ATP binding cassette transporter G1 deletion induces IL-17-dependent dysregulation of pulmonary adaptive immunity.

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

IL-35 production by inducible costimulator (ICOS)-positive regulatory T cells reverses established IL-17-dependent allergic airways disease.

BACKGROUND: Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined. OBJECTIVE: We sought to identify the regulatory cells and molecules that suppress IL-17-dependent allergic airways disease. METHODS: Mice were sensitized by means of airway instillations of ovalbumin together with low levels of LPS. Leukocyte recruitment to the lung and AHR were assessed after daily challenges with aerosolized ovalbumin. Flow cytometry, quantitative PCR, and gene-targeted mice were used to identify naturally arising subsets of regulatory T (Treg) cells and their cytokines required for the suppression of established allergic airway disease. RESULTS: Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR decreased, even though pulmonary levels of T(H)17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg cell subset expressing both forkhead box protein 3 and inducible costimulator. These Treg cells also expressed the regulatory cytokines IL-10, TGF-ß, and IL-35. Whereas IL-10 and TGF-ß were dispensable for suppression of AHR, IL-35 was required. CONCLUSION: IL-35 production by inducible costimulator-positive Treg cells can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. Targeting this pathway might therefore be of therapeutic value for treating allergic asthma in human subjects.

Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness.

RATIONALE: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant. OBJECTIVES: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum. METHODS: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR). MEASUREMENTS AND MAIN RESULTS: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17-dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR. CONCLUSIONS: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma.

High-resolution mapping of quantitative trait loci affecting increased life span in Drosophila melanogaster.

Limited life span and senescence are near-universal characteristics of eukaryotic organisms, controlled by many interacting quantitative trait loci (QTL) with individually small effects, whose expression is sensitive to the environment. Analyses of mutations in model organisms have shown that genes affecting stress resistance and metabolism affect life span across diverse taxa. However, there is considerable segregating variation for life span in nature, and relatively little is known about the genetic basis of this variation. Replicated lines of Drosophila that have evolved increased longevity as a correlated response to selection for postponed senescence are valuable resources for identifying QTL affecting naturally occurring variation in life span. Here, we used deficiency complementation mapping to identify at least 11 QTL on chromosome 3 that affect variation in life span between five old (O) lines selected for postponed senescence and their five base (B) population control lines. Most QTL were sex specific, and all but one affected multiple O lines. The latter observation is consistent with alleles at intermediate frequency in the base population contributing to the response to selection for postponed senescence. The QTL were mapped with high resolution and contained from 12 to 170 positional candidate genes.

Genomic response to selection for postponed senescence in Drosophila.

Limited lifespan and senescence are quantitative traits, controlled by many interacting genes with individually small and environmentally plastic effects, complicating genetic analysis. We performed genome wide analysis of gene expression for two Drosophila melanogaster lines selected for postponed senescence and one control, unselected line to identify candidate genes affecting lifespan as well as variation in lifespan. We obtained gene expression profiles for young flies of all lines, all lines at the time only 10% of the control lines survived, and the time at which 10% of the selected lines survived. Transcriptional responses to aging involved 19% of the genome. The transcriptional signature of aging involved the down-regulation of genes affecting proteolysis, metabolism, oxidative phosphorylation, and mitochrondrial function; and the up-regulation of genes affecting protein synthesis, immunity, defense responses, and the detoxification of xenobiotic substances. The transcriptional signature of postponed senescence involved the up-regulation of proteases and phosphatases and genes affecting detoxification of xenobiotics; and the down-regulation of genes affecting immunity, defense responses, metabolism and muscle function. Functional tests of 17 mutations confirmed 12 novel genes affecting Drosophila lifespan. Identification of genes affecting longevity by analysis of gene expression changes in lines selected for postponed senescence thus complements alternative genetic approaches.

The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens.

Allergic asthma is a complex disease characterized by eosinophilic pulmonary inflammation, mucus production and reversible airway obstruction. Exposure to indoor allergens is a risk factor for asthma, but this disease is also associated with high household levels of total and particularly Gram-negative bacteria. The ability of bacterial products to act as adjuvants suggests they might promote asthma by priming allergic sensitization to inhaled allergens. In support of this idea, house dust extracts (HDEs) can activate antigen-presenting dendritic cells (DCs) in vitro and promote allergic sensitization to inhaled innocuous proteins in vivo. It is unknown which microbial products provide most of the adjuvant activity in HDEs. A screen for adjuvant activity of microbial products revealed that the bacterial protein flagellin (FLA) stimulated strong allergic airway responses to an innocuous inhaled protein, ovalbumin (OVA). Moreover, Toll-like receptor 5 (TLR5), the mammalian receptor for FLA, was required for priming strong allergic responses to natural indoor allergens present in HDEs. In addition, individuals with asthma have higher serum levels of FLA-specific antibodies as compared to nonasthmatic individuals. Together, these findings suggest that household FLA promotes the development of allergic asthma by TLR5-dependent priming of allergic responses to indoor allergens.