotic properties of ASTEX in lung injury.

Increased Monocyte-Derived CD11b+ Macrophage Subpopulations Following Cigarette Smoke Exposure Are Associated With Impaired Bleomycin-Induced Tissue Remodelling.

RationaleThe accumulation of macrophages in the airways and the pulmonary interstitium is a hallmark of cigarette smoke-associated inflammation. Notably, pulmonary macrophages are not a homogenous population but consist of several subpopulations. To date, the manner in which cigarette smoke exposure affects the relative composition and functional capacity of macrophage subpopulations has not been elucidated.MethodsUsing a whole-body cigarette smoke exposure system, we investigated the impact of cigarette smoke on macrophage subpopulations in C57BL/6 mice using flow cytometry-based approaches. Moreover, we used bromodeoxyuridine labelling plus Il1a-/- and Il1r1-/- mice to assess the relative contribution of local proliferation and monocyte recruitment to macrophage accumulation. To assess the functional consequences of altered macrophage subpopulations, we used a model of concurrent bleomycin-induced lung injury and cigarette smoke exposure to examine tissue remodelling processes.Main ResultsCigarette smoke exposure altered the composition of pulmonary macrophages increasing CD11b+ subpopulations including monocyte-derived alveolar macrophages (Mo-AM) as well as interstitial macrophages (IM)1, -2 and -3. The increase in CD11b+ subpopulations was observed at multiple cigarette smoke exposure timepoints. Bromodeoxyuridine labelling and studies in Il1a-/- mice demonstrated that increased Mo-AM and IM3 turnover in the lungs of cigarette smoke-exposed mice was IL-1α dependent. Compositional changes in macrophage subpopulations were associated with impaired induction of fibrogenesis including decreased α-smooth muscle actin positive cells following intratracheal bleomycin treatment. Mechanistically, in vivo and ex vivo assays demonstrated predominant macrophage M1 polarisation and reduced matrix metallopeptidase 9 activity in cigarette smoke-exposed mice.ConclusionCigarette smoke exposure modified the composition of pulmonary macrophage by expanding CD11b+ subpopulations. These compositional changes were associated with attenuated fibrogenesis, as well as predominant M1 polarisation and decreased fibrotic activity. Overall, these data suggest that cigarette smoke exposure altered the composition of pulmonary macrophage subpopulations contributing to impaired tissue remodelling.

Discovery of a new class of integrin antibodies for fibrosis

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab’s yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFβ activation. In IPF patient lung fibroblasts, TGFβ treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFβ action though mechanisms beyond the inhibition of latent TGFβ activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.

The Integrin Inhibitor Cilengitide and Bleomycin-Induced Pulmonary Fibrosis : Cilengitide and Bleomycin-Induced Pulmonary Fibrosis.

Fibroproliferation and excess deposition of extracellular matrix (ECM) are the pathologic hallmarks of idiopathic pulmonary fibrosis (IPF), a chronic progressive disorder with high mortality and suboptimal treatment options. Although the etiologic mechanisms responsible for the development and progression of IPF remain unclear, cell-ECM interactions and growth factors are considered important. Cilengitide is a cyclic RGD pentapeptide with anti-angiogenic activity that targets αvβ3, αvβ5 and α5β1, integrins known to mediate cell-ECM interactions and activate the pro-fibrotic growth factor Transforming Growth Factor beta (TGF-β). Cilengitide was studied in vitro with the use of NIH/3T3 cells and primary lung fibroblasts, and in vivo in the well-characterized bleomycin-induced lung injury model. The extent of ECM deposition was determined by RT-PCR, Western blot, histologic analysis and hydroxyproline assay of lung tissue. Bronchoalveolar lavage analysis was used to determine cell counts. Cilengitide treatment of cultured fibroblasts showed decreased adhesion to vitronectin and fibronectin, both integrin-dependent events. Cilengitide also inhibited TGF-β-induced fibronectin gene expression and reduced the accumulation of mRNAs and protein for fibronectin and collagen type I. Both preventive and treatment effects of daily injections of cilengitide (20mg/kg) failed to inhibit the development of pulmonary fibrosis as determined by histological analysis (Ashcroft scoring), bronchoalveolar lavage (BAL) fluid cell counts, and hydroxyproline content. Overall, our data suggest that, despite its in vitro activity in fibroblasts, daily injections of cilengitide (20mg/kg) did not inhibit the development of or ameliorate bleomycin-induced pulmonary fibrosis in mice.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin β3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Dexamethasone fails to improve bleomycin-induced acute lung injury in mice.

Acute respiratory distress syndrome (ARDS) features an exudative phase characterized by alveolar damage, lung edema and exacerbated inflammatory response. Given their anti‐inflammatory properties, the potential therapeutic effect of corticosteroids has been evaluated in ARDS clinical trials and experimental models of ALI. These studies produced contradictory results. Therefore, our aim was to investigate the effects of dexamethasone in an animal model of bleomycin‐induced acute lung injury and then to determine if the lack of response could be related to an impairment in repair ability of alveolar epithelial cells after injury. NMRI mice were challenged with bleomycin and then treated daily with dexamethasone or saline. Bronchoalveolar lavages (BAL) and lungs were collected for assessment of the inflammatory response and wet/dry ratio (lung edema) and for histological analyses. The effect of bleomycin and dexamethasone on wound repair was also evaluated in vitro on primary alveolar epithelial cell (ATII) cultures. Our data first showed that dexamethasone treatment did not reduce the weight loss or mortality rates induced by bleomycin. Although the TNF‐α level in BAL of bleomycin‐treated mice was reduced by dexamethasone, the neutrophil infiltration remained unchanged. Dexamethasone also failed to reduce lung edema and damage scores. Finally, bleomycin elicited a time‐ and dose‐dependent reduction in repair rates of ATII cell cultures. This inhibitory effect was further enhanced by dexamethasone, which also affected the expression of β3‐ and β6‐integrins, key proteins of alveolar repair. Altogether, our data indicate that the inability of dexamethasone to improve the resolution of ALI might be due to his deleterious effect on the alveolar epithelium repair.

TAZ is required for lung alveolar epithelial cell differentiation after injury.

The lung is a relatively quiescent organ during homeostasis, but has a remarkable capacity for repair after injury. Alveolar epithelial type I cells (AEC1s) line airspaces and mediate gas exchange. After injury, they are regenerated by differentiation from their progenitors - alveolar epithelial type II cells (AEC2s) - which also secrete surfactant to maintain surface tension and alveolar patency. While recent studies showed that the maintenance of AEC2 stemness is Wnt dependent, the molecular mechanisms underlying AEC2-AEC1 differentiation in adult lung repair are still incompletely understood. Here we show that WWTR1 (TAZ) plays a crucial role in AEC differentiation. Using an in vitro organoid culture system, we found that tankyrase inhibition can efficiently block AEC2-AEC1 differentiation, and this effect was due to the inhibition of TAZ. In a bleomycin induced lung injury model, conditional deletion of TAZ in AEC2s dramatically reduced AEC1 regeneration during recovery, leading to exacerbated alveolar lesions and fibrosis. In patients with idiopathic pulmonary fibrosis (IPF), decreased blood levels of RAGE, a biomarker of AEC1 health, were associated with more rapid disease progression. Our findings implicate TAZ as a critical factor involved in AEC2 to AEC1 differentiation, and hence the maintenance of alveolar integrity after injury.

Hydrogel-based delivery of Il-10 improves treatment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a life-threatening progressive lung disorder with limited therapeutic options. While interleukin-10 (IL-10) is a potent anti-inflammatory and anti-fibrotic cytokine, its utility in treating lung fibrosis has been limited by its short half-life. We describe an innovative hydrogel-based approach to deliver recombinant IL-10 to the lung for the prevention and reversal of pulmonary fibrosis in a mouse model of bleomycin-induced lung injury. Our studies show that a hyaluronan and heparin-based hydrogel system locally delivers IL-10 by capitalizing on the ability of heparin to reversibly bind IL-10 without bleeding or other complications. This formulation is significantly more effective than soluble IL-10 for both preventing and reducing collagen deposition in the lung parenchyma after 7 days of intratracheal administration. The anti-fibrotic effect of IL-10 in this system is dependent on suppression of TGF-β driven collagen production by lung fibroblasts and myofibroblasts. We conclude that hydrogel-based delivery of IL-10 to the lung is a promising therapy for fibrotic lung disorders.

Comprehensive lipid profiling of bleomycin-induced lung injury.

Drug-induced lung injury is an adverse effect of drug treatment that can result in respiratory failure. Because lipid profiling could provide cutting-edge understanding of the pathophysiology of toxicological responses, we performed lipidomic analyses of drug-induced lung injury. We used a mouse model of bleomycin-induced lung injury and followed the physiological responses at the acute inflammatory (day 2), inflammatory-to-fibrosis (day 7) and fibrosis (day 21) phases. The overall lipid profiles of plasma, lung and bronchoalveolar lavage fluid (BALF) revealed that drastic changes in lipids occurred in the lung and BALF, but not in the plasma, after 7 and 21days of bleomycin treatment. In the lung, the levels of ether-type phosphatidylethanolamines decreased, while those of phosphatidylcholines, bismonophosphatidic acids and cholesterol esters increased on days 7 and 21. In BALF, the global lipid levels increased on days 7 and 21, but only those of some lipids, such as phosphatidylglycerols/bismonophosphatidic acids and phosphatidylinositols, increased from day 2. The lung levels of prostaglandins, such as prostaglandin D2 , were elevated on day 2, and those of 5- and 15-lipoxygenase metabolites of docosahexaenoic acid were elevated on day 7. In BALF, the levels of 12-lipoxygenase metabolites of polyunsaturated fatty acids were elevated on day 7. Our comprehensive lipidomics approach suggested anti-inflammatory responses in the inflammatory phase, phospholipidosis and anti-inflammatory responses in the inflammatory-to-fibrosis phase, and increased oxidative stress and/or cell phenotypic transitions in the fibrosis phase. Understanding these molecular changes and potential mechanisms will help develop novel drugs to prevent or treat drug-induced lung injury.

In Vivo Labeling of Plasma Proteins for Imaging of Enhanced Vascular Permeability in the Lungs.

Increased vascular permeability is an important hallmark of many diseases, including cancer, cerebral ischemia, and severe inflammatory disorders. In this regard, the noninvasive assessment of pathologically increased vascular permeability in vivo is of great interest. In this study, the potential of albumin- and transthyretin-binding radioligands was evaluated for imaging of vascular hyperpermeability. For this purpose, the bleomycin-induced lung injury model was used as a model of inflammation-associated vascular leakage. The plasma protein-binding ligands, which bind to albumin (DOTA-PPB-01) and transthyretin (DOTA-PPB-03), were radiolabeled and used for nuclear imaging and biodistribution studies. In this regard, 177Lu was employed as a surrogate nuclide for detailed preclinical investigations, including single-photon emission computed tomography (SPECT) studies, whereas 44Sc was proposed as a radionuclide for positron emission tomography (PET), which may be relevant for future clinical translation. Mice were administered with these radioligands 6-9 days after intratracheal instillation of bleomycin or saline. Bleomycin-treated mice developed pronounced lung inflammation with enhanced vascular permeability that was reflected in significantly increased lung size and weight due to edema and infiltration with inflammatory cells. Biodistribution studies revealed significantly higher accumulation of 177Lu-DOTA-PPB-01 in injured lungs as compared to lungs of control animals at all investigated time points (4-48 h p.i.). The best contrast was achieved at late time points (16.1 ± 2.91% IA/g vs 2.03 ± 1.22% IA/g, 48 h p.i.) when the blood activity levels were ∼7.5% IA/g. Injection of 177Lu-DOTA-PPB-03 also resulted in increased lung accumulation in bleomycin-treated mice at all investigated time points (2-8 h p.i.). The pharmacokinetics was significantly faster, however, resulting in good contrast already at 8 h p.i. (4.32 ± 0.85% IA/g vs 1.06 ± 0.10% IA/g) when blood activity levels were ∼2% IA/g. The absolute lung accumulation of 177Lu-DOTA-PPB-03 was significantly lower than that of 177Lu-DOTA-PPB-01. PET/CT scans performed with 44Sc-DOTA-PPB-01 distinguished injured from healthy lungs only at late time points (20 h p.i.), whereas 44Sc-DOTA-PPB-03 already allowed the differentiation at 4 h p.i. due to its faster clearance. The investigated radioligands, 44Sc/177Lu-DOTA-PPB-01 and 44Sc/177Lu-DOTA-PPB-03, hold promise for the visualization of vascular leakage in a variety of pathological conditions. 44Sc would be the radionuclide of choice for clinical application as it can be stably coordinated with a DOTA chelator and enables PET imaging over extended periods.

Identification of a T cell immunomodulatory domain in histidyl-tRNA synthetase

Abstract Histidyl-tRNA synthetase (HARS) is the autoantigen target of Jo-1 antibodies, which occur in the major form of anti-synthetase syndrome. These patients are characterized by an autoimmune myositis and interstitial lung disease. Circulating extracellular HARS is detected in healthy individuals, but is reduced or undetectable in Jo-1-positive individuals. Administration of ATYR1940, a recombinant form of HARS, ameliorates lung fibrosis and reduces T cell cytokine production in the bleomycin-induced lung injury model. Similar effects were observed with the N-terminal domain of HARS (the iMod domain) conjugated to IgG Fc, suggesting that this domain confers the immunomodulatory activity of HARS. To confirm primary immune effects of ATYR1940 and ATYR1923 (iMod.Fc), human T cells were isolated from PBMC from healthy individuals and stimulated with anti-CD3/anti-CD28. Proteins containing the HARS iMod domain reduced in vitro activation of human CD4+ and CD8+ T cells, as evidenced by reduced secretion of IL-2, IFNγ, TNFα, IL-17, IL-13, and granzyme B, as well as decreased upregulation of activation markers such as CD69 and CD40L. ATYR1940 and ATYR1923 also inhibited cytokine release after ex vivo stimulation of human memory T cells in a NSG mouse xenogeneic GVHD model. T cell inhibition by ATYR1940 was dependent on its iMod domain, as demonstrated using an iMod-specific blocking monoclonal antibody. The ATYR1940-induced T cell gene signature reflected a general inhibitory effect on activation as well as on cell cycle protein expression. These results suggest that circulating levels of HARS may act to control the threshold stimulatory signal required to activate T cells. We propose circulating HARS as a soluble immune set-point modulator.

The Selective Angiotensin II Type 2 Receptor Agonist, Compound 21, Attenuates the Progression of Lung Fibrosis and Pulmonary Hypertension in an Experimental Model of Bleomycin-Induced Lung Injury.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease characterized by scar formation and respiratory insufficiency, which progressively leads to death. Pulmonary hypertension (PH) is a common complication of IPF that negatively impacts clinical outcomes, and has been classified as Group III PH. Despite scientific advances, the dismal prognosis of IPF and associated PH remains unchanged, necessitating the search for novel therapeutic strategies. Accumulating evidence suggests that stimulation of the angiotensin II type 2 (AT2) receptor confers protection against a host of diseases. In this study, we investigated the therapeutic potential of Compound 21 (C21), a selective AT2 receptor agonist in the bleomycin model of lung injury. A single intra-tracheal administration of bleomycin (2.5 mg/kg) to 8-week old male Sprague Dawley rats resulted in lung fibrosis and PH. Two experimental protocols were followed: C21 was administered (0.03 mg/kg/day, ip) either immediately (prevention protocol, BCP) or after 3 days (treatment protocol, BCT) of bleomycin-instillation. Echocardiography, hemodynamic, and Fulton's index assessments were performed after 2 weeks of bleomycin-instillation. Lung tissue was processed for gene expression, hydroxyproline content (a marker of collagen deposition), and histological analysis. C21 treatment prevented as well as attenuated the progression of lung fibrosis, and accompanying PH. The beneficial effects of C21 were associated with decreased infiltration of macrophages in the lungs, reduced lung inflammation and diminished pulmonary collagen accumulation. Further, C21 treatment also improved pulmonary pressure, reduced muscularization of the pulmonary vessels and normalized cardiac function in both the experimental protocols. However, there were no major differences in any of the outcomes measured from the two experimental protocols. Collectively, our findings indicate that stimulation of the AT2 receptor by C21 attenuates bleomycin-induced lung injury and associated cardiopulmonary pathology, which needs to be further explored as a promising approach for the clinical treatment of IPF and Group III PH.

Transmembrane protease, serine 4 (TMPRSS4) is upregulated in IPF lungs and increases the fibrotic response in bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 μg/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 μg/lung and TMPRSS4 deficient mice: 114.1±24.2 μg/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.

Anti-inflammatory and anti-fibrotic effects of intravenous adipose-derived stem cell transplantation in a mouse model of bleomycin-induced interstitial pneumonia

Adipose-derived stem cells (AdSCs) have recently been considered a useful treatment tool for autoimmune disease because of their anti-inflammatory and immunosuppressive effects. We investigated the therapeutic effect of intravenous AdSC transplantation in a mouse model of bleomycin-induced lung injury. AdSCs accumulated in the pulmonary interstitium and inhibited both inflammation and fibrosis in the lung, markedly improving the survival rate of mice with bleomycin-induced lung injury in a cell number-dependent manner. AdSCs inhibited the production of pro-inflammatory cytokines such as TNF-α and IL-12 in activated macrophages, and AdSCs also induced the apoptosis of activated macrophages. AdSCs inhibited the differentiation and proliferation of Th2-type mCD4+ T cells but promoted the differentiation and proliferation of regulatory T cells, suggesting that the phenotypic conversion of T cells may be one of the mechanisms for the anti-inflammatory effect of AdSCs on pulmonary fibrosis. These findings suggest that intravenous AdSCs could be a promising treatment for patients with interstitial pneumonia.