Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

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.

Methods

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.

Results

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.

Conclusions

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.     

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

Background

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).

Methods

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.

Results

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.

Conclusions

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.     

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

Background

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

Methods

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

Results

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

Conclusions

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

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma

Background

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.

Methods

We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.

Results

Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.

Conclusion

These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.     

Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD

Background

Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface.

Method

Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers.

Results

In smokers with normal lung function, the expression of CD25⁺CD4⁺ was increased, whereas the proportions of FoxP3⁺ and CD127⁺ were unchanged compared to never-smokers. Among CD4⁺ cells expressing high levels of CD25, the proportion of FoxP3⁺ cells was decreased and the percentage of CD127⁺ was increased in smokers with normal lung function. CD4⁺CD25⁺ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers.

Conclusion

The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25⁺ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.     

Early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice

Background

Early-life respiratory viral infections, notably with respiratory syncytial virus (RSV), increase the risk of subsequent development of childhood asthma. The purpose of this study was to assess whether early-life infection with a species-specific model of RSV and subsequent allergen exposure predisposed to the development of features of asthma.

Methods

We employed a unique combination of animal models in which BALB/c mice were neonatally infected with pneumonia virus of mice (PVM, which replicates severe RSV disease in human infants) and following recovery, were intranasally sensitised with ovalbumin. Animals received low-level challenge with aerosolised antigen for 4 weeks to elicit changes of chronic asthma, followed by a single moderate-level challenge to induce an exacerbation of inflammation. We then assessed airway inflammation, epithelial changes characteristic of remodelling, airway hyperresponsiveness (AHR) and host immunological responses.

Results

Allergic airway inflammation, including recruitment of eosinophils, was prominent only in animals that had recovered from neonatal infection with PVM and then been sensitised and chronically challenged with antigen. Furthermore, only these mice exhibited an augmented Th2-biased immune response, including elevated serum levels of anti-ovalbumin IgE and IgG₁ as well as increased relative expression of Th2-associated cytokines IL-4, IL-5 and IL-13. By comparison, development of AHR and mucous cell change were associated with recovery from PVM infection, regardless of subsequent allergen challenge. Increased expression of IL-25, which could contribute to induction of a Th2 response, was demonstrable in the lung following PVM infection. Signalling via the IL-4 receptor α chain was crucial to the development of allergic inflammation, mucous cell change and AHR, because all of these were absent in receptor-deficient mice. In contrast, changes of remodelling were evident in mice that received chronic allergen challenge, regardless of neonatal PVM infection, and were not dependent on signalling via the IL-4 receptor.

Conclusion

In this mouse model, interaction between early-life viral infection and allergen sensitisation/challenge is essential for development of the characteristic features of childhood asthma, including allergic inflammation and a Th2-biased immune response.     

Aldose reductase deficiency in mice protects from ragweed pollen extract (RWE)-induced allergic asthma

Background

Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR^-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of ragweed pollen extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat.

Methods

The wild type (WT) and AR^-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4⁺CD25⁺ T cells population.

Results

Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR^-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4⁺CD25⁺FoxP3⁺) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat.

Conclusion

Our results using AR^-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.     

CD39+ Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

Background

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.

Methods

The numbers of both CD39⁺Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.

Results

Both CD39⁺Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39⁺Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39⁺Tregs could express latency-associated peptide on their surface. When naïve CD4⁺ T cells were cocultured with CD39⁺Tregs, Th17 cell numbers decreased as CD39⁺Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39⁺Tregs.

Conclusions

Therefore, the above results indicate that CD39⁺Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.     

Immune amplification of murine CD8 suppressor T cells induced via an immune-privileged site: quantifying suppressor T cells functionally

Background

CD8⁺ suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8⁺ suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8⁺ suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8⁺ suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells.

Methods

Suppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen.

Results

Suppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8⁺ AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH.

Conclusions

The results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8⁺ suppressor T cells are specifically amplified by antigen during an immune response.     

Lung-Homing of Endothelial Progenitor Cells and Airway Vascularization Is Only Partially Dependant on Eosinophils in a House Dust Mite-Exposed Mouse Model of Allergic Asthma

Background

Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1), a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC) to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL) whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.

Methods

Wild-type (WT) BALB/c and eosinophil deficient (PHIL) mice were sensitized to house dust mite (HDM) using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD) and airway methacholine responsiveness (AHR).

Results

Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.

Conclusions

This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.     

Chronic Low Dose Chlorine Exposure Aggravates Allergic Inflammation and Airway Hyperresponsiveness and Activates Inflammasome Pathway

Background

Epidemiologic clinical studies suggested that chronic exposure to chlorine products is associated with development of asthma and aggravation of asthmatic symptoms. However, its underlying mechanism was not clearly understood. Studies were undertaken to define the effects and mechanisms of chronic low-dose chlorine exposure in the pathogenesis of airway inflammation and airway hyperresponsiveness (AHR).

Methods

Six week-old female BALB/c mice were sensitized and challenged with OVA in the presence and absence of chronic low dose chlorine exposure of naturally vaporized gas of 5% sodium hypochlorite solution. Airway inflammation and AHR were evaluated by bronchoalveolar lavage (BAL) cell recovery and non-invasive phlethysmography, respectively. Real-time qPCR, Western blot assay, and ELISA were used to evaluate the mRNA and protein expressions of cytokines and other inflammatory mediators. Human A549 and murine epithelial (A549 and MLE12) and macrophage (AMJ2-C11) cells were used to define the responses to low dose chlorine exposure in vitro.

Results

Chronic low dose chlorine exposure significantly augmented airway inflammation and AHR in OVA-sensitized and challenged mice. The expression of Th2 cytokines IL-4 and IL-5 and proinflammatory cytokine IL-1β and IL-33 were significantly increased in OVA/Cl group compared with OVA group. The chlorine exposure also activates the major molecules associated with inflammasome pathway in the macrophages with increased expression of epithelial alarmins IL-33 and TSLP in vitro.

Conclusion

Chronic low dose exposure of chlorine aggravates allergic Th2 inflammation and AHR potentially through activation of inflammasome danger signaling pathways.     

Parasitic Nematode-Induced CD4+Foxp3+T Cells Can Ameliorate Allergic Airway Inflammation

Background

The recruitment of CD4⁺CD25⁺Foxp3⁺T (T_reg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.

Methodology/Principal Findings

We compared the effects of T_reg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced T_reg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4⁺Foxp3⁺ cells from T. spiralis-infected [Inf(+)Foxp3⁺] or uninfected [Inf(-)Foxp3⁺] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3⁺ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3⁺ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3⁺ cells migrated to inflammation sites in the lung and expressed higher levels of T_reg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3⁺ cells.

Conclusion/Significance

T. spiralis infection promotes the proliferation and functional activation of T_reg cells. Parasite-induced T_reg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced T_reg cells. The adoptive transfer of Inf(+)Foxp3⁺ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.     

miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression

Background

In humans and mice naturally occurring CD4⁺CD25⁺ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal Findings

DNA-Microarray analyses of human as well as murine conventional CD4⁺ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4⁺ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4⁺ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion

Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4⁺ Th cells to nTreg cell-mediated suppression.     

Dietary galacto-oligosaccharides prevent airway eosinophilia and hyperresponsiveness in a murine house dust mite-induced asthma model

Background

Allergic asthma is strongly associated with the exposure to house dust mite (HDM) and is characterized by eosinophilic pulmonary inflammation and airway hyperresponsiveness (AHR). Recently, there is an increased interest in using dietary oligosaccharides, also known as prebiotics, as a novel strategy to prevent the development of, or reduce, symptoms of allergy.

Aim

We investigated the preventive capacity of dietary galacto-oligosaccharides (GOS) compared to an intra-airway therapeutic treatment with budesonide on the development of HDM-induced allergic asthma in mice.

Methods

BALB/c mice were intranasally sensitized with 1 μg HDM on day 0 followed by daily intranasal challenge with PBS or 10 μg HDM on days 7 to 11. Two weeks prior to the first sensitization and throughout the experiment mice were fed a control diet or a diet containing 1% GOS. Reference mice were oropharyngeally instilled with budesonide (500 μg/kg) on days 7, 9, 11, and 13, while being fed the control diet. On day 14, AHR was measured by nebulizing increasing doses of methacholine into the airways. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lungs were collected.

Results

Sensitization and challenge with HDM resulted in AHR. In contrast to budesonide, dietary intervention with 1% GOS prevented the development of AHR. HDM sensitization and challenge resulted in a significant increase in BALF leukocytes numbers, which was suppressed by budesonide treatment and dietary intervention with 1% GOS. Moreover, HDM sensitization and challenge resulted in significantly enhanced concentrations of IL-6, CCL17, IL-33, CCL5 and IL-13 in lung tissue. Both dietary intervention with 1% GOS or budesonide treatment significantly decreased the HDM-induced increased concentrations of CCL5 and IL-13 in lung tissue, while budesonide also reduced the HDM-enhanced concentrations of IL-6 and CCL17 in lung tissue.

Conclusion

Not only did dietary intervention with 1% GOS during sensitization and challenge prevent the induction of airway eosinophilia and Th2-related cytokine and chemokine concentrations in the lung equally effective as budesonide treatment, it also prevented AHR development in HDM-allergic mice. GOS might be useful for the prevention and/or treatment of symptoms in asthmatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0171-0) contains supplementary material, which is available to authorized users.     

An alternate STAT6-independent pathway promotes eosinophil influx into blood during allergic airway inflammation

Background

Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4⁺ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.

Methodology/Principal Findings

We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4⁺ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4⁺ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4⁺ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.

Conclusions

These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.     

Histamine H4 receptor antagonism diminishes existing airway inflammation and dysfunction via modulation of Th2 cytokines

Background

Airway remodeling and dysfunction are characteristic features of asthma thought to be caused by aberrant production of Th2 cytokines. Histamine H₄ receptor (H₄R) perturbation has previously been shown to modify acute inflammation and Th2 cytokine production in a murine model of asthma. We examined the ability of H₄R antagonists to therapeutically modify the effects of Th2 cytokine production such as goblet cell hyperplasia (GCH), and collagen deposition in a sub-chronic model of asthma. In addition, effects on Th2 mediated lung dysfunction were also determined.

Methods

Mice were sensitized to ovalbumin (OVA) followed by repeated airway challenge with OVA. After inflammation was established mice were dosed with the H₄R antagonist, JNJ 7777120, or anti-IL-13 antibody for comparison. Airway hyperreactivity (AHR) was measured, lungs lavaged and tissues collected for analysis.

Results

Therapeutic H₄R antagonism inhibited T cell infiltration in to the lung and decreased Th2 cytokines IL-13 and IL-5. IL-13 dependent remodeling parameters such as GCH and lung collagen were reduced. Intervention with H₄R antagonist also improved measures of central and peripheral airway dysfunction.

Conclusions

These data demonstrate that therapeutic H₄R antagonism can significantly ameliorate allergen induced, Th2 cytokine driven pathologies such as lung remodeling and airway dysfunction. The ability of H₄R antagonists to affect these key manifestations of asthma suggests their potential as novel human therapeutics.     

Building a better neonatal mouse model to understand infant respiratory syncytial virus disease

Background

Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and severe RSV infection in infants is associated with asthma development. Today, there are still no vaccines or specific antiviral therapies against RSV. The mechanisms of RSV pathogenesis in infants remain elusive. This is partly due to the fact that the largely-used mouse model is semi-permissive for RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a chimeric virus in which the F protein of A2 strain was replaced with the F protein from the line 19 clinical isolate (rA2-19F).

Methods

Five-day-old pups were infected with the standard laboratory strain A2 or rA2-19F and various immunological and pathophysiological parameters were measured at different time points post infection, including lung histology, bronchoalveolar lavage fluid (BALF) cellularity and cytokines, pulmonary T cell profile, and lung viral load. A cohort of infected neonates were allowed to mature to adulthood and reinfected. Pulmonary function, BALF cellularity and cytokines, and T cell profiles were measured at 6 days post reinfection.

Results

The rA2-19F strain in neonatal mice caused substantial lung pathology including interstitial inflammation and airway mucus production, while A2 caused minimal inflammation and mucus production. Pulmonary inflammation was characterized by enhanced Th2 and reduced Th1 and effector CD8⁺ T cells compared to A2. As with primary infection, reinfection with rA2-19F induced similar but exaggerated Th2 and reduced Th1 and effector CD8⁺ T cell responses. These immune responses were associated with increased airway hyperreactivity, mucus hyperproduction and eosinophilia that was greater than that observed with A2 reinfection. Pulmonary viral load during primary infection was higher with rA2-19F than A2.

Conclusions

Therefore, rA2-19F caused enhanced lung pathology and Th2 and reduced effector CD8⁺ T cell responses compared to A2 during initial infection in neonatal mice and these responses were exacerbated upon reinfection. The exact mechanism is unknown but appears to be associated with increased pulmonary viral load in rA2-19F vs. A2 infected neonatal lungs. The rA2-19F strain represents a better neonatal mouse model of RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0244-0) contains supplementary material, which is available to authorized users.     

Multiple exposures to swine barn air induce lung inflammation and airway hyper-responsiveness

Background

Swine farmers repeatedly exposed to the barn air suffer from respiratory diseases. However the mechanisms of lung dysfunction following repeated exposures to the barn air are still largely unknown. Therefore, we tested a hypothesis in a rat model that multiple interrupted exposures to the barn air will cause chronic lung inflammation and decline in lung function.

Methods

Rats were exposed either to swine barn (8 hours/day for either one or five or 20 days) or ambient air. After the exposure periods, airway hyper-responsiveness (AHR) to methacholine (Mch) was measured and rats were euthanized to collect bronchoalveolar lavage fluid (BALF), blood and lung tissues. Barn air was sampled to determine endotoxin levels and microbial load.

Results

The air in the barn used in this study had a very high concentration of endotoxin (15361.75 ± 7712.16 EU/m³). Rats exposed to barn air for one and five days showed increase in AHR compared to the 20-day exposed and controls. Lungs from the exposed groups were inflamed as indicated by recruitment of neutrophils in all three exposed groups and eosinophils and an increase in numbers of airway epithelial goblet cells in 5- and 20-day exposure groups. Rats exposed to the barn air for one day or 20 days had more total leukocytes in the BALF and 20-day exposed rats had more airway epithelial goblet cells compared to the controls and those subjected to 1 and 5 exposures (P < 0.05). Bronchus-associated lymphoid tissue (BALT) in the lungs of rats exposed for 20 days contained germinal centers and mitotic cells suggesting activation. There were no differences in the airway smooth muscle cell volume or septal macrophage recruitment among the groups.

Conclusion

We conclude that multiple exposures to endotoxin-containing swine barn air induce AHR, increase in mucus-containing airway epithelial cells and lung inflammation. The data also show that prolonged multiple exposures may also induce adaptation in AHR response in the exposed subjects.     

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice

Background

Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.

Methods

AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1^-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1^-/- and WT mice was equivalent, and at 6 months of age, when the Cav1^-/- mice had established airway remodeling.

Results

Cav1^-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1^-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1^-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.

Conclusion

A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1^-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.