In this manuscript, we report the development of a double-staining immunofluorescence protocol, with which we demonstrate the presence of submucosal Th17 (CD4+IL-17+) lymphocytes in endobronchial biopsies of children with CF. This is the first direct evidence to support the hypothesis that the Th17 pathway is involved in the pathogenesis of CF lung disease. We also report increased numbers of IL-17+ cells in children newly diagnosed with CF with BALF neutrophilia, in the absence of elevation of IL-8, suggesting that IL-17+ cells are important in the very early stages of CF. Th17 cells are not the only source of IL-17; we have also demonstrated the presence of IL-17+ neutrophils, γδT cells, and NKT cells in the airway. Both established CF and non-CF bronchiectasis samples had higher numbers of IL-17+ cells, suggesting this is not a CFTR-specific phenomenon.
Our focus encompassing both the airway wall and lumen rather than just the latter, which is more commonly studied using BALF, stems from the belief that these two compartments may not be comparable and may yield different insights. In explanted lung tissue, although most of the neutrophils are seen in the surface epithelium (suggesting they may be migrating into the airway lumen), there is a lymphocyte-dominated infiltrate in the CF airway submucosa (3
), and we have reported similar findings in children (4
). In this study, the submucosal IL-17+
cell count of the endobronchial biopsies does not correlate with the levels of IL-17 or other inflammatory markers in BALF. This would seem to support our hypothesis of compartmentalization of pathogenic processes; however, we must also consider alternative explanations, including that these small proximal biopsies may not be representative of the whole of the lower airway, or that dilution of BALF is so variable that the noise in the signal masks relationships. Furthermore, not just Th17 lymphocytes can secrete IL-17 in the pediatric airway.
Of these other IL-17+
neutrophils have been shown to be present in both patients with end-stage CF undergoing lung transplantation (27
) and patients with chronic obstructive pulmonary disease undergoing surgery for lung cancer (28
). We have now also demonstrated their presence in patients with non-CF bronchiectasis as well as control subjects.
The isolation of IL-17+
γδ T cells is interesting because, in murine models, they are one of the main producers of IL-17 in response to infection with Mycobacterium tuberculosis
) and P. aeruginosa
). Furthermore, mice with chronic granulomatous disease that have pulmonary aspergillosis have also been shown to have unrestrained IL-17+
γδ T cell activity and acute inflammatory lung injury (31
The final group of IL-17+
cells we demonstrated were NKT cells in transplant tissue from adult lungs. NKT cells are a unique subset of T cells that express both NK cell markers and a T-cell receptor. Recent studies have reported that activated invariant NKT (iNKT) cells in the lung are able to produce IL-17, whereas airway neutrophilia induced by intranasal αGalCer or lipopolysaccharide instillation was significantly reduced in iNKT cell–deficient mice. These mice also produced significantly less IL-17 in their BALF compared with wild-type control mice (32
Within the airway lumen, our BALF results are consistent with previously published data showing significantly increased levels of IL-17 in the BALF of children with CF and sputum of adults with CF who are having a respiratory exacerbation with P. aeruginosa
), as well as adult patients with CF who are clinically stable (33
The exact pathogenic mechanisms of IL-17 in CF are still to be elucidated. One possible mechanism may be through priming of airway epithelial cells lacking functional CFTR, by up-regulating pattern recognition receptors, including the bacterial sensors nucleotide binding oligomerization domain (NOD) 1, NOD2, and TLR4, and its own receptors IL-17RA and IL-17RC. IL-17 priming led to much greater NOD1 agonist and Pseudomonas
diffusible material induced IL-8 secretion in ΔF508 airway epithelial cells than wild-type airway epithelial cells (34
). P. aeruginosa
is a gram-negative organism that often causes chronic infection in patients with CF. The host mounts a vigorous neutrophilic inflammatory response but is unable to clear the pathogen. Dubin and Kolls have used an agarose bead model of P. aeruginosa
infection in mice, which mimics the chronic airway infection seen in CF, and demonstrated the importance of the IL-17/IL-23 axis in this model. IL-23p19−/−
mice had significantly lower induction of IL-17, keratinocyte-derived chemokine, and IL-6, decreased BALF neutrophils, matrix metalloproteinase-9, and on histology had reduced inflammation compared with wild-type mice, but there was no difference in bacterial dissemination between the two groups (16
The importance of the Th17 pathway is supported by the patients newly diagnosed with CF who already demonstrate significantly increased numbers of IL-17+ cells in the submucosa of their endobronchial biopsies and increased neutrophil counts in their BALF compared with control subjects. Of the four patients newly diagnosed with CF in whom the diagnosis was made on newborn screening, three already showed a neutrophilia in their BALF. The one patient who had a normal cell differential had an IL-17+ cell count of 59 cells/mm2, which is comparable to that of control subjects.
The main weakness of this study, which is unavoidable, is that it is cross-sectional, and there are no longitudinal data, largely because of the ethics of bronchoscopy in children. The findings are hypothesis generating, and proof of the importance of the Th17 pathway will require intervention studies. The differences in our findings between patients with newly diagnosed and established CF, and between culture-positive and -negative patients, suggests an association between the Th17 axis and disease stage, supporting the concept that this axis is important. Our strengths are our large numbers and inclusion of the non-CF bronchiectasis group, which enable us to differentiate CFTR-specific findings from those related to inflammation in general.
Our data generate the hypothesis that early inflammation may be Th17 driven and that subsequently a vicious cycle could occur, in which the initial production of IL-17 attracts neutrophils, which are in turn stimulated to produce more IL-17. This cycle would obviously be augmented by other cytokines (e.g., IL-8), but the intriguing possibility is raised that the Th17 axis is the initiating factor.
Unanswered Questions and Future Research
One unexpected finding was the correlation between levels of IL-17 and IL-4 in BALF. There has been evidence from a murine model suggesting that Th17 cells may up-regulate Th2 cell–mediated eosinophilic airway inflammation (35
). Eosinophil cationic protein levels have been shown to be raised in the serum and sputum of patients with CF (36
). However, there was no correlation between BALF IL-17 levels and serum IgE, IL-5, IL-13, blood or BALF eosinophil counts in our study. Recently, a novel sub-population of Th17 cells that produces both IL-17 and IL-4 has been described (37
). These Th17/Th2 cells are found in very small numbers in blood from healthy subjects and are significantly increased in the circulation of patients with asthma. More work is needed to determine whether these cells have a role in CF. More work is also needed to dissect the individual roles and the relative importance of the various cells that secrete IL-17 and the complex interplay between the adaptive immune system and the innate immune system.
In summary, Th17 lymphocytes are present in the submucosa of children with CF from early in the course of the disease and could be the earliest drivers of the inflammatory response. We have also shown that IL-17+ neutrophils, γδ T cells, and NKT cells are also present in the CF airway. Both established CF and non-CF bronchiectasis samples had higher numbers of IL-17+ cells, suggesting this is not a CFTR-specific phenomenon. The Th17 pathway may be a novel therapeutic target in chronic inflammatory lung disease.