Cyp11a1, a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone.Cyp11a1 expression is increases in activated T cells.
To determine the role of Cyp11a1 activation in the development of peanut allergy and T helper cell functional differentiation.
A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and -challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on Th1, Th2, and Th17 differentiation were determined. Cyp11a1 gene silencing was performed using Cyp11a1-targeted short hairpin RNA.
Peanut sensitization and challenge resulted in diarrhea, inflammation and increased levels of Cyp11a1, IL-13, and IL-17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG-treatment decreased IL13 and IL17A mRNA expression in the small intestine without impacting Cyp11a1 mRNA or protein levels. In vitro, the inhibitor decreased levels of IL13 and IL17A mRNA and protein in differentiated Th2 and Th17 CD4 T cells, respectively, without affecting GATA3, RORγt or Th1 cells and IFNG and T-bet expression. shRNA-mediated silencing of Cyp11a1 in polarized Th2 CD4 T cells significantly decreased levels of pregnenolone, and IL13 mRNA and protein.
Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut allergic responses through effects on steroidogenesis, an essential pathway in Th2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.
Cyp11a1; peanut allergy; Th2; Th17; CD4 T cells
Increasing epidemiological data identify a link between obesity and asthma incidence and severity. Based on experimental data, it is possible that shared inflammatory mechanisms play a role in determining this linkage. Although controversial, the role of adipokines may be central to this association and the maintenance of the asthma phenotype. While leptin and adiponectin have a causal link to experimental asthma in mice, data in humans are less conclusive. Recent studies demonstrate that adipokines can regulate the survival and function of eosinophils and that these factors can affect eosinophil trafficking from the bone marrow to the airways. In addition, efferocytosis, the clearance of dead cells, by airway macrophages or blood monocytes appears impaired in obese asthmatics and is inversely correlated with glucocorticoid responsiveness. This review examines the potential mechanisms linking obesity to asthma.
Obesity; asthma; adipokines; eosinophils; macrophages; adipose tissue
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
The provirus integration site for Moloney murine leukemia virus (Pim) 1 kinase is an oncogenic serine/threonine kinase implicated in cytokine-induced cell signaling, whereas Runt-related transcription factor (Runx) has been implicated in the regulation of T-cell differentiation. The interaction of Pim1 kinase and Runx3 in the pathogenesis of peanut allergy has not been defined.
We sought to determine the effects of Pim1 kinase modulation on Runx3 expression and TH2 and TH17 cell function in an experimental model of peanut allergy. Methods: A Pim1 kinase inhibitor was administered to peanut-sensitized and challenged wild-type and Runx3+/− mice. Symptoms, intestinal inflammation, and Pim1 kinase and Runx3 mRNA expression and protein levels were assessed. The effects of Pim1 kinase inhibition on TH1, TH2, and TH17 differentiation in vivo and in vitro were also determined.
Peanut sensitization and challenge resulted in accumulation of inflammatory cells and goblet cell metaplasia and increased levels of Pim1 kinase and TH2 and TH17 cytokine production but decreased levels of Runx3 mRNA and protein in the small intestines of wild-type mice. All of these findings were normalized with Pim1 kinase inhibition. In sensitized and challenged Runx3+/− mice, inhibition of Pim1 kinase had less effect on the development of the full spectrum of intestinal allergic responses. In vitro inhibition of Pim1 kinase attenuated TH2 and TH17 cell differentiation and expansion while maintaining Runx3 expression in T-cell cultures from wild-type mice; these effects were reduced in T-cell cultures from Runx3+/− mice.
These data support a novel regulatory axis involving Pim1 kinase and Runx3 in the control of food-induced allergic reactions through the regulation of TH2 and TH17 differentiation.
Pim1 kinase; Runx3; peanut; intestinal allergy; TH2; TH17
Patients, especially young children, with atopic dermatitis are at an increased risk of developing eczema vaccinatum, a severe reaction to the smallpox vaccine, either through direct vaccination or indirect contact with a person recently vaccinated.
Using a mouse model of infection, the severity of vaccinia-induced lesions was assessed from their appearance and viral DNA content. The response to vaccinia inoculation was assessed in young and adult mice, allergen-sensitized mice, and in mast cell-deficient mice.
Young age, sensitization to an allergen prior to infection, and a mast cell deficit, accomplished by using mast cell-deficient mice, resulted in more severe viral lesions at the site of inoculation, according to lesion appearance and viral DNA content. All three factors combined demonstrated maximal susceptibility, characterized by the severity of primary lesions and the development of secondary (satellite) lesions, as occurs in eczema vaccinatum in humans. Resistance to the appearance of satellite lesions could be restored by adoptive transfer of bone marrow-derived mast cells from either wild-type or cathelicidin-related antimicrobial peptide-deficient mice. Primary lesions were more severe following the latter transfer, indicating that cathelicidin-related antimicrobial peptide does contribute to the protective activity of mast cells against infection.
The combination of young age, allergen sensitization and a mast cell deficit resulted in the most severe lesions, including satellite lesions. Understanding the factors determining the relative resistance/sensitivity to vaccinia virus will aid in the development of strategies for preventing and treating adverse reactions which can occur after smallpox vaccination.
Allergen sensitization; Eczema vaccinatum; Mast cells; Neutrophils; Vaccinia virus
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
Most of the studies investigating the effectiveness of blocking the leukotriene B4 (LTB4) receptor 1 (BLT1) have been performed in models of primary or acute allergen challenge. The role of the LTB4-BLT1 pathway in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have been established, has not been defined. We investigated the effects of blocking BLT1 on early- and late-phase development of AHR and airway inflammation in previously sensitized and challenged mice. Female BALB/c mice were sensitized (Days 1 and 14) and challenged (primary, Days 28–30) with ovalbumin. On Day 72, mice were challenged (secondary) with a single OVA aerosol, and the early and late phases of AHR and inflammation were determined. Specific blockade of BLT1 was attained by oral administration of a BLT1 antagonist on Days 70 through 72. Administration of the antagonist inhibited the secondary ovalbumin challenge–induced alterations in airway responses during the late phase but not during the early phase, as demonstrated by decreases in AHR and in bronchoalveolar lavage neutrophilia and eosinophilia 6 and 48 hours after secondary challenge. The latter was associated with decreased levels of KC protein, macrophage inflammatory protein 2, and IL-17 in the airways. These data identify the importance of the LTB4-BLT1 pathway in the development of late–phase, allergen-induced airway responsiveness after secondary airway challenge in mice with established airway disease.
LAR; EAR; established asthma; BLT1 antagonist
Cartilage-hair hypoplasia (CHH) is characterized by metaphyseal dysplasia, bone marrow failure, increased risk of malignancies, and a variable degree of immunodeficiency. CHH is caused by mutations in the RNA component of the mitochondrial RNA processing (RMRP) endoribonuclease gene, which is involved in ribosomal assembly, telomere function, and cell cycle control.
We aimed to define thymic output and characterize immune function in a cohort of patients with molecularly defined CHH with and without associated clinical immunodeficiency.
We studied the distribution of B and T lymphocytes (including recent thymic emigrants), in vitro lymphocyte proliferation, cell cycle, and apoptosis in 18 patients with CHH compared with controls.
Patients with CHH have a markedly reduced number of recent thymic emigrants, and their peripheral T cells show defects in cell cycle control and display increased apoptosis, resulting in poor proliferation on activation.
These data confirm that RMRP mutations result in significant defects of cell-mediated immunity and provide a link between the cellular phenotype and the immunodeficiency in CHH.
Cartilage-hair hypoplasia; RMRP; immunodeficiency; T lymphocytes; thymus; cell cycle; apoptosis
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.
Patients with hypomorphic nuclear factor-κB essential modulator (NEMO) mutations have extensive phenotypic variability that can include atypical infectious susceptibility.
This study may provide important insight into immunologic mechanisms of host defense.
Immunologic evaluation, including studies of Toll-like receptor (TLR) function, was performed in a 6-month-old boy with normal ectodermal development who was diagnosed with Pneumocystis pneumonia and cytomegalovirus sepsis.
Genomic and cDNA sequencing demonstrated a novel NEMO missense mutation, 337G->A, predicted to cause a D113N (aspartic acid to asparagine) substitution in the first coiled-coil region of the NEMO protein. Quantitative serum immunoglobulins, lymphocyte subset numbers, and mitogeninduced lymphocyte proliferation were essentially normal. The PBMC responses to TLR ligands were also surprisingly normal, whereas natural killer cell cytolytic activity, T-cell proliferative responses to specific antigens, and T-cell receptor–induced NF-κB activation were diminished.
Unlike the unique NEMO mutation described here, the most commonly reported mutations are clustered at the 3′ end in the tenth exon, which encodes a zinc finger domain. Because specific hypomorphic variants of NEMO are associated with distinctive phenotypes, this particular NEMO mutation highlights a dispensability of the region including amino acid 113 for TLR signaling and ectodysplasin A receptor function. This region is required for certain immunoreceptor functions as demonstrated by his susceptibility to infections as well as natural killer cell and T-cell defects.
NEMO; Toll-like receptors
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
An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-κB and MAPK pathways were the focus of investigation.
Mouse bone marrow-derived mast cells were grown in culture and stimulated with LPS. MAPKs and NF-κB were monitored by immunoblot analysis. ERK, JNK and p38 MAPK were inhibited using siRNAs or a pharmacological inhibitor. Accumulation of the p65 component of NF-κB was inhibited by siRNA and NF-κB activation was inhibited by overexpression of IκBα. MEKK2 or MEKK3 were overexpressed by transfection. The effects of all of these treatments on CRAMP gene expression were monitored by RT-PCR.
Inhibition of ERK, JNK or p38 MAPK had little discernible effect on LPS-inducible CRAMP gene expression. Overexpression of MEKK2 or MEKK3 likewise had little impact. However, inhibition of the accumulation of p65 NF-κB prevented LPS-induced CRAMP mRNA. An important role for NF-κB in CRAMP gene expression was confirmed by overexpression of IκBα, which reduced both basal and induced levels of CRAMP mRNA.
NF-κB, but not MAPKs, plays an important role in LPS-mediated induction of CRAMP gene in mast cells. Defects which inhibit NF-κB activity may increase susceptibility to bacterial and viral pathogens which are sensitive to cathelicidins.
Cathelicidin-related antimicrobial peptide; Inflammation; Mast cells; Transcription factors
Rationale: The role of airway microbiome in corticosteroid response in asthma is unknown.
Objectives: To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids.
Methods: 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation.
Measurements and Main Results: Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β–associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids.
Conclusions: A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.
microbiome; asthma; corticosteroids
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
Asthma currently affects the lives of more than 30 million Americans from infancy to the elderly. In many ways, pediatric asthma differs from adult asthma, including childhood-onset adult asthma. Despite many advances in our understanding of the disease, the natural history of asthma is not well defined, especially in different subsets of patients. For many with allergic asthma the disease has its origins in early childhood, associated with early sensitization to aeroallergens and exposure to repeated viral infections. These early life exposures, coupled with genetically determined susceptibility, have a major impact on the natural history of the disease. A number of risk factors during the critical early stages in the initiation of asthma have been associated with subsequent outcomes. In addition, protective factors linked to early life experiences have also been delineated which may impact the development of atopy and asthma and reduce the prevalence of these diseases. Cumulatively, the data highlight the critical nature of this early period in which immune/inflammatory responses in the lung are initiated and serve to maintain the disease in subsequent years.
asthma; children; predictors; persistence
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