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. Keywords: asthma; lung; immunityThe most widely used mouse model of asthma involves allergic sensitization by intraperitoneal injections of ovalbumin (OVA) complexed with the Th2 adjuvant, aluminum hydroxide (alum) (8). These animals are typically challenged by intranasal instillation, or an aerosol, of OVA. Variations of this model have been used for many years and have been valuable for studying Th2-mediated responses in allergic pulmonary inflammation. However, some features of this model are inconsistent with human asthma. For example, the level of airway eosinophils can reach 80% in this mouse model (8), whereas in human patients it is rarely greater than 5% (9). Also, in this model, neutrophil recruitment to the airway is transient (10) and dispensable for airway hyperreactivity (AHR) (11). Therefore the alum-mediated model is not useful for studying the function of neutrophils in asthma. We hypothesized that some differences between this mouse model and human asthma might reflect the impact of the unique lung environment on developing immune responses. For example, alveolar macrophages and airway epithelial cells produce the regulatory cytokine, 13), which is required for the induction of both ...

Mechanisms that protect against asthma remain poorly understood. S-nitrosoglutathione (GSNO), an endogenous bronchodilator, is depleted from asthmatic airways, suggesting a protective role. We report that, following allergen challenge, wild-type mice exhibiting airway hyperresponsivity have increased airway levels of the enzyme GSNO reductase (GSNOR) and are depleted of lung S-nitrosothiols (SNOs). In contrast, mice with genetic deletion of GSNOR exhibit increases in lung SNOs and are protected from airway hyperresponsivity. Our results indicate that endogenous SNOs, governed by GSNOR, are critical regulators of airway responsivity and may provide new therapeutic approaches to asthma.

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 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.