As an E3 ubiquitin ligase and a molecular adaptor, Cbl-b controls the activation threshold of the antigen receptor and negatively regulates CD28 co-stimulation, functioning as an intrinsic mediator of T cell anergy that maintains tolerance. However, the role of Cbl-b in the airway immune response to aeroallergens is unclear.
To determine the contribution of Cbl-b in tolerance to aeroallergens, we examined ovalbumin (OVA)-induced lung inflammation in Cbl-b deficient mice.
Cbl-b-/- mice and wildtype (WT) C57BL/6 mice were sensitized and challenged with OVA intranasally, a procedure normally tolerated by WT mice. We analyzed lung histology, BAL total cell counts and differential, cytokines and chemokines in the airway, and cytokine response by lymphocytes after re-stimulation by OVA antigen.
Compared with WT mice, OVA challenged Cbl-b-/- mice showed significantly increased neutrophilic and eosinophilic infiltration in the lung and mucus hyperplasia. The serum levels of IgG2a and IgG1, but not IgE, were increased. The levels of inflammatory mediators IFN-γ, IL-10, IL-12, IL-13, IP-10, MCP-1, MIP-1α, Eotaxin, and RANTES, but not IL-17A or IL-6, were elevated in the airway of Cbl-b-/- mice. Lymphocytes from Cbl-b-/-mice released increased amount of IFN-γ, IL-10, IL-13, and IP-10 in response to OVA re-stimulation. However, no significant changes were noted in the CD4+CD25+ Treg cell populations in the lung tissues after OVA stimulation and there was no difference between WT and Cbl-b-/- mice.
These results demonstrate that Cbl-b deficiency leads to a breakdown of tolerance to OVA allergen in the murine airways, probably through increased activation of T effector cells, indicating that Cbl-b is a critical factor in maintaining lung homeostasis upon environmental exposure to aeroallergens.
Cbl-b; Ubiquitin E3 Ligase; Aeroallergen; Allergic inflammation; Asthma
The effect of aging on several pathologic features of allergic-asthma (pulmonary inflammation, eosinophilia, mucus-hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.
To evaluate lung inflammation, mucus-metaplasia and AHR in relationship to age in murine models of allergic-asthma comparing young and older mice.
Young (6-week) and older (6-, 12- 18-month) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF) total inflammatory cell count and differential were measured. To evaluate mucus-metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by qPCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA.
AHR developed in both aged and young OVA-sensitized/challenged mice (OVA-mice), and was more significantly increased in young OVA-mice than in aged OVA-mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA-mice than in young OVA-mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA-mice. All aged OVA-mice had increased IL-5 and IFN-γ mRNA expression in the lung and IL-5 and IFN-γ protein levels from spleen cell cultures compared to young OVA-mice. OVA-IgE was elevated to a greater extent in aged OVA-mice.
Although pulmonary inflammation and mucus-metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA-mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-γ and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in aging animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.
Aging; murine; asthma; airway hyperresponsiveness; eosinophil; inflammation
Induction of nitric oxide synthase (NOS)–2 and production of nitric oxide (NO) are common features of allergic airway disease. Conditions of severe asthma are associated with deficiency of airway S-nitrosothiols, a biological product of NO that can suppress inflammation by S-nitrosylation of the proinflammatory transcription factor, NF-κB. Therefore, restoration of airway S-nitrosothiols might have therapeutic benefit, and this was tested in a mouse model of ovalbumin (OVA)-induced allergic inflammation. Naive or OVA-sensitized animals were administered S-nitrosoglutathione (GSNO; 50 μl, 10 mM) intratracheally before OVA challenge and analyzed 48 hours later. GSNO administration enhanced lung tissue S-nitrosothiol levels and reduced NF-κB activity in OVA-challenged animals compared with control animals, but did not lead to significant changes in total bronchoalveolar lavage cell counts, differentials, or mucus metaplasia markers. Administration of GSNO also altered the activation of hypoxia-inducible factor (HIF)–1, leading to HIF-1 activation in naive mice, but suppressed HIF-1 activation in OVA-challenged mice. We assessed the contribution of endogenous NOS2 in regulating NF-κB and/or HIF-1 activation and allergic airway inflammation using NOS2−/− mice. Although OVA-induced NF-κB activation was slightly increased in NOS2−/− mice, associated with small increases in bronchoalveolar lavage neutrophils, other markers of allergic inflammation and HIF-1 activation were similar in NOS2−/− and wild-type mice. Collectively, our studies indicate that instillation of GSNO can suppress NF-κB activation during allergic airway inflammation, but does not significantly affect overall markers of inflammation or mucus metaplasia, thus potentially limiting its therapeutic potential due to effects on additional signaling pathways, such as HIF-1.
nitric oxide; asthma; S-nitrosothiols; nuclear factor–κB; hypoxia inducible factor−1
The molecular determinants of the severity and persistence of allergic asthma remain poorly understood. Suppressor Of Cytokine Signaling 1 (SOCS1) is a negative regulator of interleukin (IL)-4-dependent pathways in vitro and might therefore, control T helper type 2 (Th2) immunity associated traits, such as IgE levels, mucin production, IL-5 and IL-13 induction, and eosinophilic mucosal inflammation, which are implicated in allergic asthma.
To investigate the role of SOCS1 in regulating Th2-associated disease traits in a murine sub-chronic aeroallergen-driven asthma model.
Following sensitization and challenge with ovalbumin (OVA), bronchoalveolar lavage (BAL) and serum were collected from mice lacking the Socs1 gene on an interferon γ (IFN-γ) null background (Socs1−/−Ifnγ−/−). The composition of infiltrating cells in the lung, serum IgE and IgG1 levels and cytokine levels were analysed.
Serum IgE levels and infiltrating eosinophils were greatly increased in the lungs of OVA-treated Socs1−/−Ifnγ−/− mice compared to Ifnγ−/− and C57BL/6 controls. Expression of the Th2 cytokines, IL-4, IL-5 and IL-13 was increased in CD4+ cells and lung tissue from OVA-treated Socs1−/−Ifnγ−/− mice. IgE, IL-5 levels and infiltrating eosinophils were also elevated in saline-treated Socs1−/−Ifnγ−/− mice, suggesting that in the absence of SOCS1, mice are already biased towards a Th2 response. It is at present unclear whether the elevated cytokine levels are sufficient to result in the exacerbated Th2-response to OVA challenge or whether enhanced intracellular signalling also contributes. Surprisingly, of the various IL-4/IL-13 responsive genes tested, only Arginase I appeared to be modestly up-regulated in the lungs of OVA-treated Socs1−/−Ifnγ−/− mice, suggesting that regulation by SOCS1 occurs primarily in hematopoietic cells and not in the airway epithelium.
Together these results indicate that SOCS1 is an important regulator of the Th2 response.
SOCS1; asthma; Th2 type cytokines; bronchoalveolar lavage; eosinophils
Apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen –activated protein (MAP) kinase kinase kinases (MAP3Ks) protein family, plays a crucial role in the induction of apoptosis and inflammation in some cell types. Allergic asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammatory cell infiltration, and airway remodeling. In the present study, we examined whether ASK1 is involved in the induction of bronchial asthma using a mouse model of airway inflammation.
ASK1-deficient (ASK1−/−) and wild-type (WT) control mice were sensitized with ovalbumin (OVA) in saline intraperitoneally on consecutive 7 days. Eighteen days later, mice received intranasal administration of OVA aerosol and were assayed for AHR, cytokine production, cell proliferation, antibody (Ab) production, and lung tissue histopathology at 24 hours after the last serial OVA administration. Levels of Ab and cytokines were determined by enzyme-linked immunosorbent assay (ELISA).
Control WT mice showed inflammatory infiltrates in airways in response to OVA to a greater extent than ASK1−/− mice. The number of cells, especially eosinophils accumulating in airways, was reduced in ASK1−/− mice relative to control mice. OVA-induced AHR is also compromised in ASK1−/− mice. Anti-OVA IgE Ab production in ASK1−/− mice was substantially reduced, although levels of other isotypes were comparable to those in control mice. Levels of some Th2 cytokines (IL-5 and IL-13) and pro-inflammatory cytokine TNF-a in BAL fluid from ASK1−/− mice were substantially diminished relative to control, although a comparable level of a typical Th2 cytokine IL-4 and anti-inflammatory cytokine IL-10 was produced. Although the BAL fluid TNF-a levels from ASK1−/− mice were severely diminished, lymph node cells from ASK1−/− mice produced comparable levels of TNF-a to WT in vitro. Intranasal administration of recombinant TNF-a caused a comparable increase in AHR between ASK1−/− and WT mice, whereas the TNF-a -induced accumulation of inflammatory cells was severely reduced in ASK1−/− mice.
ASK1 appears to be involved in the induction of OVA-induced bronchial asthma, probably through cytokine production such as TNF-a and IL-13. Moreover, TNF-a sensitivity in response to OVA is also regulated by ASK1.
IL-22 is a Th17/Th22 cytokine that is increased in asthma. However, recent animal studies showed controversial findings in the effects of IL-22 in allergic asthma. To determine the role of IL-22 in ovalbumin-induced allergic inflammation we generated inducible lung-specific IL-22 transgenic mice. Transgenic IL-22 expression and signaling activity in the lung were determined. Ovalbumin (OVA)-induced pulmonary inflammation, immune responses, and airway hyperresponsiveness (AHR) were examined and compared between IL-22 transgenic mice and wild type controls. Following doxycycline (Dox) induction, IL-22 protein was readily detected in the large (CC10 promoter) and small (SPC promoter) airway epithelial cells. IL-22 signaling was evidenced by phosphorylated STAT3. After OVA sensitization and challenge, compared to wild type littermates, IL-22 transgenic mice showed decreased eosinophils in the bronchoalveolar lavage (BAL), and in lung tissue, decreased mucus metaplasia in the airways, and reduced AHR. Among the cytokines and chemokines examined, IL-13 levels were reduced in the BAL fluid as well as in lymphocytes from local draining lymph nodes of IL-22 transgenic mice. No effect was seen on the levels of serum total or OVA-specific IgE or IgG. These findings indicate that IL-22 has immune modulatory effects on pulmonary inflammatory responses in allergen-induced asthma.
Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.
BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.
Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.
These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
Th2-dominated inflammatory response in the airway is an integral component in the pathogenesis of allergic asthma. Accumulating evidence supports the notion that the phosphoinositide 3-kinase (PI3K) pathway is involved in the process. We previously reported that SHIP-1, a negative regulator of the PI3K pathway, is essential in maintaining lung immunohomeostasis, potentially through regulation of innate immune cells. However, the function of SHIP-1 in adaptive immune response in the lung has not been defined. We sought to determine the role of SHIP-1 in adaptive immunity in response to aeroallergen stimulation in the airway.
SHIP-1 knockout (SHIP-1−/−) mice on BALB/c background were immunized with ovalbumin (OVA) plus aluminum hydroxide, a strong Th2-inducing immunization, and challenged with OVA. Airway and lung inflammation, immunoglobulin response, Th2 cytokine production and lymphocyte response were analyzed and compared with wild type mice. Even though there was mild spontaneous inflammation in the lung at baseline, SHIP-1−/− mice showed altered responses, including less cell infiltration around the airways but more in the parenchyma, less mucus production, decreased Th2 cytokine production, and diminished serum OVA-specific IgE, IgG1, but not IgG2a. Naïve and OVA sensitized SHIP-1−/− T cells produced a lower amount of IL-4. In vitro differentiated SHIP-1−/− Th2 cells produced less IL-4 compared to wild type Th2 cells upon T cell receptor stimulation.
These findings indicate that, in contrast to its role as a negative regulator in the innate immune cells, SHIP-1 acts as a positive regulator in Th2 cells in the adaptive immune response to aeroallergen. Thus any potential manipulation of SHIP-1 activity should be adjusted according to the specific immune response.
The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.
Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate.
In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages.
OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.
Asthma; Exacerbation; IL-13; IL-17A; M2 polarization
Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.
Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A−/− allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A−/− mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.
Fish oil (FO) is rich in n-3 polyunsaturated fatty acids (PUFA), which have been suggested to be anti-inflammatory and are associated with improvement of several inflammatory diseases. In this study, we investigated the influence of FO on allergen-induced lung inflammation and airway hyperreactivity in mice.
Male A/J mice were fed either a standard-chow (SC) or a FO diet (FO) for 8 weeks. After 4 weeks, each group was further randomized for ovalbumin (SC-OVA and FO-OVA) or saline (SC-SAL and FO-SAL) challenge. Resistance and elastance were measured at baseline and after aerosolized methacholine, 24h after the last challenge. Bronchoalveolar lavage (BAL) was performed for leukocyte counts. Lung tissue mucus deposition, peribronchiolar matrix deposition and eosinophil infiltration were quantified. Serum immunoglobulin E (IgE) and IgG1 (ref 2.2), lung IL-4, IL-5, IL-10, IL-13, IL-17, INFγ and eotaxin-1 and 2 were detected by ELISA and nuclear factor kappa B (NFκB), GATA-3 and peroxisome proliferator-activated receptor gamma (PPARγ) expression was measured by Western blot.
Levels of serum IgE and IgG1 were significantly higher in OVA sensitized mice. OVA challenge resulted in increased eosinophil infiltration, increased inflammatory cytokine production, peribronchiolar matrix and mucus deposition and airway hyperreactivity to aerosolized methacholine. Elevated lung NFκB and GATA-3 expression was noted in OVA-challenged mice. These changes were attenuated in mice fed with FO diet. Higher PPARγ expression was also detected in the lungs from the FO-fed groups.
Our results demonstrate that FO intake attenuated classical asthma features by suppressing the systemic sensitization, thus providing evidence that FO might be a prophylactic alternative for asthma prevention.
Cystic echinococcosis (CE) is a near cosmopolitan zoonosis caused by the larval stage of the dog tapeworm Echinococcus granulosus. E. granulosus infection induces a polarized T-helper type 2 (Th2) systematic immune response in its intermediate hosts. However, it is not known whether the infection modulates lung inflammation by regulating local immune response. In this study, we examined the effects of E. granulosus infection on mouse ovalbumin (OVA)-induced asthma model.
BALB/c mice were intraperitoneally transplanted with 50 small E. granulosus cysts cultured in vitro. At 3 months post-inoculation, the mice were sensitized and challenged with ovalbumin (OVA). For histopathological studies, hematoxylin eosin and periodic acid schiff staining was used to examine the inflammatory cells infiltration and goblet cells hyperplasia, respectively. Cytokine levels were measured by mouse cytometric bead array (CBA) Kit and quantitative RT-PCR and other molecular biological approaches. Airway hyperresponsiveness was assessed in response to increasing doses of methacholine. Serum immunoglobulins were determined by ELISA.
E. granulosus infection significantly increased Th2 and Treg cytokine levels in serum and lung tissues, but down-regulated the expression of IL-5 in the lungs and IL-17A in serum and lung tissues of asthmatic mice sensitized and challenged with OVA. Histological staining of lung tissues showed that E. granulosus infection significantly reduced the severity of OVA-induced airway inflammation including reduction of eosinophil cell infiltration and mucus production. The E. granulosus infection also reduced eosinophil accumulation induced by OVA in bronchoalveolar lavage fluid (BALF) and also ameliorated airway hyperresponsiveness, a hallmark symptom of asthma.
E. granulosus infection remarkably reduces the severity of OVA-induced airway inflammation likely through enhancing IL-10 and down-regulation of IL-5 and IL-17A.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-014-0522-6) contains supplementary material, which is available to authorized users.
Allergic asthma; Echinococcus granulosus; Cystic echinococcosis; IL-5; IL-10; IL-17A; Airway inflammation
Resistin-like molecule alpha (Retnla), also known as ‘Found in inflammatory zone 1’, is a secreted protein that has been found in bronchoalveolar lavage (BAL) fluid of ovalbumin (OVA)-induced asthmatic mice and plays a role as a regulator of T helper (Th)2-driven inflammation. However, the role of Retnla in the progress of Th2-driven airway inflammation is not yet clear. To better understand the function of Retnla in Th2-driven airway inflammation, we generated Retnla-overexpressing (Retnla-Tg) mice. Retnla-Tg mice showed increased expression of Retnla protein in BAL fluid and airway epithelial cells. Retnla overexpression itself did not induce any alteration in lung histology or lung function compared to non-Tg controls. However, OVA-sensitized/challenged Retnla-Tg mice had decreased numbers of cells in BAL and inflammatory cells accumulating in the lung. They also showed a reduction in mucus production in the airway epithelium, concomitant with a decreased Muc5ac level. These results were accompanied by reduced levels of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, with no effect on levels of OVA-specific immunoglobulin isotypes. Furthermore, phosphorylation of ERK was markedly reduced in the lungs of OVA-challenged Retnla-Tg mice. Taken together, these results indicates that Retnla protects against Th2-mediated inflammation in an experimental mouse model of asthma, suggesting that therapeutic approaches to enhance the production of Retnla or Retnla-like molecules could be valuable for preventing allergic lung inflammation.
In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.
Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.
Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.
Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.
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
Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4–deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-α, IL-4 −/− Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.
Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma.
C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14–16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14–17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity.
Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.
Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.
Cigarette smoke; Electrophile; Inflammation; Asthma; COPD; Nrf2; NF-κB; JNK
Asthma is a pulmonary chronic inflammatory disease characterized by airway obstruction and hyperresponsiveness. Pattern recognition receptors are known to play a key role in the development of allergic diseases as well as host defenses against microbial infection. Receptor interacting protein 2 (RIP2), a serine/threonine kinase, is an adaptor molecule of NOD1 and NOD2, and genetic variation in this receptor is known to be associated with the severity of allergic asthma in children. In this study, we examined the role of RIP2 in the development of allergic airway inflammation in a mouse model.
Airway inflammation was induced in mice through intranasal administration of ovalbumin (OVA) after 2 intraperitoneal immunizations with OVA. Lung inflammation and mucus hypersecretion were examined histologically and total cell infiltration in bronchoalveolar (BAL) fluids was determined. Levels of the Th2-related cytokines, IL-5 and IL-13, in lung extracts were measured by ELISA. Serum antigen-specific IgE and IgG1 levels were also assessed.
OVA-induced lung inflammation and mucus hypersecretion were not different between WT and RIP2-deficient mice. The IL-5 and IL-13 levels in the bronchoalveolar (BAL) fluids were also not impaired in RIP2-deficient mice compared to WT mice. Moreover, RIP2 deficiency did not affect serum OVA-specific IgG1 and IgE levels.
Our results suggest that RIP2 is not associated with the development of allergic airway inflammation.
RIP2; ovalbumin; airway inflammation; Th2; IgE
We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r–/–). Wild-type (Wt) and IL-8r–/– mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r–/– mice compared with Wt mice, whereas multiply challenged IL-8r–/– mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r–/– OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r–/– OVA/OVA mice than in Wt mice. Both the IL-8r–/– OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell– and mast cell–independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-γ in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow–derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
AHR; dendritic cells; eosinophils; mice; Syk
4-1BB (CD 137) is a costimulatory molecule expressed on activated T-cells. Repression by 4-1BB is thought to attenuate Th2-mediated allergic reactions. The aim of this study was to investigate the effect of 4-1BB on allergic airway inflammation in a murine asthma model.
BALB/c mice were sensitized to and challenged with ovalbumin (OVA). Hu.4-1BB-Fc was administered 1 day before the first OVA sensitization or 1 day after the second OVA sensitization. Following antigen challenge, airway responsiveness to methacholine was assessed and bronchoalveolar lavage (BAL) fluid was analyzed. Total immunoglobulin (Ig) E, OVA-specific IgE, IgG1, and IgG2a levels in sera were measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated.
In mice treated with Hu.4-1BB-Fc before the first OVA sensitization, there was a marked decrease in airway hyperresponsiveness, total cell count, and eosinophil count in the BAL fluid. In addition, Hu.4-1BB-Fc treatment decreased serum OVA-specific IgG1 levels and increased serum IgG2a level significantly compared with the corresponding levels in mice sensitized to and challenged with OVA. Hu.4-1BB-Fc-treated mice also showed suppressed peribronchial and perivascular inflammatory cell infiltration. In contrast, treatment with Hu.4-1BB-Fc 1 day after sensitization had no effect on airway hyperresponsiveness and showed less suppression of inflammation in lung tissue.
Administration of Hu.4-1BB-Fc can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. In addition, administration before sensitization may be more effective. These findings suggest that 4-1BB may be a useful therapeutic molecule against asthma.
4-1BB (CD137); Asthma; Allergic inflammation; Airway hyperresponsiveness; Mouse
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.
In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).
In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.
Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.
Previous studies have suggested that the asthmatic responses of airway inflammation, remodeling, and hyperresponsiveness (AHR) are interrelated; in this study, we used exercise to examine the nature of this interrelationship. Mice were sensitized and challenged with ovalbumin (OVA); mice were then exercised via running on a motorized treadmill at a moderate intensity. Data indicate that, within the lungs of OVA-treated mice, exercise attenuated the production of inflammatory mediators, including chemokines KC, RANTES, and MCP-1 and IL-12p40/p80. Coordinately, OVA-treated and exercised mice displayed decreases in leukocyte infiltration, including eosinophils, as compared with sedentary controls. Results also show that a single bout of exercise significantly decreased phosphorylation of the NFκB p65 subunit, which regulates the gene expression of a wide variety of inflammatory mediators. In addition, OVA-treated and exercised mice exhibited decreases in the levels of Th2-derived cytokines IL-5 and IL-13 and the prostaglandin PGE2, as compared with sedentary controls. In contrast, results show that a single bout of exercise had no effect on AHR in OVA-treated mice challenged with increasing doses of aerosolized methacholine (0–50 mg/ml) as compared with sedentary mice. Exercise also had no effect on epithelial cell hypertrophy, mucus production, or airway wall thickening in OVA-treated mice as compared with sedentary controls. These findings suggest that a single bout of aerobic exercise at a moderate intensity attenuates airway inflammation but not AHR or airway remodeling in OVA-treated mice. The implication of these findings for the interrelationship between airway inflammation, airway remodeling, and AHR is discussed.
asthma; aerobic exercise; airway inflammation; remodeling; hyperresponsiveness