|Home | About | Journals | Submit | Contact Us | Français|
Based on studies in mice, an important role for invariant Natural Killer T (iNKT) cells in the pathogenesis of asthma has been proposed1–4. However, the role of iNKT cells in human asthma is controversial, because there is disagreement on whether iNKT cells are present in the lungs of patients with asthma5, 6. To address this controversy, we examined bronchoalveolar lavage fluid (BALF) from asthmatics with a broad range of severity and symptom control.
The study was approved by the institutional review boards at Children’s Hospital Boston, Brigham and Women’s and Connolly Hospitals. All 18 subjects provided written informed consent and underwent fiberoptic bronchoscopy. Five had severe asthma (based on the NHLBI Severe Asthma Research Program (SARP) criteria)7, eight had well-controlled asthma (based on the NHLBI-EPR3 Guidelines) and five had no history of asthma (Table 1). BALF samples were analyzed for the presence of iNKT cells by flow cytometry (8-Color BD FACSCanto, FlowJo software), by investigators who were blinded from clinical data at the time of the analysis. BALF cells were filtered through 70 μm cell strainers to remove mucous, and examined for viability with trypan blue (mean viability 95%) before treatment with human IgG to block Fc receptors. Cells were then stained with PBS57 (analog of α-GalCer)-loaded CD1d tetramer-PE (or unloaded CD1d tetramer-PE control) (NIH tetramer facility) and anti-CD3-Alexa750 (BD bioscience). We gated on the CD3+ low side scatter population to exclude alveolar macrophages, which have high side scatter and high auto-fluorescence (Figure 1a). The percentage iNKT cells of total T cells was then analyzed (PBS57 loaded CD1d tetramer+CD3+ cells/total CD3+ cells). Background staining (unloaded CD1d tetramer+CD3+/CD3+) was then subtracted (Figure 1a, b) to calculate adjusted percentage iNKT cells of total T cells for statistical analysis (Figure 2).
We found that patients with severe asthma had a significant increase in the number of BALF iNKT cells when compared to that of non-asthmatic controls (p=0.004 Mann-Whitney test) (Figure 2). Some but not all patients with well-controlled asthma had an increase in the number of BALF iNKT cells compared to non-asthmatic controls (p=0.02). We conclude that iNKT cells are present in the BALF of some but not all patients with asthma. The specific number of iNKT cells present in the BALF was quite variable, but patients with severe asthma appeared to more consistently have an increase in BALF iNKT cells than patients with well-controlled asthma, the majority of whom did not have an increase in the number of BALF iNKT cells over that of non-asthmatic individuals.
These results suggest that asthma severity and control might be loosely related to the number of pulmonary iNKT cells. However, it is also likely that many other factors are involved in determining the number of pulmonary iNKT cells. The number of iNKT cells in the BALF appears to be extremely dynamic and fluctuates rapidly (in mice, iNKT in BAL fluid range from 1% of lung lymphocytes in naïve mice to 14% after allergen challenge). We do not yet fully understand the factors that regulate the number of iNKT cells in the lung. In our previous study of poorly controlled asthmatics, all of asthmatic patients had higher numbers of pulmonary iNKT cells compared to that in patients with sarcoidosis or normal individuals, but the specific number of iNKT cells present was much higher than in the current study. This difference in the number of pulmonary iNKT cells could be due to different patient selection criteria and possibly to improved methods for analyzing iNKT cells.
Importantly, our current results help to explain the results observed in the previous ten studies of iNKT cells in human asthma. In seven of the studies a significant increase in the number of iNKT cells in the lungs of patients with asthma compared to that in the normal individuals was observed4, 5(see supplemental reference list in the online repository). In an eighth study, there was a trend toward an increase in the iNKT cells in asthma above that of control subjects, but this did not reach statistical significance8. In two additional studies6, 9, several asthmatic patients were observed to have an increase in the number of iNKT cells in BALF (up to 2.1–2.7% of BALF lymphocytes). However, most of the patients had much fewer pulmonary iNKT cells, such that the authors concluded that iNKT cells were not important in the pathogenesis of asthma, although BALF from normal individuals was not examined6, 9. In our current study of patients with a very broad range of asthma severity and control, we observed an increase in the number of pulmonary iNKT cells in all five patients with severe asthma, and in some but not all of the patients with well controlled asthma, consistent with all ten previous studies taken together.
While addressing the major and controversial question of whether iNKT cells are present in the lungs of asthmatics, our study did not focus on other important issues regarding iNKT cells, including whether the number of iNKT cells in the lung varies over time or with exacerbations, analysis of the phenotype and the subsets of iNKT cells present in the lungs and whether distinct subsets of iNKT cells are present in different forms of asthma, as suggested by murine studies1–4. The limitations of our study were due in part to the small number of patients included in our report, and the fact that repeat bronchoscopy of patients with severe asthma cannot be easily accomplished. However, our results strongly suggest that additional studies with a larger number of subjects are warranted to examine these questions.
The observation that iNKT cells are present in some patients with asthma suggests that iNKT cells may contribute to the pathogenesis of asthma. This question is of much greater importance but must be addressed by examination of iNKT cell function in asthma. Such functional studies are difficult to perform in humans for ethical and safety reasons, but studies in mice and in non-human primates provide a very compelling argument regarding the importance of iNKT cells in the development of AHR. Thus, in three distinct models of asthma in mice, induced with allergen1, 2, virus4, or by exposure to ozone3 (a component of air pollution), AHR failed to develop in the absence of iNKT cells. In each of these models, a different subset of iNKT cell was required for AHR. Moreover, functional studies of iNKT cells, performed by direct and specific activation of iNKT cells in naïve mice10 or in cynomolgus monkeys11 resulted in the development of AHR, suggesting that even the few iNKT cells present in the normal lung, when directly activated can result in AHR. These functional studies of iNKT cells together suggest that iNKT cells may represent an important common element that links many different forms of asthma, including allergic and non-allergic asthma. These results also suggest that the absolute number of iNKT cells present in the lungs may not be as important as their functional capacity when activated to contribute to the development of AHR.
We thank the National Institutes of Health tetramer facility for providing CD1d tetramers, and Dr. Leslie Kalish, Children’s Hospital Boston for help with the statistical analysis.
Supported by grants from the National Institutes of Health (AI026322, HL63248, AI066020) and an award from the Bunning Food Allergy Project. JLF is supported by the Irish Health Research Board and the Asthma Society of Ireland.
Disclosure of potential conflict of interest: DTU is a consultant for Innate Immune, Inc. The rest of the authors have declared that they have no conflict of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.