Airway epithelial cells initiate the allergen response through transglutaminase 2 by inducing IL-33 expression and a subsequent Th2 response

Background

Transglutaminase 2 (TG2) is a post-translational protein-modifying enzyme that catalyzes the transamidation reaction, producing crosslinked or polyaminated proteins. Increased TG2 expression and activity have been reported in various inflammatory conditions, such as rheumatoid arthritis, inflammation-associated pulmonary fibrosis, and autoimmune encephalitis. In particular, TG2 from epithelial cells is important during the initial inflammatory response in the lung. In this study, we evaluated the role of TG2 in the pathogenesis of allergic asthma, particularly whether TG2 affects initial activation signaling leading to Th2 differentiation against antigens.

Methods

We induced allergic asthma by ovalbumin sensitization and intranasal challenge in wild-type (WT) BALB/c and TG2-deficient mice. Broncheoalveolar lavage fluid cells and intracellular cytokine production were analyzed by flow cytometry. Interleukin (IL)-33 and TG2 expression in lung epithelial cells was detected by confocal microscopy.

Results

Airway responsiveness was attenuated in TG2-deficient mice compared to that in the WT control. In addition, recruitment of eosinophils and Th2 and Th17 differentiation decreased in TG2-deficient mice. Treatment with cysteamine, a transglutaminase inhibitor, also reduced airway hypersensitivity, inflammatory cell recruitment, and T helper cell differentiation. TG2-deficient mice showed reduced IL-33 expression following induction of allergic asthma compared to those in the WT control.

Conclusions

We found that pulmonary epithelial cells damaged by allergens triggered TG2-mediated IL-33 expression leading to type 2 responses by recruiting both innate and adaptive arms of the immune system.     

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis

Background

Lung fibrosis is a devastating pulmonary disorder characterized by alveolar epithelial injury, extracellular matrix deposition and scar tissue formation. Due to its potent collagenolytic activity, cathepsin K, a lysosomal cysteine protease is an interesting target molecule with therapeutic potential to attenuate bleomycin-induced pulmonary fibrosis in mice. We here tested the hypothesis that over-expression of cathepsin K in the lungs of mice is protective in bleomycin-induced pulmonary fibrosis.

Methods

Wild-type and cathepsin K overexpressing (cathepsin K transgenic; cath K tg) mice were challenged intratracheally with bleomycin and sacrificed at 1, 2, 3 and 4 weeks post-treatment followed by determination of lung fibrosis by estimating lung collagen content, lung histopathology, leukocytic infiltrates and lung function. In addition, changes in cathepsin K protein levels in the lung were determined by immunohistochemistry, real time RT-PCR and western blotting.

Results

Cathepsin K protein levels were strongly increased in alveolar macrophages and lung parenchymal tissue of mock-treated cathepsin K transgenic (cath K tg) mice relative to wild-type mice and further increased particularly in cath K tg but also wild-type mice in response to bleomycin. Moreover, cath K tg mice responded with a lower collagen deposition in their lungs, which was accompanied by a significantly lower lung resistance (R_L) compared to bleomycin-treated wild-type mice. In addition, cath K tg mice responded with a lower degree of lung fibrosis than wild-type mice, a process that was found to be independent of inflammatory leukocyte mobilization in response to bleomycin challenge.

Conclusion

Over-expression of cathepsin K reduced lung collagen deposition and improved lung function parameters in the lungs of transgenic mice, thereby providing at least partial protection against bleomycin-induced lung fibrosis.     

The Role of Catalase in Pulmonary Fibrosis

Background

Catalase is preferentially expressed in bronchiolar and alveolar epithelial cells, and acts as an endogenous antioxidant enzyme in normal lungs. We thus postulated epithelial damage would be associated with a functional deficiency of catalase during the development of lung fibrosis.

Methods

The present study evaluates the expression of catalase mRNA and protein in human interstitial pneumonias and in mouse bleomycin-induced lung injury. We examined the degree of bleomycin-induced inflammation and fibrosis in the mice with lowered catalase activity.

Results

In humans, catalase was decreased at the levels of activity, protein content and mRNA expression in fibrotic lungs (n = 12) compared to control lungs (n = 10). Immunohistochemistry revealed a decrease in catalase in bronchiolar epithelium and abnormal re-epithelialization in fibrotic areas. In C57BL/6J mice, catalase activity was suppressed along with downregulation of catalase mRNA in whole lung homogenates after bleomycin administration. In acatalasemic mice, neutrophilic inflammation was prolonged until 14 days, and there was a higher degree of lung fibrosis in association with a higher level of transforming growth factor-β expression and total collagen content following bleomycin treatment compared to wild-type mice.

Conclusions

Taken together, these findings demonstrate diminished catalase expression and activity in human pulmonary fibrosis and suggest the protective role of catalase against bleomycin-induced inflammation and subsequent fibrosis.     

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Background

Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47^phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods

Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results

BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47^phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions

These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.     

Secretoglobin 3A2 Exhibits Anti-Fibrotic Activity in Bleomycin-Induced Pulmonary Fibrosis Model Mice

Objective

Secretoglobin (SCGB) 3A2 is a novel lung-enriched cytokine, previously shown to exhibit anti-inflammatory, growth factor, and anti-fibrotic activities. The latter activity was demonstrated using exogenously-administered recombinant SCGB3A2 in the bleomycin (BLM)-induced pulmonary fibrosis model. Whether SCGB3A2 exhibits anti-fibrotic activity in vivo is not known.

Methods

Mice null for the Scgb3a2 gene were subjected to the BLM-induced pulmonary fibrosis model, and the severity of pulmonary fibrosis determined using histological and biochemical methods.

Results

BLM treatment caused weight loss of both Scgb3a2-null and wild-type mice, however, the loss was far more pronounced in BLM-treated Scgb3a2-null than wild-type mice, and the weight of day 21 of BLM-treated Scgb3a2-null mice was about half of that of BLM-treated wild-type mice. Hematoxylin & Eosin, Masson Trichrome, and Sirius Red staining of lung sections, Ashcroft fibrosis scores, hydroxyproline contents, and the levels of mRNAs encoding various collagens demonstrated that BLM-treated Scgb3a2-null mouse lungs had more severe fibrosis than those of wild-type mouse lungs. Total and differential inflammatory cell numbers in bronchoalveolar lavage fluids, and levels of lung mRNAs including those encoding Th2 cytokines such as IL-4 and profibrotic cytokines such as TGFβ were higher in BLM-treated Scgb3a2-null mouse lungs as compared to those of wild-type mouse lungs. In contrast, mRNAs encoding surfactant proteins A, B, C, and D, and SCGB1A1 did not differ between BLM-treated Scgb3a2-null and wild-type mouse lungs.

Conclusion

The role of SCGB3A2 in fibrosis was revisited using Scgb3a2-null mice and littermate controls in the BLM-induced pulmonary fibrosis model. The pulmonary fibrosis in the Scgb3a2-null mice was more severe than the wild-type controls, thus establishing that SCGB3A2 has anti-fibrotic activity in vivo. Importantly, surfactant proteins and SCGB1A1 appear not to be involved in the susceptibility of Scgb3a2-null mice to BLM-induced pulmonary fibrosis.     

The Role of the Receptor for Advanced Glycation End-Products in a Murine Model of Silicosis

Background

The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated.

Methodology/Principal Findings

Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-β levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice.

Conclusions/Significance

Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.     

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis

Background

Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.

Methods

Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog^+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.

Results

Nog^+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.

Conclusions

Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog^+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.     

Interleukin-6 contributes to inflammation and remodeling in a model of adenosine mediated lung injury

Background

Chronic lung diseases are the third leading cause of death in the United States due in part to an incomplete understanding of pathways that govern the progressive tissue remodeling that occurs in these disorders. Adenosine is elevated in the lungs of animal models and humans with chronic lung disease where it promotes air-space destruction and fibrosis. Adenosine signaling increases the production of the pro-fibrotic cytokine interleukin-6 (IL-6). Based on these observations, we hypothesized that IL-6 signaling contributes to tissue destruction and remodeling in a model of chronic lung disease where adenosine levels are elevated.

Methodology/Principal Findings

We tested this hypothesis by neutralizing or genetically removing IL-6 in adenosine deaminase (ADA)-deficient mice that develop adenosine dependent pulmonary inflammation and remodeling. Results demonstrated that both pharmacologic blockade and genetic removal of IL-6 attenuated pulmonary inflammation, remodeling and fibrosis in this model. The pursuit of mechanisms involved revealed adenosine and IL-6 dependent activation of STAT-3 in airway epithelial cells.

Conclusions/Significance

These findings demonstrate that adenosine enhances IL-6 signaling pathways to promote aspects of chronic lung disease. This suggests that blocking IL-6 signaling during chronic stages of disease may provide benefit in halting remodeling processes such as fibrosis and air-space destruction.     

Resistin-like molecule alpha1 (Fizz1) recruits lung dendritic cells without causing pulmonary fibrosis

Background

Resistin-like molecule alpha or found in inflammatory zone protein (Fizz1) is increased in pulmonary epithelial cells and also in limited amounts by other lung cells during various lung injuries and fibrosis. However, the direct role of Fizz1 produced in the pulmonary epithelium has not been determined.

Methods

Fizz1 Transgenic mice (CCSP/Fizz1) were generated that overexpress Fizz1 in the lung epithelium under the control of a doxycycline (Dox) inducible lung epithelial cell specific promoter Scgb1a1 (Clara cell secretory protein, CCSP). Histology and FACS analysis of lung cells were used to identify the direct effects of Fizz1 in the transgenic mice (Dox treated) when compared with control (CCSP/-) mice. Intratracheal bleomycin sulfate or silica in saline and saline alone were used to study the role of Fizz1 during bleomycin- and silica-induced pulmonary fibrosis in CCSP/Fizz1 and CCSP/- mice. Weight change, pulmonary inflammation, and fibrosis were assessed 10 days post bleomycin or 28 days post silica challenge.

Results

When CCSP/Fizz1 mice were fed Dox food, elevated Fizz1 protein was detected in lung homogenates by western blot. Lungs of mice in which Fizz1 was induced in the epithelium contained increased lung cells staining for CD11c and F4/80 by FACS analysis consistent with increased dendritic cells however, no changes were observed in the percentage of interstitial macrophages compared to CCSP/- controls. No significant changes were found in the lung histology of CCSP/Fizz1 mice after up to 8 weeks of overexpression compared to CCSP/- controls. Overexpression of Fizz1 prior to challenge or following challenge with bleomycin or silica did not significantly alter airway inflammation or fibrosis compared to control mice.

Conclusions

The current study demonstrates that epithelial cell derived Fizz1 is sufficient to increase the bone-marrow derived dendritic cells in the lungs, but it is not sufficient to cause lung fibrosis or alter chemical or particle-induced fibrosis.     

Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice

Background

Antiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis.

Methods

C57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-β1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis.

Results

Severe lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-β1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor.

Conclusions

AF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.     

Rac2 is involved in bleomycin-induced lung inflammation leading to pulmonary fibrosis

Background

Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis.

Methods

To determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2 ^-/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content.

Results

BLM-treated rac2 ^-/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2 ^-/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2 ^-/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2 ^-/- and WT and mice that survived to day 21.

Conclusion

Rac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.     

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without Th2 associated vaccine-enhanced disease

Background

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960''s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.

Principal Findings

In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.

Conclusions

These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.     

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Background

Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods

The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results

We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt⁺ γδ T cells and to a lesser extent by CD4αβ⁺ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions

Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.     

Angiotensin II type 2 receptor antagonist reduces bleomycin-induced pulmonary fibrosis in mice

Background

The role of angiotensin II type 2 receptor (AT2) in pulmonary fibrosis is unknown. To evaluate the influence of angiotensin II type 1 receptor (AT1) and AT2 antagonists in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis.

Methods

We examined effects of the AT1 antagonist (AT1A) olmesartan medoxomil (olmesartan) and the AT2 antagonist (AT2A) PD-123319 on BLM-induced pulmonary fibrosis, which was evaluated by Ashcroft''s pathological scoring and hydroxyproline content of lungs. We also analyzed the cellular composition and cytokine levels in bronchoalveolar lavage fluid (BALF).

Results

With olmesartan, the lung fibrosis score and hydroxyproline level were significantly reduced, and lymphocyte and neutrophil counts and tumor necrosis factor (TNF)-α levels in BALF were reduced on day 7. On day 14, macrophage and lymphocyte counts in BALF were reduced, accompanied by a reduction in the level of transforming growth factor (TGF)-β₁. With PD-123319, the lung fibrosis score and hydroxyproline level were reduced. On day 7, macrophage, lymphocyte, and neutrophil counts in BALF were reduced, accompanied by reductions in TNF-α and monocyte chemoattractant protein (MCP)-1 levels. On day 14, macrophage, lymphocyte, and neutrophil counts in BALF were also reduced, accompanied by a reduction in the level of macrophage inflammatory protein (MIP)-2 level but not TGF-β₁.

Conclusion

Both AT1 and AT2 are involved in promoting interstitial pneumonia and pulmonary fibrosis via different mechanisms of action.     

Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

Background

Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH). Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6).

Methods

To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6^-/-) and wild-type (IL-6^+/+) mice exposed to hypoxia for 2 weeks.

Results

Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6^-/- mice compared to wild-type controls after 2 weeks'' hypoxia, although the pressure response to acute hypoxia was similar in IL-6^+/+ and IL-6^-/- mice. Hypoxia exposure of IL-6^+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer) mRNA levels increased after 1 and 2 weeks'' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs) and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6^-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines.

Conclusion

These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.     

HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic

Background

Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia.

Methods

Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later.

Results

In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis.

Conclusions

HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.     

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

Background

A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.

Methods

Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.

Results

Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.

Conclusions

Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.     

Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis

Background

Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).

Methods

Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.

Results

A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.

Conclusion

Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.     

Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice

Background

Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.

Methods

Wild-type, single NOS knockout and triple NOS knockout (n/i/eNOS^−/−) mice were administered bleomycin (BLM) intraperitoneally at a dose of 8.0 mg/kg/day for 10 consecutive days. Two weeks after the end of the procedure, the fibrotic and inflammatory changes of the lung were evaluated. In addition, we evaluated the effects of long-term treatment with isosorbide dinitrate, a NO donor, on the n/i/eNOS^−/− mice with BLM-induced pulmonary fibrosis.

Results

The histopathological findings, collagen content and the total cell number in bronchoalveolar lavage fluid were the most severe/highest in the n/i/eNOS^−/− mice. Long-term treatment with the supplemental NO donor in n/i/eNOS^−/− mice significantly prevented the progression of the histopathological findings and the increase of the collagen content in the lungs.

Conclusions

These results provide the first direct evidence that a lack of all three NOS isoforms led to a deterioration of pulmonary fibrosis in a BLM-treated murine model. We speculate that the entire endogenous NO and NOS system plays an important protective role in the pathogenesis of pulmonary fibrosis.