Bleomycin-induced Lung Inflammation Research Articles

Inhalation of itraconazole mitigates bleomycin-induced lung fibrosis via regulating SPP1 and C3 signaling pathway pivotal in the interaction between phagocytic macrophages and diseased fibroblasts

BackgroundIdiopathic pulmonary fibrosis (IPF) stands as a significant contributor to global mortality rates. Presently, there exists a dearth of effective anti-fibrotic treatments for this condition. While itraconazole (ITR) has exhibited potential in mitigating pulmonary fibrosis, its oral administration is hampered by unfavorable pharmacokinetics, which elevate the risk of adverse reactions, thus limiting its clinical utility.MethodsAn inhalable formulation of ITR were engineered which aimed at enhancing its pulmonary dispersion. First, pharmacokinetics were conducted to investigate the blood concentration and tissue residue of ITR after inhalation administration. In addition, bleomycin induced mouse pulmonary fibrosis model was used to compare the therapeutic effects of ITR administered by inhalation and intragastric administration. Finally, single-cell RNA sequencing (scRNAseq) was used to explore the mechanism of ITR inhalation administration.ResultsWe found that a large amount of drugs accumulated in the lung tissue for a long time after inhalation administration, thus maximizing the therapeutic effect of drugs. Inhalation of ITR daily at for 21 days significantly attenuated bleomycin-induced lung fibrosis and inflammation in murine models. Additionally, our findings revealed that ITR inhalation diminished the proportion of diseased fibroblasts while promoting reparative fibroblast populations in the murine model. Furthermore, it effectively reversed the proportion of activated phagocytic macrophages. Mechanistically, ITR inhalation exerted its effects by regulating SPP1 and C3 signaling pathway pivotal in the interaction between phagocytic macrophages and diseased fibroblasts.ConclusionsThese insights into the molecular mechanisms underlying ITR’s therapeutic effects on IPF underscore the favorable pharmacokinetic profile conferred by inhalation, thus presenting a promising formulation poised for clinical translation.

Opposite effects of systemic and local conditional CD11c+ myeloid cell depletion during bleomycin-induced inflammation and fibrosis in mice.

Elevated levels of CD11c+ myeloid cells are observed in various pulmonary disorders, including Idiopathic Pulmonary Fibrosis (IPF). Dendritic cells (DCs) and macrophages (MΦ) are critical antigen-presenting cells (APCs) that direct adaptive immunity. However, the role of CD11c+ myeloid cells in lung extracellular matrix (ECM) accumulation and pulmonary fibrosis is poorly understood. We aimed to investigate the impact of depleting CD11c+ myeloid cells, including DCs and macrophages, during bleomycin-induced pulmonary fibrosis in mice. We used a diphtheria toxin (DTx) receptor (DTR) transgenic mouse model (CD11c-DTR-Tg) to deplete CD11c+ myeloid cells through two methods: Systemic Depletion (SD) via intraperitoneal injection (i.p.) and local depletion (LD) via intranasal instillation (i.n.). We then assessed the effects of CD11c+ cell depletion during bleomycin-induced lung inflammation and fibrosis. Fourteen days after bleomycin instillation, there was a progressive accumulation of myeloid cells, specifically F4/80-MHCII+CD11c+ DCs and F4/80 + MHCII+CD11c+ MΦ, preceding mortality and pulmonary fibrosis. Systemic depletion of CD11c+ DCs and MΦ via i.p. DTx administration in CD11c-DTR-Tg mice protected against bleomycin-induced mortality and pulmonary fibrosis compared to wild-type (WT) mice. Systemic depletion reduced myeloid cells, airway inflammation (total leukocytes, neutrophils, and CD4+ lymphocytes in bronchoalveolar lavage (BAL), inflammatory and fibrogenic mediators, and fibrosis-related mRNAs (Collagen-1α1 and α-SMA). Increased anti-inflammatory cytokine IL-10 and CXCL9 levels were observed, resulting in lower lung hydroxyproline content and Ashcroft fibrosis score. Conversely, local depletion of CD11c+ cells increased mortality by acute leukocyte influx (predominantly neutrophils, DCs, and MΦ in BAL) correlated to IL-1β, with lung hyper-inflammation and early fibrosis development. Systemic depletion of CD11c+ cells confers protection against inflammation and fibrosis induced by Bleomycin, underscoring the significance of myeloid cells expressing F4/80-MHCII+CD11c+ DCs and F4/80 + MHCII+CD11c+ MΦ orchestrating the inflammatory milieu within the lungs, potentially as a source of cytokines sustaining pulmonary chronic inflammation leading to progressive fibrosis and mortality.

Zafirlukast ameliorates lipopolysaccharide and bleomycin-induced lung inflammation in mice

Acute lung injury (ALI) is the result of damage to the capillary endothelia and the alveolar epithelial cell caused by various direct and indirect factors, leading to significant pulmonary interstitial and alveolar edema and acute hypoxic respiratory insufficiency. A subset of ALI cases progresses to irreversible pulmonary fibrosis, a condition with fatal implications. Zafirlukast is a leukotriene receptor antagonist licensed for asthma prevention and long-term treatment. This study demonstrated a significant improvement in lung tissue pathology and a reduction in inflammatory cell infiltration in models of lipopolysaccharide (LPS)-induced ALI and bleomycin (BLM)-induced lung inflammation following zafirlukast administration, both in vivo and in vitro. Moreover, zafirlukast was found to suppress the inflammatory response of alveolar epithelial cells in vitro and lung inflammation in vivo by reducing the activation of the TLR4/NF-κB/NLRP3 inflammasome pathway. In conclusion, zafirlukast relieved lung injury and the infiltration of inflammatory cells in the lung by regulating the TLR4/NF-κB/NLRP3 pathway.

Carvacrol protects rats against bleomycin-induced lung oxidative stress, inflammation, and fibrosis.

The main objective of this study was to investigate the potential efficacy of carvacrol (CAR) in mitigating bleomycin (BLM)-induced pulmonary fibrosis (PF). Sixty-six male Wistar rats were assigned into two main groups of 7 and 21 days. They were divided into the subgroups of control, BLM, CAR 80 (only for the 21-day group), and CAR treatment groups. The CAR treatment groups received CAR (20, 40, and 80 mg/kg, orally) for 7 or 21 days after an instillation of BLM (5 mg/kg, intratracheally). Results indicated that BLM significantly increased total cell count in bronchoalveolar lavage fluid and the percentages of neutrophils and lymphocytes, and reduced the percentage of macrophages. CAR dose-dependently decreased total cell count and the percentage of neutrophils and lymphocytes. CAR significantly reduced thiobarbituric acid reactive substances and hydroxyproline levels and elevated the total thiol level and catalase, superoxide dismutase, and glutathione peroxidase activities in BLM-exposed rats. Furthermore, CAR decreased the transforming growth factor-β1, connective transforming growth factor, and tumor necrosis factor-α on days 7 and 21. BLM increased interferon-γ on day 7 but decreased its level on day 21. However, CAR reversed interferon-γ levels on days 7 and 21. Based on histopathological findings, BLM induced inflammation on days 7 and 21, but for induction of fibrosis, 21-day study showed more fibrotic injuries than the 7-day group. CAR showed the improvement of fibrotic injuries. The effect of CAR against BLM-induced pulmonary fibrosis is possibly due to its antioxidant, anti-inflammatory, and antifibrotic activity.

Fasting alleviates bleomycin-induced lung inflammation and fibrosis via decreased Tregs and monocytes

PurposeIdiopathic pulmonary fibrosis (IPF), a chronic and progressively worsening condition characterized by interstitial lung inflammation and fibrosis of unknown etiology, has a grim prognosis. The treatment options for IPF are limited and new therapeutic strategies are urgently needed. Dietary restriction can improve various inflammatory diseases, but its therapeutic effect on bleomycin (BLM)-induced pulmonary fibrosis mouse model remains unclear. This study aims to investigate whether intermittent fasting (IF) can alleviate BLM-induced pulmonary inflammation and fibrosis. MethodsPulmonary fibrosis mouse models were induced by BLM. The IF group underwent 24-h fasting cycles for one week prior and three weeks following BLM administration. Meanwhile, the ad libitum feeding group had unrestricted access to food throughout the experiment. The evaluation focused on lung pathology via histological staining, qPCR analysis of collagen markers, and immune cell profiling through flow cytometry. ResultsIF group significantly reduced inflammation and fibrosis in lung tissues of BLM-induced mice compared to ad libitum feeding group. qPCR results showed IF remarkably decreased the mRNA expression of Col 1a and Col 3a in the lungs of BLM-induced mouse models. IF also reduced the numbers of regulatory T cells (Tregs), T helper 17 (Th17) cells, monocytes, and monocyte-derived alveolar macrophages (MoAMs) in the lung tissues. ConclusionsIF may improve BLM-induced pulmonary fibrosis by decreasing numbers of immune cells including Treg cells, Th17 ​cells, monocytes, and MoAMs in the lungs. This study offers experimental validation for dietary intervention as a viable treatment modality in IPF management.

Immune response regulation by transduced mesenchymal stem cells with decorin gene on bleomycin-induced lung injury, fibrosis, and inflammation.

Pulmonary fibrosis is a pathological hallmark of lung injury. It is an aggressive disease that replaces normal lung parenchyma by fibrotic tissue. The transforming growth factor-beta-mothers against decapentaplegic homolog 3 (TGF-β1-Smad3) signaling pathway plays a key role in regulating lung fibrosis. Decorin (DCN), a small leucine-rich proteoglycan, has a modulatory effect on the immune system by reversibly binding with TGF-β and reducing its bioavailability. Mesenchymal stem cell (MSC) therapy is a new strategy that has an immune-modulatory capacity. The aim of this study was to introduce a new therapeutic approach to harness remodeling in injured lung. Bone marrow MSCs were isolated and transduced by decorin gene. Lung injury was induced by bleomycin and mice were treated with MSCs, MSCs-decorin, and decorin. Then, oxidative stress biomarkers, remodeling biomarkers, bronchoalveolar lavage cells, and histopathology study were conducted. Reduced catalase and superoxide dismutase increased due to treatments. Elevated malondialdehyde, hydroxyproline, TGF-β levels, and polymorphonuclear cells count decreased in the treated groups. Additionally, the histopathology of lung tissues showed controlled inflammation and fibrosis. Transfected decorin gene to MSCs and used cell therapy could control remodeling and bleomycin-induced lung injury.

Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening lung disease with high mortality rates. The limited availability of effective drugs for IPF treatment, coupled with concerns regarding adverse effects and restricted responsiveness, underscores the need for alternative approaches. Kefir peptides (KPs) have demonstrated antioxidative, anti-inflammatory, and antifibrotic properties, along with the capability to modulate gut microbiota. This study aims to investigate the impact of KPs on bleomycin-induced pulmonary fibrosis. MethodsMice were treated with KPs for four days, followed by intratracheal injection of bleomycin for 21 days. Comprehensive assessments included pulmonary functional tests, micro-computed tomography (µ-CT), in vivo image analysis using MMPsense750, evaluation of inflammation- and fibrosis-related gene expression in lung tissue, and histopathological examinations. Furthermore, a detailed investigation of the gut microbiota community was performed using full-length 16 S rRNA sequencing in control mice, bleomycin-induced fibrotic mice, and KPs-pretreated fibrotic mice. ResultsIn KPs-pretreated bleomycin-induced lung fibrotic mice, notable outcomes included the absence of significant bodyweight loss, enhanced pulmonary functions, restored lung tissue architecture, and diminished thickening of inter-alveolar septa, as elucidated by morphological and histopathological analyses. Concurrently, a reduction in the expression levels of oxidative biomarkers, inflammatory factors, and fibrotic indicators was observed. Moreover, 16 S rRNA sequencing demonstrated that KPs pretreatment induced alterations in the relative abundances of gut microbiota, notably affecting Barnesiella_intestinihominis, Kineothrix_alysoides, and Clostridium_viride. ConclusionsKefir peptides exerted preventive effects, protecting mice against bleomycin-induced lung oxidative stress, inflammation, and fibrosis. These effects are likely linked to modifications in the gut microbiota community. The findings highlight the therapeutic potential of KPs in mitigating pulmonary fibrosis and advocate for additional exploration in clinical settings.

GGA (geranylgeranylacetone) ameliorates bleomycin-induced lung inflammation and pulmonary fibrosis by inhibiting apoptosis and oxidative stress.

Fibrosis is a response to ongoing cellular injury, disruption, and tissue remodeling, the pathogenesis of which is unknown, and is characterized by extracellular matrix deposition. The antifibrotic effect of Geranylgeranylacetone (GGA), as an inducer of Heat shock protein 70 (HSP70), in liver, kidney and pulmonary fibrosis has been supported by multiple preclinical evidence. However, despite advances in our understanding, the precise roles of HSP70 in fibrosis require further investigation. The purpose of this study was to investigate whether GGA could participate in the progression of pulmonary fibrosis in mice through apoptosis, oxidative stress and inflammation. B-cell lymphoma-2(Bcl-2) and Bcl2-Associated X (Bax) are two proteins related to apoptosis. Anti-apoptotic factor Bcl-2 and pro-apoptotic factor Bax are often involved in the apoptotic process in the form of dimer. Immunofluorescence and Western blot results showed that bleomycin (BLM) and transforming growth factor-β (TGF-β) inhibited Bcl-2 expression and promoted Bax expression in vitro and in vivo, respectively. In contrast, GGA treatment reverses this change. Reactive oxygen species (ROS), Malondialdehyde (MDA) and superoxide dismutase (SOD) are markers of oxidative stress, which often reflect oxidative injury of cells. The detection of ROS, MDA and SOD expression showed that TGF-β and BLM treatment could significantly promote oxidative stress, while GGA treatment could alleviate oxidative stress damage. In addition, BLM significantly elevated Tumor necrosis factor-α(TNF-α), Interleukin1β (IL-1β) and Interleukin 6 (IL-6), while scutellarin reversed the above alterations except for that of GGA. Taken together, GGA suppressed apoptotic, oxidative stress and inflammation in BLM-induced pulmonary fibrosis.

Alcohol-mediated susceptibility to lung fibrosis is associated with group 2 innate lymphoid cells in mice.

Chronic alcohol ingestion promotes acute lung injury and impairs immune function. However, the mechanisms involved are incompletely understood. Here, we show that alcohol feeding enhances bleomycin-induced lung fibrosis and inflammation via the regulation of type 2 innate immune responses, especially by group 2 innate lymphoid cells (ILC2s). Neuroimmune interactions have emerged as critical modulators of lung inflammation. We found alcohol consumption induced the accumulation of ILC2 and reduced the production of the neuropeptide calcitonin gene-related peptide (CGRP), primarily released from sensory nerves and pulmonary neuroendocrine cells (PNECs). CGRP potently suppressed alcohol-driven type 2 cytokine signals in vivo. Vagal ganglia TRPV1+ afferents mediated immunosuppression occurs through the release of CGRP. Inactivation of the TRPV1 receptor enhanced bleomycin-induced fibrosis. In addition, mice lacking the CGRP receptor had the increased lung inflammation and fibrosis and type 2 cytokine production as well as exaggerated responses to alcohol feeding. Together, these data indicate that alcohol consumption regulates the interaction of CGRP and ILC2, which is a critical contributor of lung inflammation and fibrosis.

Inhibition of Fatty Acid Amide Hydrolase (FAAH) Regulates NF-kb Pathways Reducing Bleomycin-Induced Chronic Lung Inflammation and Pulmonary Fibrosis.

The deadly interstitial lung condition known as idiopathic pulmonary fibrosis (IPF) worsens over time and for no apparent reason. The traditional therapy approaches for IPF, which include corticosteroids and immunomodulatory drugs, are often ineffective and can have noticeable side effects. The endocannabinoids are hydrolyzed by a membrane protein called fatty acid amide hydrolase (FAAH). Increasing endogenous levels of endocannabinoid by pharmacologically inhibiting FAAH results in numerous analgesic advantages in a variety of experimental models for pre-clinical pain and inflammation. In our study, we mimicked IPF by administering intratracheal bleomycin, and we administered oral URB878 at a dose of 5 mg/kg. The histological changes, cell infiltration, pro-inflammatory cytokine production, inflammation, and nitrosative stress caused by bleomycin were all reduced by URB878. Our data clearly demonstrate for the first time that the inhibition of FAAH activity was able to counteract not only the histological alteration bleomycin-induced but also the cascade of related inflammatory events.

Arginine concentration in arterial vs venous blood in a bleomycin-induced lung inflammation model in mice.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

Extracellular superoxide dismutase (EC-SOD) R213G variant reduces mitochondrial ROS and preserves mitochondrial function in bleomycin-induced lung injury: EC-SOD R213G variant and intracellular redox regulation

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the lung and vasculature. A common human single nucleotide polymorphism (SNP) in the matrix binding region of EC-SOD leads to a single amino acid substitution, R213G, and alters EC-SOD tissue binding affinity. The change in tissue binding affinity redistributes EC-SOD from tissue to extracellular fluids. Mice (R213G mice) expressing a knock-in of this EC-SOD SNP exhibit elevated plasma and reduced lung EC-SOD content and activity and are protected against bleomycin-induced lung injury and inflammation. It is unknown how the redistribution of EC-SOD alters site-specific redox-regulated molecules relevant for protection. In this study, we tested the hypothesis that the change in the local EC-SOD content would influence not only the extracellular redox microenvironment where EC-SOD is localized but also protect the intracellular redox status of the lung. Mice were treated with bleomycin and harvested 7 days post-treatment. Superoxide levels, measured by electron paramagnetic resonance (EPR), were lower in plasma and Bronchoalveolar lavage fluid (BALF) cells in R213G mice compared to wild-type (WT) mice, while lung cellular superoxide levels in R213G mice were not elevated post-bleomycin compared to WT mice despite low lung EC-SOD levels. Lung glutathione redox potential (EhGSSG), determined by HPLC and fluorescence, was more oxidized in WT compared to R213G mice. In R213G mice, lung mitochondrial oxidative stress was reduced shown by mitochondrial superoxide level measured by EPR in lung and the resistance to bleomycin-induced cardiolipin oxidation. Bleomycin treatment suppressed mitochondrial respiration in WT mice. Mitochondrial function was impaired at baseline in R213G mice but did not exhibit further suppression in respiration post-bleomycin. Collectively, the results indicate that R213G variant preserves intracellular redox state and protects mitochondrial function in the setting of bleomycin-induced inflammation.

PLG nanoparticles target fibroblasts and MARCO+ monocytes to reverse multiorgan fibrosis.

Systemic sclerosis (SSc) is a chronic, multisystem orphan disease with a highly variable clinical course, high mortality rate, and a poorly understood complex pathogenesis. We have identified an important role for a subpopulation of monocytes and macrophages characterized by surface expression of the scavenger receptor macrophage receptor with collagenous structure (MARCO) in chronic inflammation and fibrosis in SSc and in preclinical disease models. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in topographic proximity to activated myofibroblasts in patients with SSc and in the bleomycin-induced mouse model of SSc. Short-term treatment of mice with a potentially novel nanoparticle, poly(lactic-co-glycolic) acid (PLG), which is composed of a carboxylated, FDA-approved, biodegradable polymer and modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture, PLG nanoparticles inhibited TGF-dependent fibrotic responses in vitro. Thus, MARCO+ monocytes are potent effector cells of skin and lung fibrosis and can be therapeutically targeted in SSc using PLG nanoparticles.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin

PurposeGrowth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice.MethodsWe tested whether MIA-602 (5 μg or vehicle given subcutaneously [SC] on days 1–21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28.ResultsInflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal–regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production.ConclusionsMIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

Local inflammation alters the lung disposition of a drug loaded pegylated liposome after pulmonary dosing to rats

The development of inhalable ‘nanomedicines’ based on biocompatible lipids and polymers is attracting increasing interest worldwide. Our understanding of how pulmonary inflammation impacts on lung distribution and clearance kinetics however, is limited. Similarly, there is limited information on how the inhaled delivery of biocompatible nanomaterials affects existing respiratory disease. We have addressed these knowledge gaps by describing and comparing the pulmonary pharmacokinetic behaviour of a 3H-labelled PEGylated liposome loaded with a model drug (ciprofloxacin) after intratracheal administration to healthy rats and rats with bleomycin-induced lung inflammation by following both 3H label and drug. Cell- and cytokine-based markers of lung inflammation were used to evaluate the response of healthy and inflamed lungs to the liposome. Liposomes were initially cleared more rapidly from inflamed lungs than from healthy lungs, but exhibited similar rates of lung clearance after 3 days. This was interesting given that mucociliary clearance was more efficient from healthy lungs, despite evidence of higher mucus retention in inflamed lungs and reduced association of the liposome with lung tissue. Although the plasma pharmacokinetics of ciprofloxacin did not differ between rats with healthy or inflamed lungs after pulmonary administration, the plasma pharmacokinetics of 3H-phosphatidylcholine suggested higher liposome bioavailability and more prolonged absorption from inflamed lungs. Concentrations of the pro-inflammatory cytokine IL-1β were increased in bronchoalveolar lavage fluid after a single pulmonary dose of liposomes to rats with inflamed lungs, but no other significant changes in lung inflammatory markers were identified in healthy or bleomycin-challenged rats.

Role of phospholipase D in bleomycin-induced mitochondrial reactive oxygen species generation, mitochondrial DNA damage, and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by alveolar epithelial apoptosis, dysregulated repair of epithelial injury, scar formation, and respiratory failure. In this study, we identified phospholipase D (PLD)-generated phosphatidic acid (PA) signaling in the development of pulmonary fibrosis (PF). Of the PLD isoenzymes, the protein expression of PLD2, but not PLD1, was upregulated in lung tissues from IPF patients and bleomycin challenged mice. Both PLD1 (Pld1-/-)- and PLD2 (Pld2-/-)-deficient mice were protected against bleomycin-induced lung inflammation and fibrosis, thereby establishing the role of PLD in fibrogenesis. The role of PLD1 and PLD2 in bleomycin-induced lung epithelial injury was investigated by infecting bronchial airway epithelial cells (Beas2B) with catalytically inactive mutants of PLD (hPLD1-K898R or mPld2-K758R) or downregulation of expression of PLD1 or PLD2 with siRNA. Bleomycin stimulated mitochondrial (mt) superoxide production, mtDNA damage, and apoptosis in Beas2B cells, which was attenuated by the catalytically inactive mutants of PLD or PLD2 siRNA. These results show a role for PLD1 and PLD2 in bleomycin-induced generation of mt reactive oxygen species, mt DNA damage, and apoptosis of lung epithelial cells in mice. Thus, PLD may be a novel therapeutic target in ameliorating experimental PF in mice.

Deficiency of CRTH2, a Prostaglandin D2 Receptor, Aggravates Bleomycin-induced Pulmonary Inflammation and Fibrosis.

Chemoattractant receptor homologous with T-helper cell type 2 cells (CRTH2), a receptor for prostaglandin D2, is preferentially expressed on T-helper cell type 2 lymphocytes, group 2 innate lymphoid cells, eosinophils, and basophils, and elicits the production of type 2 cytokines, including profibrotic IL-13. We hypothesized that lack of CRTH2 might protect against fibrotic lung disease, and we tested this hypothesis using a bleomycin-induced lung inflammation and fibrosis model in CRTH2-deficient (CRTH2-/-) or wild-type BALB/c mice. Compared with wild-type mice, CRTH2-/- mice treated with bleomycin exhibited significantly higher mortality, enhanced accumulation of inflammatory cells 14-21 days after bleomycin injection, reduced pulmonary compliance, and increased levels of collagen and total protein in the lungs. These phenotypes were associated with decreased levels of IFN-γ, IL-6, IL-10, and IL-17A in BAL fluid. Adoptive transfer of splenocytes from wild-type, but not CRTH2-/-, mice 2 days before injection of bleomycin resolved the sustained inflammation as well as the increased collagen and protein accumulation in the lungs of CRTH2-/- mice. We consider that the disease model is driven by γδT cells that express CRTH2; thus, the adoptive transfer of γδT cells could ameliorate bleomycin-induced alveolar inflammation and fibrosis.

A STAT6 Inhibitor AS1517499 Reduces Preventive Effects of Apoptotic Cell Instillation on Bleomycin-Induced Lung Fibrosis by Suppressing PPARγ

Background/Aims: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. Methods: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. Results: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. Conclusion: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Cutting Edge: Anti-TIM-3 Treatment Exacerbates Pulmonary Inflammation and Fibrosis in Mice.

Promising results of immune checkpoint inhibitors have indicated the use of immunotherapy against malignant tumors. However, they cause serious side effects, including autoimmune diseases and pneumonitis. T cell Ig and mucin domain (TIM)-3 is a new candidate immune checkpoint molecule; however, the potential toxicity associated with anti-TIM-3 treatment is unknown. In this study, we investigated the pathological contribution of anti-TIM-3 mAb in a bleomycin-induced lung inflammation and fibrosis model. Anti-TIM-3-treated mice showed more severe inflammation and peribronchiolar fibrosis compared with control IgG-treated mice. Anti-TIM-3 mAb was associated with increased numbers of myofibroblasts, collagen deposition, and TGF-β1 production in lungs. TIM-3 expression was only detected on alveolar macrophages that protect against fibrosis by apoptotic cell clearance. Treatment with anti-TIM-3 mAb inhibited the phagocytic ability of alveolar macrophages in vivo, resulting in the defective clearance of apoptotic cells in lungs. In summary, anti-TIM-3 mAb treatment might cause pneumonitis and it should be used with caution in clinical settings.

The SOD3 R213G Polymorphism Promotes Resolution of Innate Immune Activation and Blocks Induction of miR29b in Bleomycin Induced Lung Inflammation and Fibrosis

A naturally occurring single nucleotide polymorphism (SNP) (R213G) in extracellular superoxide dismutase (SOD3), a key lung and vascular antioxidant enzyme, decreases tissue binding and increases SOD3 in epithelial lining fluid and plasma. Mice harboring the R213G SNP are protected against bleomycin-induced alveolar inflammation and fibrosis. We hypothesized that the redistribution of SOD3 due to the R213G SNP, through site specific changes in the redox environment, modulates key miRNA and mRNA signaling networks responsible for macrophage activation and fibrosis. Plasma, lung and BALF O2•‒ was measured by electron paramagnetic resonance using the CMH spin probe. Bleomycin increased O2•‒ in all three compartments; the increase in O2•‒ was attenuated in the blood of R213G mice. We performed high throughput RNAseq and bioinformatics analyses in lung mRNA and miRNA 7 days post-bleomycin in WT and R213G mice. We identified distinct mRNA and miRNA profiles between strains in response to bleomycin. By pathway analysis, key immune and inflammatory pathways were activated in WT mice but suppressed in R213G mice at 7 days, the timepoint associated with resolution of inflammation in the R213G mice. Predicted upstream regulators included multiple signaling molecules within the TLR4/NF-ks/IKKe/IRF3 pathway. Distinct strain specific miRNA responses were observed, including miR29, a key miRNA implicated in fibrosis, which decreased only in R213G mice. In bone marrow derived macrophages (BMDM) from WT and R213G mice, the response to classic “M1” LPS + IFN polarizing stimuli was similar between strains but attenuated by pretreatment with exogenous extracellular SOD. Using bioinformatics and molecular approaches, we conclude that the redistribution of ecSOD, due to its impact on O2•‒ , can suppress innate immune responses by regulating macrophage plasticity, accounting for the ability of the R213G SNP to protect against bleomycin-induced lung fibrosis. The role of miRNA regulation in this process requires further study.