BACKGROUND:Asthma is a complex lung disease resulting from the interplay of genetic and environmental factors. To understand the molecular changes that occur during the development of allergic asthma without genetic and environmental confounders, an experimental model of allergic asthma in mice was used. Our goals were to (1) identify changes at the small molecule level due to allergen exposure, (2) determine perturbed pathways due to disease, and (3) determine whether small molecule changes correlate with lung function.METHODS:In this experimental model of allergic asthma, matched bronchoalveolar lavage (BAL) fluid and plasma were collected from three groups of C57BL6 mice (control vs sensitized and/or challenged with ovalbumin, n=3-5/group) 6 hour, 24 hour, and 48 hour after the last challenge. Samples were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Airway hyper-responsiveness (AHR) measurements and differential cell counts were performed.RESULTS:In total, 398 and 368 dysregulated metabolites in the BAL fluid and plasma of sensitized and challenged mice were identified, respectively. These belonged to four, interconnected pathways relevant to asthma pathogenesis: sphingolipid metabolism (P=6.6×10-5 ), arginine and proline metabolism (P=1.12×10-7 ), glycerophospholipid metabolism (P=1.3×10-10 ), and the neurotrophin signaling pathway (P=7.0×10-6 ). Furthermore, within the arginine and proline metabolism pathway, a positive correlation between urea-1-carboxylate and AHR was observed in plasma metabolites, while ornithine revealed a reciprocal effect. In addition, agmatine positively correlated with lung eosinophilia.CONCLUSION:These findings point to potential targets and pathways that may be central to asthma pathogenesis and can serve as novel therapeutic targets.

IL-13-producing effector memory CD8+ T cells play a pivotal role in the development of airway hyperresponsiveness (AHR) and inflammation in severe asthmatics and experimental models. The mechanism underlying the phenotypic conversion of CD8+ Teff cells from IFN-gamma-producing to IL-13-producing cells has not been defined. Mononuclear cells were isolated from the spleen of OT-1 mice and CD8+ T cells were differentiated in culture for 4 days with IL-2 or IL-2 plus IL-4. Cytokine production and lineage-specific transcription factors were analyzed. In vivo, anti-IL-4 was injected into sensitized CD8 KO mice prior to receiving differentiated CD8+ T cells and three consecutive days of airway allergen challenge. Airway function and inflammation were monitored. In the presence of IL-2, differentiated CD8+ T effector cells were exclusively IFN-gamma producers. Differentiation in the presence of IL-2 plus IL-4 followed by antigen re-stimulation resulted in CD8+ T cells that predominantly produced IL-13 rather than IFN-gamma, and was associated with changes in levels of expression of T-bet (reduced) and GATA3 (increased). IFN-gamma-producing CD8+ T effector cells adoptively transferred into sensitized CD8 KO recipients prior to challenge, converted in vivo to IL-13 producers and restored the development of AHR and inflammation, these changes were prevented by treating the recipients with anti-IL-4. IL-4 plays a pivotal role in the conversion of CD8+ T cells from IFN-gamma-producing to pathogenic IL-13-producing cells which mediate AHR and inflammation in sensitized and challenged animals.

RATIONALE: CD8+ effector memory T cells (Teff) restore airway hyperresponsiveness (AHR) and inflammation in CD8-deficient (CD8-/-) mice (Nat Med 2004:865), and was dependent on CD4+IL-4+ T cells (J Immunol 2007:2787). IL-6 is important for T helper cell differentiation and allergic asthma. (Nat Med 2008:565). We investigated the role of IL-6 in CD8+ Teff cell-mediated AHR and inflammation. METHODS: CD8+ Teff cells were differentiated in culture with IL-2 or IL-2 plus IL-4. IL-6 mRNA levels were evaluated by real-time PCR. Cellular content and protein levels of IL-6 were assessed by intracellular staining and ELISA, respectively. In vivo, CD8+ Teff cells were adoptively transferred into OVA-sensitized CD8-/- mice before challenge. IL-6 neutralizing antibody was given during the challenge phase. 48 hrs after the last challenge, AHR and eosinophilic inflammation were evaluated. RESULTS: IL-6 mRNA levels, cellular content, and protein levels increased in CD8+ Teff cells after culture with IL-2 plus IL-4, when compared to CD8+ Teff cells cultured with IL-2 alone. In vivo, recipients of adoptively transferred CD8+ Teff cells, differentiated in the presence of IL-2, restored AHR and eosinophilic inflammation. Following treatment with IL-6 antibody, CD8+ Teff cells failed to restore AHR. IFN-γ levels in the BAL were increased in the recipients of anti-IL-6. Intracellular staining of recovered lung CD8+ T cells after allergen challenge demonstrated their ability to produce IL-6. CONCLUSIONS: These data demonstrate that CD8+ Teff cells are an important source of IL-6 which appears essential in triggering of AHR. Their ability to produce IL-6 appears dependent on IL-4.

Background and purpose: Montelukast and S‐carbocysteine have been used in asthmatic patients as an anti‐inflammatory or mucolytic agent respectively. S‐carbocysteine also exhibits anti‐inflammatory properties. Experimental approach: Ovalbumin (OVA) sensitized BALB/c mice were challenged with OVA for 3 days followed by single OVA re‐challenge (secondary challenge) 2 weeks later. Forty‐eight hours after secondary challenge, mice were assessed for airway hyperresponsiveness (AHR) and cell composition in bronchoalveolar lavage (BAL) fluid. Suboptimal doses of 10 mg·kg −1 of S‐carbocysteine by intraperitoneal injection (ip), 20 mg·kg −1 of montelukast by gavage, the combination of S‐carbocysteine and montelukast or 3 mg·kg −1 of dexamethasone as a control were administered from 1 day before the secondary challenge to the last experimental day. Isolated lung cells were cultured with OVA and montelukast to determine the effects on cytokine production. Key results: Treatment with S‐carbocysteine or montelukast reduced both AHR and the numbers of eosinophils in BAL fluid. Neutralizing IFN‐γ abolished the effects of S‐carbocysteine on these airway responses. Combination of the two drugs showed further decreases in both AHR and eosinophils in the BAL fluid. Goblet cell metaplasia and Th2‐type cytokines, interleukin (IL)‐4, IL‐5 and IL‐13, in BAL fluid were decreased with montelukast treatment. Conversely, S‐carbocysteine increased Th1‐type cytokines, IFN‐γ and IL‐12 in BAL fluid. Conclusions and inplications: The combination of two agents, montelukast and S‐carbocysteine, demonstrated additive effects on AHR and airway inflammation in a secondary allergen model most likely through independent mechanisms of action.

RATIONALE: The high-affinity IgE receptor, FcεRI, is expressed on mast cells and mediates a number of allergic responses. IL-13 is a central mediator of allergic inflammation and disease. The contributions of FcεRI and IL-13 to the pathogenesis of peanut-induced intestinal allergy are not well defined. METHODS: Sensitized wild-type (WT) and FcεRI-deficient (FcεRI-/-) mice received peanut orally every day for 1 week. Symptoms were monitored and intestinal inflammation assessed. Bone marrow-derived mast cells (BMMC) from WT, FcεRI-/- and IL-13-/- mice were differentiated and transferred into WT and FcεRI-/- recipients. Specific in vivo blockade of IL-13 was obtained after i.p. injection of soluble IL-13Ra2-IgG fusion protein (sIL-13Ra2Fc) on peanut challenge days. RESULTS: Compared to sensitized and challenged WT mice, FcεRI-/- mice showed decreased symptoms, weight loss, allergic responses, and intestinal inflammation, reduced goblet cell metaplasia and decreased 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 symptoms and intestinal inflammation whereas transfer of FcεRI-/- or IL-13-/- BMMC was without effect. Sensitized and challenged FcεRI-/- mice exhibited lower IL-13 levels in jejunal tissue homogenates compared to WT mice and treatment of WT mice with the IL-13 inhibitor prevented peanut-induced symptoms and intestinal inflammation. CONCLUSIONS: These data indicate that the development of peanut-induced intestinal allergy is mediated by 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.

RATIONALE: The high affinity IgE receptor (FcɛRI) is expressed on mast cells and plays a key role in many allergic diseases. However, the role of FcɛRI in the development of peanut-induced food allergy has not been well defined. METHODS: Balb/c mice (WT) and FcɛRI-deficient (FcɛRI−/−) mice were sensitized to peanut by intraperitoneal injection with alum as adjuvant and subsequently challenged orally with peanut for seven consecutive days. Diarrhea was assessed by visually monitoring mice for release of liquid stool up to 1 hour after intragastric challenge. Symptom score assessment was as previously described (Li XM et al., JACI 2000;106:158). Twenty-four hours after the last peanut challenge, serum and intestinal tissue was collected for immunohistological analysis. Controls were sham-sensitized (PBS) but challenged orally with peanut. RESULTS: Levels of total IgE and peanut-specific IgE, IgG1 and IgG2a in serum from WT and FcɛRI−/− mice were significantly increased following sensitization and challenge with peanut. Peanut sensitized and challenged WT mice demonstrated a higher incidence of diarrhea and greater symptom scores than similarly sensitized and challenged FcɛRI−/− mice. Peanut sensitized and challenged WT mice showed a significant weight loss, but FcɛRI−/− mice maintained their body weight. Further, allergen sensitized and challenged FcɛRI−/− mice showed less mast cell accumulation in the intestinal tissue compared to WT mice. CONCLUSIONS: These results indicate that FcɛRI plays an important role in the development of peanut-induced food allergy.

Effector memory CD8+ T cells (TEFF), not central memory CD8+ T cells (TCM), play an important role in airway hyperresponsiveness (AHR) and allergic airway inflammation. Examining transcription levels, we found a strong induction of Notch1 in TEFF compared to TCM. Notch signaling is important for T cell activation and differentiation, though the role of Notch in TEFF generation and development of AHR is not defined. To generate TEFF, mononuclear cells from spleen and lymph nodes of OT-1 mice were cultured with IL-2 after OVA peptide (OVA257-264) stimulation. After 7 days culture, TEFF were treated with an inhibitor of Notch signaling (gamma secretase inhibitor: GSI) or DMSO as control. OVA sensitized and challenged C57Bl/6 and CD8-/- mice received TEFF treated with GSI or vehicle prior to secondary OVA challenge. 48 hrs after secondary challenge, AHR, BAL cell composition and cytokine levels were determined. Both C57Bl/6 and CD8-/- mice, recipients of GSI-treated TEFF, developed lower levels of AHR and reduced numbers of eosinophils and IL-13, accompanied by much higher levels of IFN-γ in BAL fluid, compared to recipients of DMSO-treated TEFF. Moreover, these effects were linked to high levels of Delta1 expression, a Notch ligand. Treatment of WT mice with the Notch ligand, Delta1-Fc, resulted in decreased AHR and airway inflammation accompanied by higher levels of IFN-γ in BAL fluid. Notch receptor expression on CD8+ TEFF together with its ligand Delta1 represent an important signaling module in the functional regulation of the development of AHR and allergic airway inflammation.

RATIONALE: Allergen-specific (ANR) and allergen non-specific (NNR) nasal responses have been investigated separately, and it remains unclear how the NNR component influences early- and late-phase nasal reactivity. METHODS: We investigated the contributions of ANR and NNR during early- and late-phase nasal responses. BALB/c mice, sensitized to OVA, were exposed to OVA via the nose for 3, 5, or 12 days in the absence of anesthesia to prevent lower airway disease. Mice were challenged with OVA, ragweed, or glucose 24 hrs after the last OVA exposure. Nasal responses were assessed by monitoring respiratory frequency, which correlated with measurements of nasal resistance (Miyahara et al. 2005 JACI). RESULTS: After 3 days of intranasal OVA exposure, OVA-sensitized mice showed nasal hyperreactivity to subsequent nasal challenge not only with OVA, but to ragweed and glucose. The extent and time course of these responses was similar, suggesting a predominant NNR component in this early-phase response. No response was seen in non-sensitized or non-exposed mice. As the number of allergen exposures increased (5-12), there was a progressive diminution in NNR with increased ANR; the late-phase response was almost entirely ANR. CONCLUSIONS: NNR is a major contributor to the early-phase nasal response particularly at the beginning of allergen exposure. Additional allergen exposure results in allergen-specific early- and late-phase nasal responses, converting the nasal response from one that is predominantly NNR to one which is essentially ANR. Contributions of NNR and ANR have important implications for study design and assessing the response to therapy.

RATIONALE: Respiratory syncytial virus (RSV) infection in early life is a risk factor for the subsequent development of persistent wheezing and asthma. RSV-specific IgE antibodies enhance airway hyperresponsiveness (AHR) in mice (AJRCCM 2004, 170:952) and neonatal RSV infection predisposes to the development of enhanced AHR on subsequent RSV infection (JI 2005, 175:1876). The present study was conducted to investigate the role of IgE in the enhancement of RSV-induced AHR following re-infection of newborn mice. METHODS: Newborn BALB/c mice were infected with RSV during the first week of life and re-infected 5 weeks later, a protocol that elicits enhanced AHR, mucus hyperproduction and airway eosinophilia on re-infection. Non-anaphylactogenic anti-mouse IgE antibody was administered after neonatal RSV infection, twice/week for 4 weeks. Control groups were treated with normal rat IgG. Assays were carried out on day 6 following re-infection. RESULTS: Neonatal RSV infection resulted in the development of a significant RSV-specific IgE response in wild-type (WT) but not in IL-4/IL-13 double knockout (KO) BALB/c mice. Unlike WT mice, neither IL-4/IL-13 double KO nor FcεRI KO mice developed an enhanced AHR on re-infection after neonatal RSV infection, suggesting the involvement of IgE in these altered airway responses. Treatment of RSV-infected newborn WT mice with anti-IgE prior to re-infection diminished the development of enhanced AHR after re-infection. CONCLUSIONS: Collectively, these results suggest a role for IgE in the enhancement of the response to re-infection with RSV and a potential benefit of anti-IgE in preventing post-RSV wheezing and asthma.