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
Sphingosine-1-phosphate (S1P) produced by two sphingosine kinase isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution and the role that S1P plays in vivo in a mast cell and IgE-dependent mouse model of allergic asthma has not yet been examined.
We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.
Allergic airway inflammation and AHR were examined in a mast cell-dependent mouse model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I prior to sensitization and challenge with OVA or only prior to challenge.
SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of NF-κB, a master transcription factor that regulates expression of pro-inflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, which develop mast cell-dependent allergic inflammation, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, 5, 6, 13, IFN-γ, and TNF-α and the chemokines eotaxin, and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation as well as activation of NF-κB in the lungs.
S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
sphingosine-1-phosphate; sphingosine kinase; mast cells; NF-kB; airway hyperresponsiveness; asthma
Allergic subjects produce relatively low amounts of IFN-γ, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-γ gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-γ pDNA nanoparticles (CIN) for in situ production of IFN-γ and its in vivo effects.
CIN were administered to OVA-sensitized mice to investigate the possibility of using gene transfer to modulate ovalbumin (OVA)-induced inflammation and AHR.
Mice treated with CIN exhibit significantly lower AHR to methacholine challenge and less lung histopathology. Production of IFN-γ is increased after CIN treatment while the Th2-cytokines, IL-4 and IL-5, and OVA-specific serum IgE are reduced compared to control mice. AHR and eosinophilia are also significantly reduced by CIN therapy administered therapeutically in mice with established asthma. CIN was found to inhibit epithelial inflammation within 6 hours of delivery by inducing apoptosis of goblet cells. Experiments performed on STAT4-defective mice do not show reduction in AHR with CIN treatment, thus implicating STAT4 signaling in the mechanism of CIN action.
These results demonstrate that mucosal CIN therapy can effectively reduce established allergen-induced airway inflammation and AHR.
Chronic allergic asthma is the result of a Th2- biased immune status. Current asthma therapies control symptoms in some patients, but a long lasting therapy has not been established. ASHMI™, a Chinese herbal formula improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma.
We evaluated the persistence of ASHMI™ beneficial effects following therapy in a murine model of persistent asthma and the immunological mechanisms underlying such effects. BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyperreactivity (AHR) and inflammation (OVA mice). Additional OVA mice were treated with ASHMI™ (OVA/ASHMI™) or water (OVA/Sham) for 4 weeks, and then challenged immediately and eight weeks post-therapy. In other experiments OVA mice received ASHMI™ treatment with concomitant neutralization of IFN-γ or TGF-β. Effects on airway responses, cytokine and OVA-specific IgE levels were determined 8 weeks post-therapy.
Prior to treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI™ and did not re-occur following OVA re-challenge up to 8 wks post-therapy. Reduced allergen-specific IgE and Th2 cytokine levels, and increased IFN-γ levels also persisted at least 8 wks post-therapy. ASHMI™ effects were eliminated by neutralization of IFN-γ, but not TGF-β, during therapy.
ASHMI™ induced long-lasting post-therapy tolerance to antigen-induced inflammation and AHR. IFN-γ is a critical factor in ASHMI™ effects.
Allergic asthma; Mice; Traditional Chinese Medicine; Th-2 cytokines; Interferon-γ; IgE
IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-γ-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-γ over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-γ-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-γ expression were impaired in allergen-challenged IL-12Rβ2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Rα-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-γ. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-γ axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
asthma; interferon-γ; interleukin-12; interleukin-13; respiratory hypersensitivity; Th2 cells
Asthma is characterized by bronchial inflammation causing increased airway hyperresponsiveness and eosinophilia. The interaction between airway epithelium and inflammatory mediators plays a key role in the asthmatic pathogenesis. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid found in apples and many berries, on inflammation in human airway epithelial BEAS-2B cells. Nontoxic kaempferol at ≤20 μM suppressed the LPS-induced IL-8 production through the TLR4 activation, inhibiting eotaxin-1 induction. The in vivo study explored the demoting effects of kaempferol on asthmatic inflammation in BALB/c mice sensitized with ovalbumin (OVA). Mouse macrophage inflammatory protein-2 production and CXCR2 expression were upregulated in OVA-challenged mice, which was attenuated by oral administration of ≥10 mg/kg kaempferol. Kaempferol allayed the airway tissue levels of eotaxin-1 and eotaxin receptor CCR3 enhanced by OVA challenge. This study further explored the blockade of Tyk-STAT signaling by kaempferol in both LPS-stimulated BEAS-2B cells and OVA-challenged mice. LPS activated Tyk2 responsible for eotaxin-1 induction, while kaempferol dose-dependently inhibited LPS- or IL-8-inflamed Tyk2 activation. Similar inhibition of Tyk2 activation by kaempferol was observed in OVA-induced mice. Additionally, LPS stimulated the activation of STAT1/3 signaling concomitant with downregulated expression of Tyk-inhibiting SOCS3. In contrast, kaempferol encumbered STAT1/3 signaling with restoration of SOCS3 expression. Consistently, oral administration of kaempferol blocked STAT3 transactivation elevated by OVA challenge. These results demonstrate that kaempferol alleviated airway inflammation through modulating Tyk2-STAT1/3 signaling responsive to IL-8 in endotoxin-exposed airway epithelium and in asthmatic mice. Therefore, kaempferol may be a therapeutic agent targeting asthmatic diseases.
Atopic dermatitis (AD) is characterized by local and systemic Th2 responses to cutaneously introduced allergens and is a risk factor for asthma. Blockade of Th2 cytokines has been suggested as therapy for AD.
To examine the effect of the absence of IL-4 and IL-13 on the Th-17 response to epicutaneous (EC) sensitization in a mouse model of allergic skin inflammation with features of AD.
Wild-type (WT), IL-4KO, IL-13KO and IL-4/13 double KO (DKO) mice were subjected to EC sensitization with ovalbumin (OVA) or saline and airway challenged with OVA. Systemic immune responses to OVA, skin and airway inflammation, and airway hyperresponsiveness (AHR) were examined.
OVA sensitized DKO mice exhibited impaired Th2 driven responses with undetectable OVA specific IgE and severely diminished eosinophil infiltration at sensitized skin sites, but intact dermal infiltration with CD4+ cells. DKO mice mounted an exaggerated IL-17A, but normal IFN-γ and IL-5 systemic responses. Airway challenge of these mice with OVA caused marked upregulation of IL-17 mRNA expression in the lungs, increased neutrophilia in bronchoalveolar lavage fluid (BALF), airway inflammation characterized by mononuclear cell infiltration with no detectable eosinophils, and bronchial hyperresponsiveness to methacholine that were reversed by IL-17 blockade. IL-4, but not IL-13, was identified as the major Th2 cytokine that downregulates the IL-17 response in EC sensitized mice.
EC sensitization in the absence of IL-4/IL-13 induces an exaggerated Th17 response systemically, and in lungs following antigen challenge that results in airway inflammation and AHR.
Blockade of IL-4 may promote IL-17-mediated airway inflammation in AD.
IL-17; Th2 cytokines; atopic dermatitis; asthma
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
To determine the roles of breast regression protein-39 (BRP-39) in regulating dendritic cell maturation and in pathology of acute asthma.
Mouse bone marrow-derived dendritic cells (BMDCs) were prepared, and infected with adenovirus over-expressing BRP-39. Ovalbumin (OVA)-induced murine model of acute asthma was made in female BALB/c mice by sensitizing and challenging with chicken OVA and Imject Alum. The transfected BMDCs were adoptively transferred into OVA-treated mice via intravenous injection. Airway hyperresponsiveness (AHR), inflammation and pulmonary histopathology were characterized.
The expression of BRP-39 mRNA and protein was significantly increased in lung tissues of OVA-treated mice. The BMDCs infected with adenovirus BRP-39 exhibited greater maturation and higher activity in vitro. Adoptive transfer of the cells into OVA-treated mice significantly augmented OVA-induced AHR and eosinophilic inflammation. Meanwhile, BRP-39 further enhanced the production of OVA-induced Th2 cytokines IL-4, IL-5 and IL-13, but significantly attenuated OVA-induced IFN-γ production in bronchoalveolar lavage fluid.
In OVA-induced murine model of acute asthma, BRP-39 is over-expressed in lung tissue and augments Th2 inflammatory response and AHR. BRP-39 promotes dendritic cell maturation in vitro. Therefore, BRP-39 may be a potential therapeutic target of asthma.
asthma; ovalbumin; bone marrow-derived dendritic cells (BMDCs); breast regression protein-39 (BRP-39); YKL-40; Th2 inflammation; airway hyperresponsiveness; bronchoalveolar lavage fluid
Viral infections are the most frequent cause of asthma exacerbations and are linked to increased airway reactivity (AR) and inflammation. Mice infected with respiratory syncytial virus (RSV) during ovalbumin (OVA)-induced allergic airway inflammation (OVA/RSV) had increased AR compared to OVA or RSV mice alone. Further, IL-17A was only increased in OVA/RSV mice.
To determine if IL-17A increases AR and inflammation in the OVA/RSV model.
Wild-type BALB/c and IL-17A KO mice underwent mock, RSV, OVA, or OVA/RSV protocols. Lungs, bronchoalveolar lavage (BAL) fluid, and/or mediastinal lymph nodes (MLNs) were harvested post infection. Cytokine expression was determined by flow cytometry and ELISA in the lungs or BAL fluid. MLNs were restimulated with either OVA (323–229) peptide or RSV M2 (127–135) peptide and IL-17A protein expression was analyzed. AR was determined by methacholine challenge.
RSV increased IL-17A protein expression by OVA-specific T cells 6 days post infection. OVA/RSV mice had decreased IFN-α and IFN-β protein expression compared to RSV mice. OVA/RSV mice had increased IL-23 mRNA expression in lung homogenates compared to mock, OVA, or RSV mice. Unexpectedly, IL-17A KO OVA/RSV mice had increased AR compared to WT OVA/RSV mice. Further, IL-17A KO OVA/RSV mice had increased eosinophils, lymphocytes, and IL-13 protein expression in BAL fluid compared to WT OVA/RSV mice.
IL-17A negatively regulated AR and airway inflammation in OVA/RSV mice. This finding is important because IL-17A has been identified as a potential therapeutic target in asthma, and inhibiting IL-17A in the setting of virally induced asthma exacerbations may have adverse consequences.
IL-17A; airway reactivity; CD4+ T cells; allergic inflammation; RSV
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.
Objectives: To determine the contribution of TGF-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
lung; mice; hypersensitivity; cytokines
Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.
CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.
Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.
Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.
We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.
chemokine receptor; CXCR3; CD8+ T lymphocyte; airway inflammation; airway hyperresponsiveness
Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6−/− mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6−/− mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6−/− mice even when STAT6+/+ BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2−/− mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4+CD25+ Foxp3+ T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6−/− mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6−/− mice were equally efficient in suppressing CD4+ T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.
A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.
Approximately 50% of asthmatics have non-eosinophilic inflammation, and 20% of these patients have severe neutrophilic inflammation and increased IL-8 levels. These so-called neutrophilic asthmatics have persistent airway colonization with bacteria, and Haemophilus influenzae is one of the bacteria most commonly isolated. However, how H. influenzae is associated with the pathogenesis of neutrophilic asthma is unknown. In this study we used mouse models to investigate the relationship between H. influenzae infection and allergic airways disease (AAD). We showed that infection promoted the development of hallmark features of neutrophilic asthma. Infection suppressed Th2 cytokines, eosinophilic inflammation, and AHR in AAD, while increasing neutrophilic inflammation and IL-17 responses. Importantly, inhibition of IL-17 during AAD reduced airway neutrophils and neutrophil chemokines, suggesting that infection drives the development of neutrophilic inflammation through an IL-17-mediated mechanism. This provides novel insights into the mechanisms that may underpin infection-induced neutrophilic asthma. These data also suggest that treatments targeting infection may lead to improved management of neutrophilic asthma.
Epidemiological studies have already shown that females are dominant in terms of the sex ratio of adult asthma prevalence and severe asthma. It has also been reported that female mice are more susceptible to the development of allergic airway inflammation and airway hyperresponsiveness (AHR) than males. However, there have been few reports of studies on sex difference in the pathogenesis of severe asthma, especially airway remodeling in an animal model. In this study, we investigated sex difference in formation of airway remodeling using a long-term antigen challenged asthma model.
Following ovalbumin (OVA)/alum intraperitoneal injection, male or female mice (BALB/c) were challenged with aerosolized 1% OVA on 3 days/week for 5 weeks, and we investigated the sex difference in AHR, airway inflammation, as well as airway remodeling.
In OVA-sensitized and -challenged (OVA/OVA) female mice, AHR, the number of eosinophils and lymphocytes, as well as Th2 cytokines and growth factors in BAL fluid were increased compared with OVA/OVA male mice. On the other hand, there is no significant difference in the level of eotaxin in BAL fluid. The histological features of airway remodeling, including goblet cell hyperplasia, subepithelial fibrosis and myofibroblast hypertrophy, were also increased in OVA/OVA female mice. Moreover, serum total and OVA-specific IgE were significantly elevated in OVA/OVA female mice.
These results indicate that female mice are dominant in terms of forming airway remodeling as compared with male mice. The involvement of sex difference for sensitization and growth factor release in lung tissue based on inflammatory cell infiltration is indicated for the mechanism of sex difference of airway remodeling.
Obesity has been suggested to be linked to asthma. However, it is not yet known whether obesity directly leads to airway hyperreactivity (AHR) or obesity-induced airway inflammation associated with asthma. We investigated obesity-related changes in adipokines, AHR, and lung inflammation in a murine model of asthma and obesity.
Materials and Methods
We developed mouse models of chronic asthma via ovalbumin (OVA)-challenge and of obesity by feeding a high-fat diet, and then performed the methacholine bronchial provocation test, and real-time PCR for leptin, leptin receptor, adiponectin, adiponectin receptor (adipor1 and 2), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) β, and tumor necrosis factor (TNF) α in lung tissue. We also measured cell counts in bronchoalveolar lavage fluid.
Both obese and lean mice chronically exposed to OVA developed eosinophilic lung inflammation and AHR to methacholine. However, obese mice without OVA challenge did not develop AHR or eosinophilic inflammation in lung tissue. In obese mice, lung mRNA expressions of leptin, leptin receptor, VEGF, TGF, and TNF were enhanced, and adipor1 and 2 expressions were decreased compared to mice in the control group. On the other hand, there were no differences between obese mice with or without OVA challenge.
Diet-induced mild obesity may not augment AHR or eosinophilic lung inflammation in asthma.
Adipokine; asthma; high fat; vascular endothelial growth factor; transforming growth factor beta; tumor necrosis factor alpha; obesity; airway hyperresponsiveness
Th2 immune responses are linked primarily to mild and moderate asthma, while Th17 cells, Interleukin-17A (IL-17) and neutrophilia have been implicated in more severe forms of disease. How Th2-dependent allergic reactions are influenced by Th17 and IL-17-γδ T cells is poorly understood. In murine models, under some conditions, IL-17 promotes Th2-biased airway inflammatory responses. However, IL-17-γδ T cells have been implicated in the inhibition and resolution of allergic airway inflammation and hyperresponsiveness (AHR).
We compared airway responses in Balb/c mice sensitized to OVA with (and without) a Th2-skewing aluminum-based adjuvant and the IL-17 skewing, complete Freund’s adjuvant (CFA). AHR was measured invasively by flexiVent, while serum OVA-IgE was quantified by an enzyme immunoassay. Airway inflammatory and cytokine profiles, and cellular sources of IL-17 were assessed from bronchoalveolar lavage and/or lungs. The role of γδ T cells in these responses was addressed in OVA/CFA sensitized mice using a γδ T cell antibody.
Following OVA challenge, all mice exhibited mixed eosinophilic/neutrophilic airway inflammatory profiles and elevated serum OVA-IgE. Whereas OVA/alum sensitized mice had moderate inflammation and AHR, OVA/CFA sensitized mice had significantly greater inflammation but lacked AHR. This correlated with a shift in IL-17 production from CD4+ to γδ T cells. Additionally, OVA/CFA sensitized mice, given a γδ TCR stimulatory antibody, showed increased frequencies of IL-17-γδ T cells and diminished airway reactivity and eosinophilia.
Thus, the conditions of antigen sensitization influence the profile of cells that produce IL-17, the balance of which may then modulate the airway inflammatory responses, including AHR. The possibility for IL-17-γδ T cells to reduce AHR and robust eosinophilic inflammation provides evidence that therapeutic approaches focused on stimulating and increasing airway IL-17-γδ T cells may be an effective alternative in treating steroid resistant, severe asthma.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0090-5) contains supplementary material, which is available to authorized users.
Asthma; Interleukin-17; γδ T cell; Adjuvant; Complete Freund’s adjuvant
Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2–associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood.
Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS).
Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR).
Results: Mice instilled with OVA together with very low doses (≤ 10–3 μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10–1 μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10–1 μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS.
Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics.
Citation: Whitehead GS, Thomas SY, Cook DN. 2014. Modulation of distinct asthmatic phenotypes in mice by dose-dependent inhalation of microbial products. Environ Health Perspect 122:34–42; http://dx.doi.org/10.1289/ehp.1307280
An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease.
We used ovalbumin (OVA)-based mouse models of asthma and primary CD4+ T cells. C57BL/6J WT or PARP-1−/− mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge.
Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4+ T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1−/− mice. Adoptive transfer of Th2-skewed OT-II-WT CD4+ T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1−/−mice suggesting a role for PARP-1 in CD4+ T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4+ T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4+ T cells.
Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.
PARP inhibition; Olaparib (AZD2281); Allergen-induced eosinophilia; Adoptive transfer; Th2 cytokines; Allergen-specific IgE
Obesity is a known risk factor for allergic asthma. It has been recognized as a key player in the pathogenesis of several inflammatory disorders via activation of macrophages, which is also vital to the development of allergic asthma. We investigated the mechanism of obesity-related asthma and whether treating obesity through exercise or diet ameliorates the severity of asthma in the obesity-related asthma model. We generated diet-induced obesity (DIO) in C57BL/6 mice by high-fat-feeding and ovalbumin-induced asthma (lean-OVA or DIO-OVA). The DIO-OVA mice were then treated with tumor necrosis factor (TNF)-α neutralizing antibody as a TNF-α blockade or a Cl2MDP-containing liposome to induce an alveolar macrophage deficiency. To treat obesity, the DIO-OVA mice were under dietary restrictions or exercised. The pathophysiological and immunological responses were analyzed. Airway hyperresponsiveness (AHR), serum IgE and TNF-α levels in the lung tissue increased in the DIO-OVA mice compared to the lean-OVA mice. Both the TNF-α blockade and depletion of alveolar macrophages in the DIO-OVA mice decreased AHR compared to the DIO-OVA mice. Treating obesity by exercise or through dietary means also reduced pulmonary TNF-α levels and AHR in the DIO-OVA mice. These results suggest that restoring normal body weight is an appropriate strategy for reducing TNF-α levels, and controlling inflammation may help improve asthma severity and control in obesity-related asthma.
Janus kinases (JAK) are regulators of signaling through cytokine receptors. The importance of JAK1/3 signaling on Th2 differentiation and development of lung allergic responses has not been investigated.
To examine a selective JAK1/3 inhibitor (R256) on differentiation of Th subsets in vitro and on development of ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in an experimental model of asthma.
A selective JAK1/3 inhibitor was used to assay the importance of this pathway on induction of Th1, Th2, and Th17 differentiation in vitro. In vivo, the effects of inhibiting JAK1/3 signaling were examined by administering the inhibitor during the sensitization or during allergen challenge phases in the primary challenge model or just prior to provocative challenge in the secondary challenge model. Airway inflammation and AHR were examined after the last airway challenge.
In vitro, R256 inhibited differentiation of Th2 but not Th1 or Th17 cells, associated with downregulation of STAT6 and STAT5 phosphorylation. However, once polarized, Th2 cells were unaffected by the inhibitor. In vivo, R256 administered during the OVA sensitization phase prevented development of AHR, airway eosinophilia, mucus hypersecretion, and Th2 cytokine production without changes in Th1 and Th17 cytokine levels, indicating that selective blockade of Th2 differentiation was critical. Inhibitor administration after OVA sensitization but during the challenge phases in the primary or secondary challenge models similarly suppressed AHR, airway eosinophilia, and mucus hypersecretion without any reduction in Th2 cytokine production, suggesting the inhibitory effects were downstream of Th2 cytokine receptor signaling pathways.
Targeting the Th2-dependent JAK-STAT activation pathway represents a novel therapeutic approach for the treatment of asthma.
Targeting JAK1/3 signaling pathways provides a novel intervention for preventing allergen-induced alterations in lung function.
JAK1/3 signaling pathways are essential for initiation of Th2 differentiation and the development of lung allergic responses.
JAK1/3; asthma; Th2
Airways hyperresponsiveness (AHR) is usually produced within days of first antigen exposure in mouse models of asthma. Furthermore, continual antigen challenge eventually results in the resolution of the AHR phenotype. Human asthma also waxes and wanes with time, suggesting that studying the time course of AHR in the allergic mouse would offer insights into the variation in symptoms seen in asthmatics.
Mice were sensitized with ovalbumin (OVA) on days 0 and 14. As assessed by airway resistance (Rn), lung elastance (H) and tissue damping (G), AHR was measured post an OVA inhalation on day 21 (Short Challenge group), after three days of OVA inhalation on day 25 (Standard Challenge group) and following an OVA inhalation on day 55 in mice previously challenged on days 21–23 (Recall Challenge group). Bronchoalveolar lavage was analyzed for inflammatory cells, cytokines and protein.
AHR in the Short Challenge group was characterized by an increase in Rn and neutrophil accumulation in the lavage. AHR in the Standard Challenge group was characterized by increases in H and G but by only a modest response in Rn, while inflammation was eosinophilic. In the Standard Challenge protocol, mice lacking fibrinogen were no different from control in their AHR response. AHR in the Recall Challenge group was characterized by increases only in G and H and elevated numbers of both neutrophils and eosinophils. Lavage cytokines were only elevated in the Recall Challenge group. Lavage protein was significantly elevated in all groups.
The phenotype in allergically inflamed mice evolves distinctly over time, both in terms of the nature of the inflammation and the location of the AHR response. The study of mouse models of AHR might be better served by focusing on this variation rather than simply on a single time point at which AHR is maximal.
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.
Immunoglobulins (Ig), particularly IgE, are believed to be crucially involved in the pathogenesis of asthma and, equally, in allergic models of the disease. To validate this paradigm we examined homozygous mutant C57BL/6 mice, which are B cell deficient, lacking all Ig. Mice were immunized intraperitoneally with 10 μg ovalbumin (OVA) plus alum, followed by daily (day 14–20) 30 min exposures to OVA aerosol (OVA/OVA group). Three control groups were run: OVA intraperitoneally plus saline (SAL) aerosol (OVA/SAL group); saline intraperitoneally plus saline aerosol; saline intraperitoneally plus OVA aerosol (n = 6–7). Lung and large airway tissues obtained 24 h after the last OVA or SAL exposure were examined by light microscopy and transmission electron microscopy (TEM). The Ig-deficient mice receiving OVA/ OVA treatment had swollen and discolored lungs and exhibited marked eosinophilia both in large airway subepithelial tissue (49.2 ± 12.0 cells/mm basement membrane [BM] versus OVA/ SAL control 1.2 ± 0.3 cells/mm BM; P <0.001), and perivascularly and peribronchially in the lung (49.3 ± 9.0 cells/unit area versus OVA/SAL control 2.6 ± 0.6 cells/unit area; P <0.001). The eosinophilia extended to the regional lymph nodes. TEM confirmed the subepithelial and perivascular localization of eosinophils. Mucus cells in large airway epithelium increased from 1.5 ± 0.8 (OVA/SAL mice) to 39.5 ± 5.7 cells/mm BM in OVA/OVA treated mice (P <0.001). OVA/SAL mice never differed from the other control groups. Corresponding experiments in wild-type mice (n = 6–7 in each group) showed qualitatively similar but less pronounced eosinophil and mucus cell changes. Macrophages and CD4+ T cells increased in lungs of all OVA/OVA-treated mice. Mast cell number did not differ but degranulation was detected only in OVA/OVA-treated wild-type mice. Immunization to OVA followed by OVA challenges thus cause eosinophil-rich inflammation in airways and lungs of mice without involvement of B cells and Ig.