Gregory S. Whitehead

In utero supplementation with methyl donors enhances allergic airway disease in mice

Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.

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.

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-β, and IL-35. Whereas IL-10 and TGF-β 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.

Pulmonary CD103(+) dendritic cells prime Th2 responses to inhaled allergens.

Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103+ DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103+ DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11bhi DCs primed Th1 differentiation. Moreover, mice lacking CD103+ DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103+ DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103+ DCs have a significant role in priming Th2 responses to inhaled allergens.

TLR4 Signaling Attenuates Ongoing Allergic Inflammation

The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure.

Mechanisms of allergy and clinical immunologyIL-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-β, and IL-35. Whereas IL-10 and TGF-β 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.

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.

Migratory properties of pulmonary dendritic cells are determined by their developmental lineage.

The chemokine receptor, CCR7, directs the migration of dendritic cells (DCs) from peripheral tissue to draining lymph nodes (LNs). However, it is unknown whether all pulmonary DCs possess migratory potential. Using novel Ccr7gfp reporter mice, we found that Ccr7 is expressed in CD103+ and a CD14med/lo subset of CD11bhi classical (c)DCs but not in monocyte-derived (mo)DCs, including Ly-6ChiCD11bhi inflammatory DCs and CD14hiCD11bhi DCs. Consequently, cDCs migrated to lung-draining LNs but moDCs did not. Mice lacking the chemokine receptor, CCR2, also lacked inflammatory DCs in the lung after lipopolysaccharide inhalation but retained normal levels of migratory DCs. Conversely, the lungs of fms-like tyrosine kinase 3 ligand (Flt3L)-deficient mice lacked cDCs but retained moDCs, which were functionally mature but did not express Ccr7 and were uniformly non-migratory. Thus, the migratory properties of pulmonary DCs are determined by their developmental lineage.

Lung function and airway inflammation in rats following exposure to combustion products of carbon-graphite/epoxy composite material: comparison to a rodent model of acute lung injury.

Pulmonary function and inflammation in the lungs of rodents exposed by inhalation to carbon/graphite/epoxy advanced composite material (ACM) combustion products were compared to that of a rodent model of acute lung injury (ALI) produced by pneumotoxic paraquat dichloride. This investigation was undertaken to determine if short-term exposure to ACM smoke induces ALI; and to determine if smoke-related responses were similar to the pathogenic mechanisms of a model of lung vascular injury. We examined the time-course for mechanical lung function, infiltration of inflammatory cells into the lung, and the expression of three inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Male Fischer-344 rats were either exposed to 26.8-29.8 g/m(3) nominal concentrations of smoke or were given i.p. injections of paraquat dichloride. Measurements were determined at 1, 2, 3, and 7 days post exposure. In the smoke-challenged rats, there were no changes in lung function indicative of ALI throughout the 7-day observation period, despite the acute lethality of the smoke atmosphere. However, the animals showed signs of pulmonary inflammation. The expression of TNF-alpha was significantly increased in the lavage fluid 1 day following exposure, which preceded the maximum leukocyte infiltration. MIP-2 levels were significantly increased in lavage fluid at days 2, 3, and 7. This followed the leukocyte infiltration. IFN-gamma was significantly increased in the lung tissue at day 7, which occurred during the resolution of the inflammatory response. The paraquat, which was also lethal to a small percentage of the animals, caused several physiologic changes characteristic of ALI, including significant decreases in lung compliance, lung volumes/capacities, distribution of ventilation, and gas exchange capacity. The expression of TNF-alpha and MIP-2 increased significantly in the lung tissue as well as in the lavage fluid. Increased MIP-2 levels also preceded the maximum neutrophil infiltration. The differences in the time-course and primary site of TNF-alpha, MIP-2, and IFN-gamma expression; and the differences in the temporal relationship between their expression and infiltration of inflammatory cells may have accounted for the differences in lung function between paraquat treated and ACM smoke exposed animals.

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.