In this report, we describe the early response to a single exposure of GC frass and show that GC frass directly activated DC chemokine production in resident airway cells leading to the preferential accumulation of mDCs into the lungs. In addition, these mDCs were activated as determined by increased co-stimulatory molecule expression. In the whole-lung cultures, we detected cytokine skewing towards a Th2/Th17 phenotype which occurred within 18 h of GC frass exposure. We confirmed that GC frass could directly activate BMDCs in vitro by releasing high levels of IL-6, along with increased levels of both IL-23 and IL-12p70. To confirm the importance of DC activation on the initiation of allergic airway disease, we adoptively transferred BMDCs treated ex vivo with GC frass and found that a single exposure of GC frass-treated mDCs was sufficient to induce airway inflammation in naïve mice. Lastly, we present compelling evidence that TLR stimulation is crucial for GC frass-induced BMDC activation since MyD88-deficient BMDCs do not respond to GC frass stimulation. This is important as GC frass has been shown to contain both endotoxin and a TLR2 agonist [6
], both of which require a functional MyD88 for signaling. Collectively, these data show that GC frass is sufficient to alter the cytokine/chemokine expression to induce cellular recruitment and alter the local cellular milieu within the lung and this is due in part to activation of TLRs.
An early response to GC frass exposure is the significant upregulation of chemokines and cytokines (CCL20, MIP-1α, GM-CSF and G-CSF) involved in DC recruitment and differentiation. Importantly, the fact that these mediators are synthesized by the airway epithelium following allergen exposure implicates the airway epithelium as an initiator of the adaptive immune response. Ambient particulate matter [29
] and HDM [30
] have been shown to increase CCL20 mRNA and protein secretion from human airway epithelium. GM-CSF and G-CSF release was also shown to be significantly increased in primary human bronchial epithelial cells and epithelial cell lines following HDM exposure [31
]. Thus, the chemokine milieu present in the lung following allergen exposure may be set up very rapidly by epithelial cells, hence implicating the importance of the epithelium in shaping the nature of the adaptive immune response.
Classically it was thought that only Th2 cytokines drove allergic airway disease. However, Th17 cells may also contribute to the pathogenesis of T cell-mediated allergic reactions. Our data show that GC frass induced a mixed Th2/Th17 cytokine profile within 18 h of a single exposure. We have previously shown that GC frass induced significant airway neutrophilia within 18 h of a single exposure [6
], and while we did not quantify the amount of IL-17A released by neutrophils, a recent study has confirmed that neutrophils are a significant source of IL-17A [33
]. Recent studies have implicated Th17 in allergic asthma. HDM-driven AHR was associated with a mixed Th2/Th17 cytokine profile [18
]. Allergic sensitization through the airways was shown to prime Th17 cells to release IL-17 into the airway when challenged with allergen [34
]. IL-17 was shown to regulate allergic airway inflammation in mouse models [35
]. In humans, sputum IL-17A and IL-17F levels have been shown to correlate with AHR [23
]. Thus, it is not surprising that GC frass exposure was sufficient to induce IL-17A in the airways of mice. It is possible that activation of Th17 cells may amplify allergic airway inflammation and that activation of Th17 may not be sufficient to induce asthma, but may be required for increased severity of symptoms.
Interestingly, we found a trend towards increased IFN-γ levels following a single exposure to GC frass, as well as a significant increase in IFN-γ levels in the lung following adoptive transfer of GC frass-pulsed BMDCs and challenge with GC frass. This is in contrast to our other work showing that sensitization and challenge to GC frass lead to decreased IFN-γ levels in the lungs [5
]. It is possible that a single exposure of GC frass is sufficient to induce IFN-γ but that these levels are not maintained long term. In our previous studies, mice were sensitized and challenged with multiple exposures to GC frass, whereas in the adoptive transfer experiment, the sensitization was to GC frass-pulsed BMDC and there was only a single challenge with GC frass. It is possible that an early increase in Th1 is common and may be suppressed by high levels of Th2 or Th17 cytokines following multiple allergen exposures. This concept is supported by a recent study showing that ragweed extract concurrently upregulated Th1-associated genes with Th2-associated genes and the authors propose that allergen-induced airway inflammation is activated by both Th1 and Th2 gene regulation [40
]. They argue that an unopposed Th1 gene upregulation would resolve allergic airway inflammation. In our current studies, we find that sensitization of naïve mice with GC frass-pulsed BMDCs induced a Th1/Th2/Th17 profile of cytokines in the lung.
GC frass exposure resulted in an increased percentage of mDCs and no change in the percentage of pDCs in the lung. This would suggest that GC frass can trigger the preferential recruitment or proliferation of the subtype of DCs important in the promotion of asthma. In this report, we did not study the proliferation of resident lung DCs, but others have shown that GM-CSF suppressed the development of pDCs [41
], while increasing the proliferation of mDCs. mDCs can induce a variety of T cell response (Th1, Th2, Th17) depending on where they are isolated and what stimuli they received [42
], while pDC can induce Th1, Th2 or regulatory T cell development depending on the stimulus [44
]. Our recent work revealed that susceptibility to AHR was associated with mDC allergen uptake while resistance to allergen-derived AHR was associated with pDC allergen uptake [18
]. Interestingly, however, in human data, the percentage of pDCs was higher than mDCs following allergen challenge [46
]. Thus, further understanding the mechanism by which mDCs and pDCs are recruited into the lungs may be of therapeutic potential.
The airway response to GC frass highlights the complex interactions incorporated in the regulation of the inflammatory responses seen in respiratory diseases, particularly asthma. Furthermore, our data suggest that an allergen exposure may influence the maturation of airway mucosal DCs and also provides a potential mechanism by which airway epithelial cells may directly affect the dynamics of the DCs. Finally, the evidence presented here provides compelling evidence for the role of the innate immune response in the development of the adaptive Th2 immune response. Further understanding of the complexity of the allergen and its interaction with the respiratory mucosa will help to further elucidate the molecular interplay between airway epithelial cells and DCs.