Inflammation and tissue remodeling with pathologic fibrosis are common consequences of Th2 responses in the lung and other organs. Interleukin (IL)-13 and transforming growth factor-β1 (TGF-β1) are frequently coexpressed in these responses and are believed to play important roles in the pathogenesis of Th2-induced pathologies. To shed light on the mechanisms of these responses, overexpression transgenic approaches were used to selectively target each of these cytokines to the murine lung. IL-13 proved to be a potent stimulator of eosinophilic inflammation, mucus metaplasia, tissue fibrosis, and alveolar remodeling. CC chemokines, specific chemokine receptors (CCR2, CCR1), adenosine metabolism, vascular endothelial growth factor, and IL-11 contributed to the genesis of these responses. IL-13 also induced tissue fibrosis, at least in part, via its ability to induce and activate TGF-β1. In the TGF-β1 transgenic mouse, epithelial apoptosis preceded the onset of tissue fibrosis and alveolar remodeling. In addition, chemical (Z-VAD-fmk) and genetic (null mutations of early growth response gene 1) interventions blocked apoptosis and ameliorated TGF-β1–induced fibrosis and alveolar restructuring. These studies define an IL-13–TGF-β1 pathway of tissue remodeling that regulates inflammation, mucus metaplasia, apoptosis, vascular responses, and fibrosis in the lung. They also highlight the intimate relationship between apoptosis and fibrosis induced by TGF-β1. By defining the complexities of this pathway, these studies highlight sites at which therapies can be directed to control these important responses.
asthma; fibrosis; interleukin-13; transforming growth factor-β; 1; transgenic
Pulmonary emphysema is characterized by alveolar destruction and persistent inflammation of the airways. Although IL-17A contributes to many chronic inflammatory diseases, it’s role in the inflammatory response of elastase-induced emphysema remains unclear.
In a model of elastase-induced pulmonary emphysema we examined the response of IL-17A-deficient mice, monitoring airway inflammation, static compliance, lung histology and levels of neutrophil-related chemokine and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) fluid.
Wild-type mice developed emphysematous changes in the lung tissue on day 21 after elastase treatment, whereas emphysematous changes were decreased in IL-17A-deficient mice compared to wild-type mice. Neutrophilia in BAL fluid, seen in elastase-treated wild-type mice, was reduced in elastase-treated IL-17A-deficient mice on day 4, associated with decreased levels of KC, MIP-2 and IL-1 beta. Elastase-treated wild-type mice showed increased IL-17A levels as well as increased numbers of IL-17A+ CD4 T cells in the lung in the initial period following elastase treatment.
These data identify the important contribution of IL-17A in the development of elastase-induced pulmonary inflammation and emphysema. Targeting IL-17A in emphysema may be a potential therapeutic strategy for delaying disease progression.
IL-17; Elastase; Emphysema; Chronic obstructive pulmonary disease
Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation, alveolar destruction, and airway and vascular remodeling. However, the mechanisms that lead to these diverse alterations have not been defined.
Objectives: We hypothesized that IL-18 plays a central role in the pathogenesis of these lesions.
Methods: We generated and characterized lung-specific, inducible IL-18 transgenic mice.
Measurements and Main Results: Here we demonstrate that the expression of IL-18 in the mature murine lung induces inflammation that is associated with the accumulation of CD4+, CD8+, CD19+, and NK1.1+ cells; emphysema; mucus metaplasia; airway fibrosis; vascular remodeling; and right ventricle cardiac hypertrophy. We also demonstrate that IL-18 induces type 1, type 2, and type 17 cytokines with IFN-γ–inhibiting macrophage, lymphocyte, and eosinophil accumulation while stimulating alveolar destruction and genes associated with cell cytotoxicity and IL-13 and IL-17A inducing mucus metaplasia, airway fibrosis, and vascular remodeling. We also highlight interactions between these responses with IL-18 inducing IL-13 via an IL-17A–dependent mechanism and the type 1 and type17/type 2 responses counterregulating each another.
Conclusions: These studies define the spectrum of inflammatory, parenchymal, airway, and vascular alterations that are induced by pulmonary IL-18; highlight the similarities between these responses and the lesions in COPD; and define the selective roles that type 1, type 2, and type 17 responses play in the generation of IL-18–induced pathologies.
IL-18; chronic obstructive pulmonary disease; airway fibrosis; mucus metaplasia; vascular remodeling
Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2′-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2′-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.
eosinophil; asthma; emphysema; alveolar macrophage; adenosine deaminase
Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.
Chronic inflammation containing CD8+ lymphocytes, neutrophils, and macrophages, and pulmonary emphysema coexist in lungs from patients with chronic obstructive pulmonary disease. Although this inflammatory response is believed to cause the remodeling that is seen in these tissues, the mechanism(s) by which inflammation causes emphysema have not been defined. Here we demonstrate that interferon γ (IFN-γ), a prominent product of CD8+ cells, causes emphysema with alveolar enlargement, enhanced lung volumes, enhanced pulmonary compliance, and macrophage- and neutrophil-rich inflammation when inducibly targeted, in a transgenic fashion, to the adult murine lung. Prominent protease and antiprotease alterations were also noted in these mice. They included the induction and activation of matrix metalloproteinase (MMP)-12 and cathepsins B, H, D, S, and L, the elaboration of MMP-9, and the selective inhibition of secretory leukocyte proteinase inhibitor. IFN-γ causes emphysema and alterations in pulmonary protease/antiprotease balance when expressed in pulmonary tissues.
chronic obstructive pulmonary disease; matrix metalloproteinase; cathepsin; neutrophil; secretory leukocyte proteinase inhibitor
Infection with the parasitic helminth Schistosoma mansoni causes significant liver fibrosis and extracellular matrix (ECM) remodeling. Matrix metalloproteinases (MMP) are important regulators of the ECM by regulating cellular inflammation, extracellular matrix deposition, and tissue reorganization. MMP12 is a macrophage-secreted elastase that is highly induced in the liver and lung in response to S. mansoni eggs, confirmed by both DNA microarray and real-time PCR analysis. However, the function of MMP12 in chronic helminth-induced inflammation and fibrosis is unclear. In this study, we reveal that MMP12 acts as a potent inducer of inflammation and fibrosis after infection with the helminth parasite S. mansoni. Surprisingly, the reduction in liver and lung fibrosis in MMP12-deficient mice was not associated with significant changes in cytokine, chemokine, TGF-β1, or tissue inhibitors of matrix metalloproteinase expression. Instead, we observed marked increases in MMP2 and MMP13 expression, suggesting that Mmp12 was promoting fibrosis by limiting the expression of specific ECM-degrading MMPs. Interestingly, like MMP12, MMP13 expression was highly dependent on IL-13 and type II–IL-4 receptor signaling. However, in contrast to MMP12, expression of MMP13 was significantly suppressed by the endogenous IL-13 decoy receptor, IL-13Rα2. In the absence of MMP12, expression of IL-13Rα2 was significantly reduced, providing a possible explanation for the increased IL-13-driven MMP13 activity and reduced fibrosis. As such, these data suggest important counter-regulatory roles between MMP12 and ECM-degrading enzymes like MMP2, MMP9, and MMP13 in Th2 cytokine-driven fibrosis.
Cathepsin K, a cysteine protease predominantly expressed in osteoclasts, is a major drug target for the treatment of osteoporosis. Recent findings, however, indicate that cathepsin K is also involved in non-skeletal metabolism. The development of fibrotic phenotypes in lung and skin is a concern for cathepsin K inhibitors presently evaluated in clinical trials. Cathepsin K is expressed in lung tissue and has been implicated in lung fibrosis. However, little is known about the role of cathepsin K in airway development and its effect on TGF-β1 degradation.
We investigated the effects of cathepsin K-deficiency on alterations in airway integrity, extracellular matrix composition, and TGF-β1 expression and degradation. Lung homogenates of wild-type and cathepsin K-deficient mice were used to evaluate their contents of collagen, glycosaminoglycans, and TGF-β1. The accessibility of TGF-β1 to cathepsin K-mediated degradation was determined in vitro and lung fibroblast proliferations in wild-type and cathepsin K-deficient cells were evaluated.
Lung airway cathepsin K expression in wild-type mice remained constant between 1 and 6 months of age and the airway integrity was maintained. In contrast, after 2 months of age, all Ctsk-/- mice demonstrated increased airway epithelium thickness by 16-28%, a lower structural airway integrity (1-2 score units lower), elevated cytokeratin expression of 12%, increased α-actin and vimentin expression by 50% and 70%, increased area of smooth muscle cells by 15%, elevated hydroxyproline and GAGs content by 20% and 25%, and increased TGF-β1 expression by 25%. TGF-β1 proved an efficient substrate of cathepsin K and TGF-β1 protein content in lung was increased by a potent cathepsin inhibitor. Lung fibroblasts from Ctsk-/- mice after TGF-β1 treatment showed increased proliferation rates, increased levels of TGF-β1 by 30%, and increased ECM secretion.
This study suggests that airway development is partly regulated by cathepsin K and that its expression contributes to the maintenance of the airway structural integrity. The anticipated use of therapeutic cathepsin K inhibitors needs to take potential changes in human lungs into consideration.
lung airway; cathepsin K; TGF-β1; extracellular matrix; protease inhibitors
To identify the physiological role of Hck, a functionally redundant member of the Src family of tyrosine kinases expressed in myelomonocytic cells, we generated HckF/F “knock-in” mice which carry a targeted tyrosine (Y) to phenylalanine (F) substitution of the COOH-terminal, negative regulatory Y499-residue in the Hck protein. Unlike their Hck−/− “loss-of-function” counterparts, HckF/F “gain-of-function” mice spontaneously acquired a lung pathology characterized by extensive eosinophilic and mononuclear cell infiltration within the lung parenchyma, alveolar airspaces, and around blood vessels, as well as marked epithelial mucus metaplasia in conducting airways. Lungs from HckF/F mice showed areas of mild emphysema and pulmonary fibrosis, which together with inflammation resulted in altered lung function and respiratory distress in aging mice. When challenged transnasally with lipopolysaccharide (LPS), HckF/F mice displayed an exaggerated pulmonary innate immune response, characterized by excessive release of matrix metalloproteinases and tumor necrosis factor (TNF)α. Similarly, HckF/F mice were highly sensitive to endotoxemia after systemic administration of LPS, and macrophages and neutrophils derived from HckF/F mice exhibited enhanced effector functions in vitro (e.g., nitric oxide and TNFα production, chemotaxis, and degranulation). Based on the demonstrated functional association of Hck with leukocyte integrins, we propose that constitutive activation of Hck may mimic adhesion-dependent priming of leukocytes. Thus, our observations collectively suggest an enhanced innate immune response in HckF/F mice thereby skewing innate immunity from a reversible physiological host defense response to one causing irreversible tissue damage.
knock-in mutation; LPS; endotoxemia; macrophage; neutrophil
IL-18 plays a key role in the pathogenesis of pulmonary inflammatory diseases including pulmonary infection, pulmonary fibrosis, lung injury and chronic obstructive pulmonary disease (COPD). However, it is unknown whether IL-18 plays any role in the pathogenesis of asthma. We hypothesized that overexpression of mature IL-18 protein in the lungs may exacerbate disease activities of asthma. We established lung-specific IL-18 transgenic mice on a Balb/c genetic background. Female mice sensitized– and challenged– with antigen (ovalbumin) were used as a mouse asthma model. Pulmonary inflammation and emphysema were not observed in the lungs of naïve transgenic mice. However, airway hyperresponsiveness and airway inflammatory cells accompanied with CD4+ T cells, CD8+ T cells, eosinophils, neutrophils, and macrophages were significantly increased in ovalbumin-sensitized and challenged transgenic mice, as compared to wild type Balb/c mice. We also demonstrate that IL-18 induces IFN-γ, IL-13, and eotaxin in the lungs of ovalbumin-sensitized and challenged transgenic mice along with an increase in IL-13 producing CD4+ T cells. Treatment with anti-CD4 monoclonal antibody or deletion of the IL-13 gene improves ovalbumin-induced airway hyperresponsiveness and reduces airway inflammatory cells in transgenic mice. Overexpressing the IL-18 protein in the lungs induces type 1 and type 2 cytokines and airway inflammation, and results in increasing airway hyperresponsiveness via CD4+ T cells and IL-13 in asthma.
BACKGROUND--Emphysema is currently defined as "a condition of the lung characterised by abnormal, permanent enlargement of the airspaces distal to the terminal bronchiole, accompanied by destruction of their walls, and without obvious fibrosis." The functional and morphological changes that occur in emphysema have largely been attributed to changes in alveolar elastin rather than in collagen. A study was performed to determine whether the amount of collagen in the alveolar wall changes with age in the lungs of non-smokers and of smokers with different types of macroscopically defined emphysema in relation to a microscopic measurement of lung structure. METHODS--Total alveolar wall collagen was measured (as hydroxyproline) in known volumes of distended lung tissue (by reverse phase high pressure liquid chromatography) in the lungs of non-smokers (n = 23) and in regions sampled away from emphysematous lesions in the lungs of 36 smokers (four with no emphysema, 13 with centriacinar emphysema (CAE), nine with panacinar emphysema (PAE), and 10 with a mixture (MIX) of both PAE and CAE). Mean lung airspace wall surface area per unit volume (AWUV) was calculated from at least six random blocks per lung and on histological sections immediately adjacent to those prepared for collagen measurement with a rapid scanning device (fast interval processor). RESULTS--In non-smokers there was no significant correlation between the amount of collagen in the alveolar wall tissue and either mean lung AWUV or increasing patient age when amounts of collagen were expressed either per unit volume of distended lung (40 mm3 sample) or per unit surface area of airspace wall tissue. Smokers without emphysema had similar amounts of collagen to non-smokers. Lungs with PAE and MIX, but not CAE alone, contained significantly more collagen than normal when expressed per unit volume of airspace wall tissue whereas all groups, including CAE, contained significantly raised amounts of collagen when expressed per unit surface area. CONCLUSIONS--There is no significant age related change in the collagen content of the lungs of non-smokers which suggests that, as AWUV is lost with age, the main collagenous framework is maintained. However, in smokers with emphysema there is a loss of airspace wall tissue in regions remote from the macroscopic lesions that is accompanied by a net increase in collagen mass. The greater accumulation of collagen in MIX lungs than in CAE lungs suggests a greater degree of structural damage, indicative of an alternative pathogenetic mechanism operating between the different types of emphysema. Our results suggest an active alveolar wall fibrosis in emphysema as a consequence of cigarette smoking. It is suggested that the definition of emphysema may require further revision to include such change.
Obliterative bronchiolitis (OB) is a major cause of allograft dysfunction after lung transplantation and is thought to result from immunologically mediated airway epithelial destruction and luminal fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the regulation of lung inflammation, airway epithelial repair, and extracellular matrix remodeling and therefore may participate in the pathogenesis of OB. The goals of this study were to determine the expression profiles of MMPs and TIMPs and the role of TIMP-1 in the development of airway obliteration using the murine heterotopic tracheal transplant model of OB. We demonstrate the selective induction of MMP-3, MMP-9, MMP-12, and TIMP-1 in a temporally restricted manner in tracheal allografts compared with isografts. In contrast, the expression of MMP-7, TIMP-2, and TIMP-3 was decreased in allografts relative to isografts during the period of graft rejection. TIMP-1 protein localized to epithelial, mesenchymal, and inflammatory cells in the tracheal grafts in a temporally and spatially restricted manner. Using TIMP-1–deficient mice, we demonstrate that the absence of TIMP-1 in the donor trachea or the allograft recipient reduced luminal obliteration and increased re-epithelialization in the allograft compared with wild-type control at 28 d after transplantation. Our findings provide direct evidence that TIMP-1 contributes to the development of airway fibrosis in the heterotopic tracheal transplant model, and suggest a potential role for this proteinase inhibitor in the pathogenesis of OB in patients with lung transplant.
heterotopic tracheal transplant; matrix metalloproteinase; obliterative bronchiolitis; tissue inhibitor of metalloproteinase
In lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue-resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process.
Short term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and brochoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU+ and BrdU- cells were characterized by hematopoietic (CD45+), pluripotency (TTF1+, Oct3/4+, SSEA-3+, SSEA-4+, Sca1+, Lin-, CD34+, CD31+), and lung lineage-specific (SPC+, AQP-5+, CC-10+) markers. Clonogenic potential of LRCs were measured by CFU-c assays.
STA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21.LTA- Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU+ (LRC) were the putative stem cells. BrdU+CD45+ cells increased by 0.7-fold and SPC+CC10+ bronchoalveolar stem cells (BASC), decreased by ~40-fold post-bleomycin. BrdU+VEGF+ cells decreased by 1.8-fold while BrdU-VEGF+ cells increased 4.6-fold. Most BrdU- cells were CD45-. BrdU- BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU+ cells remained similar in control and bleomycin-treated lungs.
STA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/- but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) - significance is unknown. LTA- The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF+ LRC may indicate a signal for progenitor mobilization from niches. BrdU- BASC population may be a small quiescent population that remains as a reserve for more severe lung injury. Increase in VEGF+ non-LRC may indicate a checkpoint to counterbalance the mobilization of VEGF+ cells from the stem cell niche.
Rationale: There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of α1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke–induced emphysema.
Objectives: We propose that, in addition to its antielastolytic effects, α1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis.
Methods, Measurements, and Main Results: Transduction of human α1-antitrypsin via replication-deficient adeno-associated virus attenuated airspace enlargement and emphysema caused by inhibition of vascular endothelial growth factor (VEGF) receptors with SU5416 in mice, a model of apoptosis-dependent emphysema lacking neutrophilic inflammation. The overexpressed human serine protease inhibitor accumulated in lung cells and suppressed caspase-3 activation and oxidative stress in lungs treated with the VEGF blocker or with VEGF receptor-1 and -2 antibodies. Similar results were obtained in SU5416-treated rats given human α1-antitrypsin intravenously.
Conclusions: Our findings suggest that inhibition of structural alveolar cell apoptosis by α1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of α1-antitrypsin.
antiprotease; caspase; chronic obstructive pulmonary disease; oxidative stress; serpin
Inhalation of antigen in immunized mice induces an infiltration of eosinophils into the airways and increased bronchial hyperreactivity as are observed in human asthma. We employed a model of late-phase allergic pulmonary inflammation in mice to address the role of leukotrienes (LT) in mediating airway eosinophilia and hyperreactivity to methacholine. Allergen intranasal challenge in OVA-sensitized mice induced LTB4 and LTC4 release into the airspace, widespread mucus occlusion of the airways, leukocytic infiltration of the airway tissue and broncho-alveolar lavage fluid that was predominantly eosinophils, and bronchial hyperreactivity to methacholine. Specific inhibitors of 5- lipoxygenase and 5-lipoxygenase-activating protein (FLAP) blocked airway mucus release and infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation. The role of leukotrienes or eosinophils in mediating airway hyperresponsiveness to aeroallergen could not be established, however, in this murine model.
Siglec-F is a sialic acid-binding Ig superfamily receptor that is highly expressed on eosinophils. We have investigated whether administration of an anti-Siglec-F Ab to OVA-challenged wild-type mice would reduce levels of eosinophilic inflammation and levels of airway remodeling. Mice sensitized to OVA and challenged repetitively with OVA for 1 mo who were administered an anti-Siglec-F Ab had significantly reduced levels of peribronchial eosinophilic inflammation and significantly reduced levels of subepithelial fibrosis as assessed by either trichrome staining or lung collagen levels. The anti-Siglec-F Ab reduced the number of bone marrow, blood, and tissue eosinophils, suggesting that the anti-Siglec-F Ab was reducing the production of eosinophils. Administration of a F(ab′)2 fragment of an anti-Siglec-F Ab also significantly reduced levels of eosinophilic inflammation in the lung and blood. FACS analysis demonstrated increased numbers of apoptotic cells (annexin V+/CCR3+ bronchoalveolar lavage and bone marrow cells) in anti-Siglec-F Ab-treated mice challenged with OVA. The anti-Siglec-F Ab significantly reduced the number of peribronchial major basic protein+/TGF-β+ cells, suggesting that reduced levels of eosinophil-derived TGF-β in anti-Siglec-F Ab-treated mice contributed to reduced levels of peribronchial fibrosis. Administration of the anti-Siglec-F Ab modestly reduced levels of periodic acid-Schiff-positive mucus cells and the thickness of the smooth muscle layer. Overall, these studies suggest that administration of an anti-Siglec-F Ab can significantly reduce levels of allergen-induced eosinophilic airway inflammation and features of airway remodeling, in particular subepithelial fibrosis, by reducing the production of eosinophils and increasing the number of apoptotic eosinophils in lung and bone marrow.
As passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model, in which ovalbumin (OVA) + ETS induce significantly increased levels of eosinophilic airway inflammation and remodeling compared to either stimulus alone, to determine whether a Toll-like receptor-9 (TLR-9) agonist could reduce levels of airway inflammation, airway remodeling and airway hyperreactivity (AHR).
Mice treated with or without a TLR-9 agonist were sensitized to OVA and challenged with OVA + ETS for 1 month. AHR to methacholine was assessed in intubated and ventilated mice. Lung Th2 cytokines and TGF-β1 were measured by ELISA. Lungs were processed for histology and immunohistology to quantify eosinophils, mucus, peribronchial fibrosis and smooth muscle changes using image analysis.
Administration of a TLR-9 agonist to mice coexposed to chronic ETS and chronic OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial fibrosis, the thickness of the peribronchial smooth muscle layer, and AHR. The reduced airway remodeling in mice treated with the TLR-9 agonist was associated with significantly reduced numbers of peribronchial MBP+ and peribronchial TGF-β1+ cells, and with significantly reduced levels of lung Th2 cytokines [interleukin-5 and interleukin-13] and TGF-β1.
These studies demonstrate that TLR-9-based therapies inhibit airway inflammation, remodeling and AHR in mice coexposed to ETS and allergen who exhibit enhanced airway inflammation and remodeling.
Toll-like receptor-9; Airway hyperreactivity; Airway inflammation; Airway remodeling; Eosinophils
Eosinophil and mast cell infiltrations are consistent findings in nasal polyp tissue. Previous studies have shown that matrix metalloproteinases (MMPs) may be involved in eosinophil infiltration in airway mucosa of asthmatic patients, and that transforming growth factor-beta1 (TGF-beta1) induces extracellular matrix deposition in nasal polyp tissue. The aim of this study was to evaluate the role of MMPs and tissue-inhibitor of metalloproteinase-1 (TIMP-1) in association with TGF-beta1, eosinophils and mast cell activation in nasal polyp tissue. Nasal polyp tissues from 20 patients who underwent polypectomies were collected and prepared into tissue homogenate. Eosinophil cationic protein (ECP) and tryptase levels were measured by CAP system (Pharmacia, Sweden). MMP-2, MMP-9, TIMP-1 and TGF-beta1 levels were measured by enzyme-liked immunosorbent assay. MMP-2 was the predominant form of MMPs, followed by MMP-9 and TIMP-1. There were significant correlations between ECP, and MMP-9, MMP-2, TGF-beta1 and tryptase, but not with TIMP-1. Significant correlations were noted between tryptase, and MMP-2, MMP-9, and TGF-beta1, but not with TIMP-1. Close correlations were noted between TGF-beta1, and MMP-9 and MMP-2, but not with TIMP-1. MMP-2, MMP-9, and TGF-beta1 may contribute to eosinophil and mast cell migrations into nasal polyp tissue.
IL-13 potently stimulates eosinophilic and lymphocytic inflammation and alveolar remodeling in the lung, effects that depend on the induction of various matrix metalloproteinases (MMPs). Here, we compared the remodeling and inflammatory effects of an IL-13 transgene in lungs of wild-type, MMP-9–deficient, or MMP-12–deficient mice. IL-13–induced alveolar enlargement, lung enlargement, compliance alterations, and respiratory failure and death were markedly decreased in the absence of MMP-9 or MMP-12. Moreover, IL-13 potently induced MMPs-2, -12, -13, and -14 in the absence of MMP-9, while induction of MMPs-2, -9, -13, and -14 by IL-13 was diminished in the absence of MMP-12. A deficiency in MMP-9 did not alter eosinophil, macrophage, or lymphocyte recovery, but increased the recovery of total leukocytes and neutrophils in bronchoalveolar lavage (BAL) fluids from IL-13 transgenic mice. In contrast, a deficiency in MMP-12 decreased the recovery of leukocytes, eosinophils, and macrophages, but not lymphocytes or neutrophils. These studies demonstrate that IL-13 acts via MMPs-9 and -12 to induce alveolar remodeling, respiratory failure, and death and that IL-13 induction of MMPs-2, -9, -13, and -14 is mediated at least partially by an MMP-12–dependent pathway. The also demonstrate that MMPs-9 and -12 play different roles in the generation of IL-13–induced inflammation, with MMP-9 inhibiting neutrophil accumulation and MMP-12 contributing to the accumulation of eosinophils and macrophages.
The mechanisms that initiate allergic lung inflammation are relevant to expression of diseases such as asthma, but the factors underlying resolution of inflammation are equally important. Previously, we demonstrated the importance of matrix metalloproteinase 2 (MMP2) for airway egression of lung eosinophils, a critical anti-inflammatory mechanism without which mice are rendered highly susceptible to lethal asphyxiation. Here we show that leukocyte MMP9 is the dominant airway MMP controlling inflammatory cell egression. The allergic lung phenotype of MMP9−/− mice was similar to WT and was not altered by concomitant deletion of the MMP2 gene (double knockout; dko). However, inflammatory cells accumulated aberrantly in the lungs of allergen-challenged MMP9−/− and dko mice and fewer eosinophils and neutrophils were present in bronchoalveolar lavage. These aberrant cellular trafficking patterns were explained by disruption of transepithelial chemokine gradients, in MMP2−/− mice affecting only eotaxin (CCL11), but in MMP9−/− and dko mice involving eotaxin, MARC (CCL7), and TARC (CCL17). Thus, by establishing multiple transepithelial chemokine gradients, MMP9 is broadly implicated in the resolution of allergic inflammation, an essential protective mechanism that overlaps with a more limited role played by MMP2.
chemotaxis; cytokines; gelatinase B; matrix metalloproteinase-9
Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 in Porphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8−/−) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses. P. gingivalis infection induced accelerated site-specific alveolar bone loss in both MMP8−/− and WT mice relative to uninfected mice. The most extensive bone degradation took place in the P. gingivalis-infected MMP8−/− group. Surprisingly, MMP-8 significantly attenuated (P < 0.05) P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss in P. gingivalis-infected MMP8−/− and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, the MMP8−/− mice underwent a shift toward a smaller HDL/VLDL particle sizes. P. gingivalis infection increased the HDL/VLDL particle size in the MMP8−/− mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response to P. gingivalis were significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially in MMP8−/− mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.
Rationale: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model.
Objectives: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone.
Methods: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14–73 and montelukast or dexamethasone or placebo from Days 73–163.
Measurements and Main Results: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration.
Conclusions: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.
eosinophils; fibrosis; mucus; smooth muscle
Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A3 adenosine receptor (A3R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A3R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A3R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A3R-deficient mice exposed to bleomycin, an observation suggesting that the A3R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A3R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-β1 expression, but not a concomitant increase in TGF-β1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-β1, in alveolar macrophages and airway mast cells in the lungs of A3R-deficient mice. Together, these results suggest that the A3R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.
pulmonary fibrosis; adenosine receptors; inflammation; eosinophil; extracellular matrix
Periostin was originally identified as a secreted factor during screening of a mouse osteoblastic library. In a recent study, periostin was found to directly regulate eosinophil accumulation in allergic mucosal inflammation. Chronic eosinophilic inflammation is related to the development of remodeling. The present study examined the expression of periostin and evaluated its role in the inflammatory process and remodeling associated with allergic rhinitis.
A murine model of allergic rhinitis was established in periostin knockout mice. We analyzed the expression of periostin, manifestation of nasal symptoms, eosinophilic inflammation, and subepithelial fibrosis as well as the expression of MMP-2, TIMP-1, and type 1 collagen in nasal tissue.
Periostin was mainly distributed in the subepithelial tissue of the nasal mucosa. The subepithelial tissue was thinner in the knockout group than in the control group. No differences in the expression of MMP-2 or TIMP-1 were found in the knockout group. However, after a month of allergen challenge, type I collagen in the nasal tissue was lower in the knockout group than in the control group. The number of eosinophils and the symptom score were also lower in the knockout group.
Periostin is expressed in nasal tissues of murine models of allergic rhinitis. Periostin deficiency may affect the remodeling of nasal tissue with reduced subepithelial fibrosis, and lead to less eosinophilic inflammation.
Airway remodeling; allergy; eosinophils; mice; periostin protein; rhinitis
Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.