Glucocorticoid-induced TNFR family-related protein (GITR)-mediated activation of c-Jun N-terminal kinase (JNK) was shown to regulate the suppressive activity of naturally occurring CD4+CD25+ T regulatory cells (nTregs) in wild-type (WT) hosts. Here, CD4+CD25+ T cells were shown capable of becoming pathogenic effector cells in sensitized and challenged CD8−/− recipient mice. Only GITR-expressing CD4+CD25+ T cells but neither GITR knocked-in CD4+CD25− T cells or GITR-silenced CD4+CD25+ T cells enhanced development of lung allergic responses. Inhibition of JNK in WT nTregs or nTregs from GITR−/− and JNK2−/− mice failed to enhance lung allergic responses in sensitized and challenged CD8−/− recipient mice. The failure to enhance responses was associated with increased bronchoalveolar lavage (BAL) fluid levels of IL-10 and TGF-β and decreased levels of IL-5, IL-6, and IL-13. In contrast, nTregs from JNK1−/− mice, similar to WT nTregs, were fully effective in enhancing responses. Thus, GITR stimulation of nTregs and signaling through JNK2 but not JNK1 triggered the loss of regulatory function while concomitantly gaining pathogenic CD4+ T effector cell function responsible for exacerbating asthma-like immunopathology.
CD4+CD25+ T regulatory cells; plasticity; c-Jun amino-terminal kinase 1 and 2 (JNK1 and JNK2); asthma
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. Both RSV-G and RSV-F glycoproteins play pathogenic roles during infection with RSV. The objective of this study was to compare the effects of anti–RSV-G and anti–RSV-F monoclonal antibodies (mAbs) on airway hyperresponsiveness (AHR) and inflammation after primary or secondary RSV infection in mice. In the primary infection model, mice were infected with RSV at 6 weeks of age. Anti–RSV-G or anti–RSV-F mAbs were administered 24 hours before infection or Day +2 postinfection. In a secondary infection model, mice were infected (primary) with RSV at 1 week (neonate) and reinfected (secondary) 5 weeks later. Anti–RSV-G and anti–RSV-F mAbs were administered 24 hours before the primary infection. Both mAbs had comparable effects in preventing airway responses after primary RSV infection. When given 2 days after infection, anti–RSV-G–treated mice showed significantly decreased AHR and airway inflammation, which persisted in anti–RSV-F–treated mice. In the reinfection model, anti–RSV-G but not anti–RSV-F administered during primary RSV infection in neonates resulted in decreased AHR, eosinophilia, and IL-13 but increased levels of IFN-γ in bronchoalveolar lavage on reinfection. These results support the use of anti–RSV-G in the prevention and treatment of RSV-induced disease.
airway; inflammation; respiratory syncytial virus; anti–respiratory syncytial virus–G; anti–respiratory syncytial virus–F
Changes made in the 8th edition of the Guide for the Care and Use of Laboratory Animals included new recommendations for the amount of space for breeding female mice. Adopting the new recommendations required, in essence, the elimination of trio breeding practices for all institutions. Both public opinion and published data did not readily support the new recommendations. In response, the National Jewish Health Institutional Animal Care and Use Committee established a program to directly compare the effects of breeding format on mouse pup survival and growth. Our study showed an overall parity between trio and pairwise breeding formats on the survival and growth of the litters, suggesting that the housing recommendations for breeding female mice as stated in the current Guide for the Care and Use of Laboratory Animals should be reconsidered.
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
The functional plasticity of CD8+ T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness (AHR) and inflammation and yet remains mechanistically undefined. We demonstrate that CD8+ T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8+ T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the step-wise molecular conversion of CD8+ T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA Pol II recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage following TCR stimulation, indicating that IL-4 induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro an atopic (IL-4) environment poises CD8+ T cells via step-wise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.
CD8 T cells; IL-4; plasticity; IL-13; asthma
Histamine H4 receptor (H4R)-deficient mice (H4R−/−), H4R antagonist-treated WT mice, and WT mice depleted of basophils failed to develop early (EPR) or late phase (LPR) nasal responses following allergen sensitization and challenge. Basophil transfer from WT but not H4R−/− mice restored the EPR and LPR in H4R−/− mice. Following passive sensitization with OVA-specific IgE, FcεRI−/− recipients of WT basophils plus OVA and histamine developed an EPR and LPR. OVA-IgE passively sensitized FcεRI−/− recipients of H4R−/− basophils and OVA and histamine challenge failed to develop an EPR or LPR, and basophils were not detected in nasal tissue. In contrast, recipients of basophils from IL-13−/− and IL-4−/−/IL-13−/− mice developed an EPR but not LPR. These results demonstrate the development of allergic rhinitis proceeded in two distinct stages: histamine release from FcεRI-activated mast cells, followed by histamine-mediated recruitment of H4R-expressing basophils to the nasal cavity and activation through FcεRI.
Basophils; mast cells; histamine H4 receptor; FcεRI; rhinitis
Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in TH2 priming and polarization.
We sought to determine the importance of the TSLP-OX40L axis in neonatal respiratory syncytial virus (RSV) infection.
Mice were initially infected with RSV as neonates or adults and reinfected 5 weeks later. Anti-OX40L or anti-TSLP were administered during primary or secondary infection. Outcomes included assessment of airway function and inflammation and expression of OX40L, TSLP, and IL-12.
OX40L was expressed mainly on CD11c+MHC class II (MHCII)+CD11b+ DCs but not CD103+ DCs. Treatment of neonates with OX40L antibody during primary RSV infection prevented the subsequent enhancement of airway hyperresponsiveness and the development of airway eosinophilia and mucus hyperproduction on reinfection. Administration of anti-TSLP before neonatal RSV infection reduced the accumulation of lung DCs, decreased OX40L expression on lung DCs, and attenuated the enhancement of airway responses after reinfection.
In mice initially infected as neonates, TSLP expression induced by RSV infection is an important upstream event that controls OX40L expression, lung DC migration, and TH2 polarization, accounting for the enhanced response on reinfection.
Respiratory syncytial virus; OX40 ligand; thymic stromal lymphopoietin
Heat shock proteins (HSPs), produced in response to stress are suppressive in disease models. We previously showed that Mycobacterium leprae HSP65 prevented development of airway hyperresponsiveness and inflammation in mice. Our goal here was to define the mechanism responsible for the suppressive effects of HSP. In one in vivo approach, BALB/c mice were sensitized to ovalbumin (OVA) followed by primary OVA challenges. Several weeks later, HSP65 was administered prior to a single, provocative secondary challenge. In a second in vivo approach, the secondary challenge was replaced by intratracheal instillation of allergen-pulsed bone marrow-derived dendritic cells (BMDCs). The in vitro effects of HSP65 on BMDCs were examined in co-culture experiments with CD4+ T cells. In vivo, HSP65 prevented development of airway hyperresponsiveness and inflammation. As well, Th1 cytokine levels in bronchoalveolar lavage (BAL) fluid were increased. In vitro, HSP65 induced notch receptor ligand Delta1 expression on BMDCs and HSP65-treated BMDCs skewed CD4+ T cells to Th1 cytokine production. Thus, HSP65-induced effects on allergen-induced airway hyperresponsiveness and inflammation were associated with increased Delta 1 expression on DCs, modulation of DC function, and CD4+ Th1 cytokine production.
HSP65; asthma; dendritic cells; T cells
Pim kinases are a family of serine/threonine kinases whose activity can be induced by cytokines involved in allergy and asthma. These kinases play a role in cell survival and proliferation, but have not been examined, to the best of our knowledge, in the development of allergic disease. This study sought to determine the role of Pim1 kinase in the development of allergic airway responses. Mice were sensitized and challenged with antigen (primary challenge), or were sensitized, challenged, and rechallenged with allergen in a secondary model. To assess the role of Pim1 kinase, a small molecule inhibitor was administered orally after sensitization and during the challenge phase. Airway responsiveness to inhaled methacholine, airway and lung inflammation, cell composition, and cytokine concentrations were assessed. Lung Pim1 kinase concentrations were increased after ovalbumin sensitization and challenge. In the primary allergen challenge model, treatment with the Pim1 kinase inhibitor after sensitization and during airway challenges prevented the development of airway hyperresponsiveness, eosinophilic airway inflammation, and goblet cell metaplasia, and increased Th2 cytokine concentrations in bronchoalveolar fluid in a dose-dependent manner. These effects were also demonstrated after a secondary allergen challenge, where lung allergic disease was established before treatment. After treatment with the inhibitor, a significant reduction was evident in the number of CD4+ and CD8+ T cells and concentrations of cytokines in the airways. The inhibition of Pim1 kinase was effective in preventing the development of airway hyperresponsiveness, airway inflammation, and cytokine production in allergen-sensitized and allergen-challenged mice. These data identify the important role of Pim1 kinase in the full development of allergen-induced airway responses.
airway hyperresponsiveness; inflammation; Pim1 kinase; T cells
Activation of the alternative pathway of complement plays a critical role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in mice. Endogenous factor H, a potent inhibitor of the alternative pathway, is increased in the airways of sensitized and challenged mice, but its role in regulating inflammation or AHR has been unknown. We found that blocking the tissue-binding function of factor H with a competitive antagonist increased complement activation and tissue inflammation after allergen challenge of sensitized mice. Conversely, administration of a fusion protein that contains the iC3b/C3d binding region of complement receptor 2 (CR2) linked to the inhibitory region of factor H (CR2-fH), a molecule directly targeting complement activating surfaces, protected mice in both primary and secondary challenge models of AHR and lung inflammation. Thus, although endogenous factor H does play a role in limiting the development of AHR, strategies to deliver the complement regulatory region of factor H specifically to the site of inflammation provide greater protection than that afforded by endogenous regulators. Such an agent may be an effective therapy for the treatment of asthma.
Alternative pathway; factor H; airway hyperresponsiveness; inflammation
Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation.
Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR.
Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge.
These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.
Asthma; Dendritic cells; Endotoxin; Notch ligands
Rationale: Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway.
Objectives: To determine the effects of montelukast (MK), a cysteinyl leukotriene (cysLT) receptor antagonist, in primary and secondary RSV-infected newborn and adult mice.
Methods: BALB/c mice were infected with RSV at 1 week (neonate) or 6 to 8 weeks (adult) of age and reinfected 5 weeks later. MK was administered 1 day before the initial infection and through Day 6 after infection. Seven days after primary or secondary infection, airway function was assessed by lung resistance to increasing doses of inhaled methacholine; lung inflammation, goblet cell metaplasia, and cytokine levels in bronchoalveolar lavage fluid were monitored.
Measurements and Main Results: RSV infection induced cysLT release in bronchoalveolar lavage fluid. MK decreased RSV-induced AHR, airway inflammation, and increased IFN-γ production in primary infected adult and neonatal mice. MK, administered during initial infection of neonates but not during secondary infection, prevented subsequent enhancement of AHR, airway eosinophilia, and mucus hyperproduction upon reinfection.
Conclusions: MK attenuated the initial responses to primary RSV infection in both age groups and altered the consequences of RSV reinfection in mice initially infected as neonates. These data support an important role for cysLT in RSV-induced AHR and inflammation.
airway; inflammation; RSV; cysteinyl leukotrienes
Although implicated in the disease, the specific contributions of FcεRI and IL-13 to the pathogenesis of peanut-induced intestinal allergy are not well defined.
To determine the contributions of FcεRI, IL-13, and mast cells to the development of intestinal mucosal responses in a mouse model of peanut-induced intestinal allergy.
Sensitized wild-type (WT), FcεRI-deficient (FcεRI−/−), and mast cell-deficient (KitW-sh/W-sh) mice received peanut orally every day for 1 week. Bone marrow-derived mast cells (BMMC) from WT, FcεRI−/−, IL- 4−/−, IL-13−/−, and IL- 4/IL-13−/− mice were differentiated and transferred into WT, FcεRI−/−, and KitW-sh/W-sh recipients. BMMC from WT and UBI-GFP/BL6 mice were differentiated and transferred into WT and KitW-sh/W-sh mice. Blockade of IL-13 was achieved using IL- 13Ra2-IgG fusion protein.
FcεRI−/− mice showed decreased intestinal inflammation (mast cell and eosinophil numbers) and goblet cell metaplasia, and reduced levels of IL-4, IL-6, IL-13, and IL-17A mRNA expression in the jejunum. Transfer of WT BMMC to FcεRI−/− recipients restored their ability to develop intestinal allergic responses compared to transfer of FcεRI−/−, IL-13−/−, or IL-4/IL-13−/−BMMC. FcεRI−/− mice exhibited lower IL-13 levels and treatment of WT mice with IL-13Rα2 prevented peanut-induced intestinal allergy and inflammation.
These data indicate that the development of peanut-induced intestinal allergy is mediated through a mast cell-dependent, IgE-FcεRI-IL-13 pathway. Targeting IL-13 may be a potential treatment for IgE-mediated peanut allergic responses in the intestine.
Peanut; intestinal allergy; mast cell; IgE; FcεRI; IL-13
Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract diseases (bronchiolitis and pneumonia) during infancy and early childhood. There is increasing evidence which indicates that severe pulmonary disease caused by RSV infection in infancy is associated with recurrent wheezing and development of asthma later in childhood. However, the underlying mechanisms linking RSV infection to persistent airway hyperresponsiveness and dysfunction are not fully defined. To study these processes in ways which are not available in humans, animal models have been established and have provided valuable insight into the pathophysiology of RSV-induced disease. In this paper, we discuss experimental models of RSV infection in mice and highlight a new investigative approach in which mice are initially infected as neonates and then reinfected later in life. The findings shed light on the mechanisms underlying the association between early severe RSV infection and development of asthma later in childhood.
Naturally occurring CD4+CD25+Foxp3+ T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-β. nTregs from CD8−/− mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-β, and high levels of IL-6. Administration of anti–IL-6 or anti–IL-6R to wild-type recipients prior to transfer of CD8−/− nTregs restored suppression. nTregs from IL-6−/− mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8+ cells. Transfer of nTregs from CD8−/− donors reconstituted with CD8+ T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8–T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.
Dendritic cells (DCs) are considered to be the most efficient antigen-presenting cells. Intratracheal administration of allergen-pulsed bone marrow–derived dendritic cells (BMDCs) before allergen challenge induces airway hyperresponsiveness (AHR) and inflammation. Ovalbumin (OVA)-pulsed BMDCs from wild-type (WT) mice were transferred into naive WT, CD4−/−, CD8−/−, or IL-13−/− mice. Two days (short protocol) or 10 days (long protocol) after BMDC transfer, mice were challenged with 1% OVA for 3 days and assayed 2 days later. Transfer of OVA-primed BMDCs into BALB/c or C57BL/6 mice elicited AHR in both protocols. Airway eosinophilia, Th2 cytokines, or goblet cell metaplasia were increased in the long but not short protocol. Lung T cells from both protocols produced Th2 cytokines in response to OVA in vitro. Carboxyfluorescein diacetate succinimidylester–labeled BMDCs were observed in bronchoalveolar lavage (BAL) fluid and lung parenchyma at early time points, and were detected in draining lymph nodes 48 hours after transfer. CD8−/− mice developed AHR comparable to WT mice in the short protocol, but decreased levels of AHR, airway eosinophilia, Th2 cytokines in BAL fluid, and goblet cell metaplasia compared with WT mice in the long protocol. CD4−/− or IL-13−/− mice did not develop AHR or airway inflammation in either protocol. These data suggest that allergen-pulsed BMDCs initiate development of AHR that is dependent initially on CD4+ T cells, and at later time periods on CD8+ T cells and IL-13. Thus, the timing of allergen challenge after transfer of allergen-pulsed BMDC affects the development of AHR and airway inflammation.
dendritic cells; CD8+ T cells; airway hyperresponsiveness
Previous studies have shown that leukotriene B4 (LTB4), a proinflammatory lipid mediator, is linked to the development of airway hyperresponsiveness through the accumulation of IL-13–producing CD8+ T cells, which express a high affinity receptor for LTB4, BLT1 (Miyahara et al., Am J Respir Crit Care Med 2005;172:161–167; J Immunol 2005;174:4979–4984). By using leukotriene A4 hydrolase–deficient (LTA4H−/−) mice, which fail to synthesize LTB4, we determined the role of this lipid mediator in allergen-induced airway responses. Two approaches were used. In the first, LTA4H−/− mice and wild-type (LTA4H+/+) mice were systemically sensitized and challenged via the airways to ovalbumin. In the second, mice were passively sensitized with anti-ovalbumin IgE and exposed to ovalbumin via the airways. Mast cells were generated from bone marrow of LTA4H+/+ mice or LTA4H−/− mice. After active sensitization and challenge, LTA4H−/− mice showed significantly lower airway hyperresponsiveness compared with LTA4H+/+ mice, and eosinophil numbers and IL-13 levels in the bronchoalveoloar lavage of LTA4H−/− mice were also significantly lower. LTA4H−/− mice also showed decreased airway reactivity after passive sensitization and challenge. After LTA4H+/+ mast cell transfer, LTA4H−/− mice showed increased airway reactivity after passive sensitization and challenge, but not after systemic sensitization and challenge. These data confirm the important role for LTB4 in the development of altered airway responses and suggest that LTB4 secretion from mast cells is critical to eliciting increased airway reactivity after passive sensitization with allergen-specific IgE.
rodent; T cells; cytokines; lipid mediators; lung
γδ T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of γδ T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total γδ T cells or specific subsets of γδ T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-δ−/− mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of γδ T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vγ1+, but not of Vγ4+ T cells, reduced O3-induced AHR, and transfer of total γδ T cells or Vγ1+ T cells to TCR-δ−/− mice restored AHR. After transfer of Vγ1+ cells to TCR-δ−/− mice, restoration of AHR after O3 exposure was blocked by anti–TNF-α. However, AHR could be restored in TCR-δ−/−mice by transfer of γδ T cells from TNF-α–deficient mice, indicating that another cell type was the source of TNF-α. These results demonstrate that TNF-α and activation of Vγ1+ γδ T cells are required for the development of AHR after O3 exposure.
ozone; airway responsiveness; γδ T cells; TNF-α
Asthma is a complex syndrome with many clinical phenotypes in children and adults. Despite the rapidly increasing prevalence, clinical investigation and epidemiological studies of asthma, the successful introduction of new drugs has been limited due to the different disease phenotypes and ethical issues. Mouse models of asthma replicate many of the features of human asthma, including airway hyperreactivity, and airway inflammation. Therefore, examination of disease mechanisms in mice has been used to elucidate asthma pathology and to identify and evaluate new therapeutic agents. In this article, we discuss the various animal models of asthma with a focus on mouse strains, allergens, protocols, and outcome measurements.
animal model; asthma; mouse
The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17β-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1–dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.
estrogen; sex; airway hyperresponsiveness; EFS; neuronal activation
Rationale: Severe respiratory syncytial virus (RSV) bronchiolitis has been associated with deficient IFN-γ production in humans, but the role of this cytokine in determining the outcome of reinfection is unknown.
Objectives: To define the role of IFN-γ in the development of RSV-mediated airway hyperresponsiveness (AHR) and lung histopathology in mice.
Methods: Wild-type (WT) and IFN-γ knockout mice were infected with RSV in the newborn or weaning stages and reinfected 5 weeks later. Airway responses were assessed on Day 6 after the primary or secondary infection.
Measurements and Main Results: Both WT and IFN-γ knockout mice developed similar levels of AHR and airway inflammation after primary infection. After reinfection, IFN-γ knockout mice, but not WT mice, developed AHR, airway eosinophilia, and mucus hyperproduction. Intranasal administration of IFN-γ during primary infection but not during reinfection prevented the development of these altered airway responses on reinfection in IFN-γ knockout mice. Adoptive transfer of WT T cells into IFN-γ knockout mice before primary infection restored IFN-γ production in the lungs and prevented the development of altered airway responses on reinfection. Treatment of mice with IFN-γ during primary neonatal infection prevented the enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction on reinfection.
Conclusions: IFN-γ production during primary RSV infection is critical to the development of protection against AHR and lung histopathology on reinfection. Provision of IFN-γ during primary infection in infancy may be a potential therapeutic approach to alter the course of RSV-mediated long-term sequelae.
respiratory syncytial virus; interferon-γ; asthma; airway hyperresponsiveness; mice
Adoptive transfer of in vivo–primed CD8+ T cells or in vitro–generated effector memory CD8+ T (TEFF) cells restores airway hyperresponsiveness (AHR) and airway inflammation in CD8-deficient (CD8−/−) mice. Examining transcription levels, there was a strong induction of Notch1 in TEFF cells compared with central memory CD8+ T cells. Treatment of TEFF cells with a γ-secretase inhibitor (GSI) strongly inhibited Notch signaling in these cells, and after adoptive transfer, GSI-treated TEFF cells failed to restore AHR and airway inflammation in sensitized and challenged recipient CD8−/− mice, or to enhance these responses in recipient wild-type (WT) mice. These effects of GSI were also associated with increased expression of the Notch ligand Delta1 in TEFF cells. Treatment of sensitized and challenged WT mice with Delta1-Fc resulted in decreased AHR and airway inflammation accompanied by higher levels of interferon γ in bronchoalveolar lavage fluid. These results demonstrate a role for Notch in skewing the T cell response from a T helper (Th)2 to a Th1 phenotype as a consequence of the inhibition of Notch receptor activation and the up-regulation of the Notch ligand Delta1. These data are the first to show a functional role for Notch in the challenge phase of CD8+ T cell–mediated development of AHR and airway inflammation, and identify Delta1 as an important regulator of allergic airway inflammation.
IL-18 is known to induce IFN-γ production, which is enhanced when combined with IL-2. In the present study, we investigated whether the combination of exogenous IL-2 and IL-18 alters airway hyperresponsiveness (AHR) and airway inflammation. Sensitized mice exposed to ovalbumin (OVA) challenge developed AHR, inflammatory cells in the bronchoalveolar lavage (BAL) fluid, and increases in levels of Th2 cytokines and goblet cell numbers. The combination of IL-2 and IL-18, but neither alone, prevented these changes while increasing levels of IL-12 and IFN-γ. The combination of IL-2 and IL-18 was ineffective in IFN-γ–deficient and signal transducer and activator of transcription (STAT)4-deficient mice. Flow cytometry analysis showed significant increases in numbers of IFN-γ–positive natural killer (NK) cells in the lung after treatment with the combination therapy, and transfer of lung NK cells isolated from sensitized and challenged mice treated with the combination significantly suppressed AHR and BAL eosinophilia. These data demonstrate that the combination of IL-2 and IL-18 prevents AHR and airway inflammation, likely through IL-12–mediated induction of IFN-γ production in NK cells.
IL-2; IL-18; STAT4; IFN-γ; airway hyperresponsiveness
RANTES (CC chemokine ligand 5) contributes to airway inflammation through accumulation of eosinophils, but the exact role of RANTES (CCL5) is not defined. C57BL/6 mice, sensitized by injection of ovalbumin (OVA) on Days 1 and 14, were challenged with OVA on Days 28, 29, and 30 (3 challenges, short-term–challenge model) or on Days 28, 29, 30, 36, 40, 44, and 48 (7 challenges, repeated–challenge model) and evaluated 48 h later. Anti-mouse RANTES was given intravenously, and recombinant mouse RANTES or PBS was given intratracheally. These reagents were given on Days 28, 29, and 30 in the short-term–challenge study and on Days 44 and 48 in the repeated-challenge study. After short-term challenge, there were no effects after administration of anti-RANTES or RANTES. In the repeated-challenge study, although control mice showed a decrease in airway hyperresponsiveness, administration of anti-RANTES sustained and enhanced airway hyperresponsiveness and increased goblet cell numbers. In contrast, administration of RANTES normalized airway function but reduced goblet cell numbers. IL-12 and IFN-γ levels in BAL decreased in the anti-RANTES group and increased in the RANTES group. IFN-γ–producing CD4 T cells in lung, and IFN-γ production from lung T cells in response to OVA in the anti-RANTES group, were significantly decreased but were increased in the RANTES group. Anti–IFN-γ, administered with RANTES, decreased the effects of RANTES on AHR after repeated challenge. These data indicate that RANTES plays a role in the regulation of airway function after repeated allergen challenge, in part through modulation of levels of IFN-γ and IL-12.
airway hyperresponsiveness; IFN-γ; IL-12; RANTES (CCL5)