Discussion
In this study, we used a standardized nasal allergen provocation test with a single threshold dose that led to an acute self-limited allergic respiratory inflammatory response. Here we showed for the first time that NK cells are recruited to the nasal mucosa shortly after the initiation of the allergic inflammatory response and correlate with eosinophilic infiltration.
Natural killer (NK) cells are key members of the innate lymphocyte family with essential roles in host defense against pathogens as well as in malignancies (20). However, a role for NK cells in the regulation of type 2 immune responses and allergic diseases has been recently suggested. Indeed, experimental studies using murine models have shown that NK cells can participate in the regulation of allergen-induced eosinophilic airway inflammation (11, 21-23). Moreover, the presence of activated NK cells has been noted in patients with type 2 diseases, including asthma (12, 24), allergic chronic rhinosinusitis (25, 26) and atopic dermatitis (27). Whether NK cells enhance or limit type 2 immune responses is still debating.
In human in vitro experiments, it has been shown that human NK cells can promote apoptosis of autologous eosinophils but the molecular mechanisms underlying these events were only partially addressed (13, 28). In the present study, we confirmed that peripheral blood NK cells from healthy donors are able to induce the apoptosis of autologous peripheral blood eosinophils. As eosinophils have the capacity to release a broad range of toxic mediators into surrounding host tissues with consequent tissue damage, eosinophil apoptotic cell death followed by their subsequent phagocytosis by surrounding phagocytes is therefore critical for the successful resolution of allergic and asthmatic inflammation (29). Moreover, as suggested earlier (13, 28), we confirmed that NK cell-induced eosinophil apoptosis needed a direct contact between eosinophils and NK cells.
Besides potential inflammation pro-resolving effects, activator effects of NK cells on eosinophils have also been reported. In a previous study, human NK cells were shown to induce a dose-dependent in vitroactivation of autologous eosinophils after 3 and 12 hours of co-culture (13). Upon activation, eosinophils are able to release many mediators, including highly reactive oxygen species that can damage surrounding tissues and worsen inflammation (30). In the current study, we observed that among different leukocyte populations involved in type 2 allergic inflammation, healthy donor eosinophils from were by far the most important producers of superoxide anion. After 1 hr of co-incubation with NK cells, the release of superoxide by eosinophils was significantly reduced. This inhibitor effect was overwhelmed after 4 hrs of co-incubation, confirming a partial activator effect of NK cells on eosinophils.
Several classes of natural pro-resolving and anti-inflammatory chemical mediators are produced at sites of inflammation, including the respiratory mucosa (4). LXA4 is a lead member of a newly described class of pro-resolving lipid mediators derived from arachidonic acid that signals through ALX/FPR2 receptors (5). LXA4 has been reported to decrease allergic and asthmatic responses in mouse models (6). In the current study, we showed that LXA4 was already present in the nasal mucosa at baseline and significantly increased in the nasal lavage fluid shortly after the nasal allergen challenge.
LXA4 is produced by multistep enzymatic process resulting from lipoxygenase (LO) activities in different cell types. One of the main pathways of LXA4 synthesis involves lipoxygenation of arachidonic acid by 15-LO in epithelial cells to generate 15(S)-hydroxyeicosatetranoic acid (15S-HETE) that can then be utilized as a substrate by the 5-LO expressed in neutrophils to synthesize LXA4 (31). In our study, neutrophils were the most abundant population recruited during the nasal allergic response, peaking 1 hr after the allergen challenge and returning to baseline levels at 24 hrs. A such transient increase of neutrophils has been documented earlier after allergen challenge in animal models and human asthma but their role in allergic disease has been questioned (32-34). Interestingly, we observed an association between the LXA4 levels and the peak of nasal neutrophil infiltration, suggesting the involvement of neutrophils in LXA4 biosynthesis during the early respiratory allergic inflammatory response. After falling back to baseline levels, LXA4 significantly re-increased at 48 and 72 hrs post-allergen challenge at time points where monocytes were present in the nasal lavage samples. An increasing number of studies have linked efferocytosis by macrophages and monocytes with their further production of pro-resolving mediators, including LXA4 (35).
Both NK cells (12) and eosinophils (36) express the LXA4receptor ALX/FPR2. Since anti-inflammatory effects of LXA4 on eosinophils were previously reported (19), we next determined whether LXA4 was able to modulate eosinophil superoxide release. Activated eosinophils significantly reduced their superoxide release in a dose-dependent manner when exposed to LXA4. When eosinophils were co-incubated with NK cells in the presence of LXA4, superoxide release was still inhibited in a dose-dependent manner 4 hrs later, in contrast with the previously observed partial activator effect of NK cells on eosinophils 4 hrs later.
Our study underlines the complex network between cellular and molecular actors during resolution of allergic airway inflammation. Our data suggested that NK cells could represent an endogenous counterregulatory response to type 2 inflammation, as NK cells are recruited to the nasal mucosa during an allergic inflammatory response in parallel to eosinophil accumulation an are able to promote eosinophil apoptosis. The accumulation of neutrophils along with monocytes during the allergic inflammatory response may furthermore be an important regulatory feedback to initiate and promote resolution of allergic inflammation, as our data suggest involvement of these cells in LXA4biosynthesis, a specific pro-resolving lipid mediator. Finally, LXA4 appeared to be essential in limiting superoxide release by eosinophils in the presence of NK cells.
Current treatment strategies in allergic airway diseases have focused on combating pathologic type 2 inflammation using either broad or selective immunosuppression. In the next future, the therapeutic paradigm could shift to the use of pharmacologic agents that enhance endogenous pro-resolving mechanisms. This presupposes a better understanding of the roles and interactions between cellular and molecular processes that drive the resolution of allergic inflammation.
In conclusion, here we report for the first time that NK cells are recruited to the nasal mucosa of subjects with allergy in response to nasal allergen challenge and we identify a synergistic role for NK cells and LXA4 in mediating resolution of allergic eosinophilic inflammation.