Allergic sensitization through the airway is likely the first in a cascade of events that ultimately leads to allergic asthma. To fully understand the mechanisms underlying the initiation of allergic asthma, it is essential to study early events in the lung and draining lymph nodes that determine the commitment to either immunotolerance or allergic sensitization. Sensitization through the peritoneum using alum as an adjuvant has been used for many years, and has been very useful for studying Th2-mediated responses to allergen challenge. However, the unique features of the lung suggest that inhaled allergens might provoke immune responses that are qualitatively different from those arising in the peritoneum. Accordingly, we have adopted a model in which mice are sensitized to OVA through the airway using LPS as an adjuvant. Previous descriptions of this model have shown that LPS-mediated sensitization is dependent on TLR4 and MyD88, whereas alum-mediated sensitization through the peritoneum is not (15
). Here, we show that immune responses arising from these two methods are also substantially different. Whereas the latter are characterized by very strong Th2 responses and sustained inflammation, the former display only modest Th2 responses but very strong Th17 responses. It is likely that the different adjuvants used in these two approaches are at least partially responsible for these immunologic differences. However, our data suggest that the route of sensitization itself also affects immune responses. This question is difficult to fully address experimentally because alum would cause asphyxiation if administered to the airways of mice. However, in the converse experiment, we found that neither low nor high doses of LPS were effective adjuvants in the peritoneum. It is possible that the lung is particularly prone to develop Th17 responses because one of the cytokines required for the development of these cells, TGF-β, is constitutively produced by alveolar macrophages (13
). On its own, TGF-β primes the development of Treg cells. However, in the presence of IL-6, TGF-β supports the differentiation of Th17 cells. By inducing the production of IL-6, LPS might convert the lung from a tolerogenic environment to one that is particularly supportive of Th17 development.
We cannot rule out an involvement of IL-17–producing cells other than Th17 cells in the model used here. For example, macrophages, γδ TCR T cells, and CD1d-restricted (i)NKT cells have previously been shown to be important sources of IL-17 in the lung (26
). However, intracellular staining for IL-17 revealed that these cell types were represented at very low frequencies compared with IL-17–containing αβ TCR T cells. Moreover, airway-sensitized CD1d-deficient mice developed neutrophilia and AHR on a single OVA challenge, indicating that iNKT cells are not required for AHR in this model (Wilson, personal communication). Thus, LPS-mediated sensitization through the airway appears to efficiently prime conventional Th17 cells that home back to the lung and are poised to release IL-17 into the airway on subsequent encounter with the sensitizing allergen. It seems possible, therefore, that inhaled aeroallergens might also provoke similar Th17-dominated immune responses in humans.
Regardless of the relative impacts of the different adjuvants and routes of sensitizations in the two models used here, comparisons between the elicited responses themselves allow several conclusions to be drawn regarding the molecular and cellular requirements for AHR. The high levels of IL-4, IL-5, and IL-13 and the robust eosinophil accumulation in the airways of intraperitoneally sensitized mice after a single challenge indicate that efficient priming had occurred. This interpretation is consistent with the previous finding that mediastinal lymph nodes, which drain the lung, also drain the peritoneal cavity and contain antigen-responsive T cells within 2 days of intraperitoneal sensitization (35
). However, the absence of AHR in intraperitoneally sensitized mice challenged on a single occasion, despite their relatively robust Th2 responses, suggests that these Th2 responses are not sufficient for the development of AHR. The presence of AHR in airway-sensitized mice, which display only modest Th2 responses but strong Th17 responses, suggested that both Th2 and Th17 responses are required for AHR. This was confirmed by the absence of AHR in airway-sensitized IL-17ra−/−
mice, which cannot respond to IL-17, and with airway-sensitized Stat6−/−
mice, which cannot respond to IL-4 or IL-13. Moreover, airway delivery of exogenous IL-17 was sufficient to provoke AHR in intraperitoneally sensitized mice undergoing Th2 responses, but not in unchallenged mice.
An increasing body of evidence has revealed that neutrophils are associated with severe asthma, although the functional relevance of these cells to disease progression remains unclear (5
). To date, animal models have not been particularly helpful in this regard because although mice sensitized through the peritoneum undergo a very transient influx of neutrophils within 8 hours of intranasal allergen challenge, these cells are no longer evident at 48 hours post challenge (10
). Here, we confirm and extend these previous findings. The transient neutrophilia seen in intraperitoneally sensitized mice was associated with a similarly transient production of CXCL1, which was not dependent on IL-17RA. In contrast, the more prolonged neutrophilia seen in airway-sensitized mice after a single challenge was associated with a sustained increase in a different chemokine, CXCL5. This production of CXCL5 was in turn dependent on IL-17RA. Therefore, the transient neutrophil recruitment to the airway in intraperitoneally sensitized mice and more prolonged accumulation of these cells in airway-sensitized mice result from different signaling pathways. Moreover, blockade of neutrophil recruitment to the airway with neutrophil-depleting antibodies, or through genetic deletion of Cxcr2
, prevented the development of AHR in airway-sensitized mice. However, neutrophil accumulation in the airway cannot be sufficient for AHR, even in the presence of eosinophils, because intraperitoneally sensitized mice that received CXCL1 or CXCL5 developed robust neutrophilia, but not AHR. This finding suggests that AHR requires the activation of neutrophils, in addition to their recruitment to the lung.
The phenotype of airway-sensitized mice appears to have been shaped by the interactions of both Th2 and Th17 immune responses. IL-17 has been previously reported to inhibit Th2 responses. Consistent with these previous observations, we found that eosinophil accumulation in the lung was increased in Il17ra−/−
mice compared with WT mice. In light of this result, it was surprising that IL-17 synergized with ongoing Th2 responses to promote airway neutrophilia and AHR. This synergistic interaction was seen in both gain-of-function experiments, in which IL-17 promoted increased neutrophilia and AHR in the setting of ongoing Th2 inflammation, and in loss-of-function experiments, in which neutrophilia and AHR was reduced and abolished, respectively, in Stat6−/−
mice. It is unlikely that the reduced neutrophilia seen in Stat6−/−
mice is due to impaired Th17 development because these mice are reported to have normal Th17 responses (22
). The absence of IL-13 signaling might account for the absence of AHR in Stat6−/−
mice, but the reduction in neutrophilia seen in these mice is more difficult to explain. One possibility is that ongoing Th2 responses promote neutrophil recruitment indirectly. For example, these responses might alter the physical properties of the airway, thereby making it more accessible to neutrophils. Alternatively, one or more cytokines present during ongoing Th2 responses might act directly or indirectly on neutrophils to promote their recruitment.
It should be noted that allergic asthma is a chronic disease, whereas in the experiments here the mice were challenged on a single occasion. Thus, we do not yet know whether the model used here will also be useful for studying chronic responses to allergen challenge. Our preliminary evidence suggests that unlike the heightened inflammation and AHR seen with continued challenges of intraperitoneally sensitized mice, continued OVA challenges suppress the acute responses seen after a single challenge of airway-sensitized mice. However, if these allergen challenges were temporarily discontinued and then resumed, robust inflammatory responses develop once again (Whitehead, personal communication). This aspect of our findings might be particularly relevant to individuals who experience infrequent, but severe, exacerbations. It is likely that in these patients, the actions of effector T cells are usually constrained, perhaps by regulatory T cells (Tregs). Interestingly, in addition to its well-described ability to induce innate immune responses, inhaled LPS can also lead to a delayed accumulation of Treg cells in the lung (Landon King, personal communication). Thus, in our model of LPS-mediated allergic sensitization and prolonged OVA challenge, Tregs might suppress the actions of Th2 and Th17 cells. The extent to which inhaled LPS determines the balance between Th17 and Treg responses in humans is not known. However, LPS is ubiquitous in the environment and it is possible that a propensity to develop Th17 responses rather than Treg responses after exposure to LPS might be one factor that contributes to asthma susceptibility. If so, this model might provide insight into the mechanisms that give rise to exacerbations in individuals whose asthma is normally asymptomatic.