End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis

Cystic fibrosis (CF) is the most prevalent autosomal recessive disorder in the Caucasian population and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1, 2]. Loss of CFTR-mediated chloride and bicarbonate transport leads to dehydration of the airway surface fluid layer and impaired mucociliary clearance. As a result, desiccated secretions obstruct the airways and prevent bacterial elimination, establishing chronic infection, commonly with Pseudomonas aeruginosa, and leading to bronchiectasis [3, 4]. These events are followed by vigorous airway inflammation and may eventually result in severe lung damage, the principal cause of death or reason for transplantation in these patients [4‐6].

Neutrophil-dominated airway inflammation of the CF lung environment is a well-established concept: bacterial pathogens interact with epithelial cells and alveolar macrophages, which are part of the lung’s innate cellular surveillance system, causing them to release pro-inflammatory mediators leading to excessive recruitment of neutrophils into the bronchoalveolar compartments of the lung [7, 8]. When chronic infection is established, dendritic cells and T cells also become activated, the latter releasing even more cytokines contributing to the excessive and perpetual recruitment of neutrophils. This immune response is unable to resolve the infection and massive amounts of oxidants and proteases are released [7, 8]. The inflammatory cellular pattern of CF lung disease is presumably diverse, given the many different mediators present, but remains insufficiently studied. Besides the key players listed above, data on other immune cells within the CF lung such as eosinophils, mast cells, cytotoxic T cells and B cells, are scarce [9‐12]. A diverse inflammatory pattern in CF may also be anticipated based on the genotypic variation and the broad range of bacteria and fungi attacking the lungs, which may result in a pathogen-specific CD4 T cell response [7].

Another phenomenon indicative of the diversity of CF lung disease is the CF ‘gender gap’. There seems to be a discrepancy between the median survival of male and female CF patients. This CF ‘gender gap’ was first described in the 1990’s [13]. A large retrospective cohort analysis of the US CF registry, comprising over 20 000 patients between 1988 and 1992, showed that females had a 60% greater chance of dying compared to males, and this effect remained significant even after adjusting for lung function, pancreatic insufficiency and age at diagnosis [14]. Harness-Brumley et al. demonstrated that in 2014, CF females still have a decreased median life expectancy (36.0 years versus 38.7 years in males) and that female gender is a risk factor for death [15]. Morbidity is also increased in female CF patients, with a significantly higher use of intravenous (I.V.) antibiotics, macrolides and steroids, and an increased number of days spent in the hospital [16].

In this study, we aimed to describe the diversity of the inflammatory pattern in the CF lung by quantifying and localising the different immune cells within end-stage CF lungs. Furthermore, we wanted to investigate whether there is a different inflammatory signature in male versus female CF patients.

The present study demonstrated a significant increase of neutrophils, mast cells, CD1a and CD207 dendritic cells, macrophages, CD8 and CD4 T cells in CF lung tissue. Concomitantly, we found a disproportionate increase of neutrophils around the CF airways. The number of single B cells was not increased in the CF lung tissue. The number, but not the size of lymphoid follicles in CF lungs was increased and there was a skewed B cell/T cell composition. We also found some gender-specific changes in our CF population. Eosinophil, mast cell and CD4 T cell counts were specifically increased in lung tissue of female CF patients. Lastly, lymphoid follicles had less B cells and more CD8 T cells in females.

In this study, we have shown that the inflammatory signal in end stage CF lung disease is diverse and involves an increased abundance of both myeloid and lymphoid cells. As more and more players involved in CF lung inflammation are being identified, it becomes increasingly clear how diverse and disturbed the inflammatory pattern really is. This may be the consequence of the presence of mutated CFTR channels on various inflammatory cells. The absence of functional CFTR leads to impaired bacterial killing in murine and human macrophages and a deregulated release of pro-inflammatory cytokines by macrophages during the innate immune response [7]. As the CF lung is liable to infection by a myriad of pathogens, this may also promote the diversity of the immune response. By demonstrating an increase in T cells and lymphoid follicles in CF, we have confirmed the involvement of the cellular adaptive immune response in CF lung disease. A role for T cells in the pathophysiology of CF has already been suggested as CFTR present on circulating T cells participates in immune cell signalling. This CFTR-mediated signalling is disturbed in CF [7].

A striking finding was the large amount of lymphoid follicles found in all compartments of the CF lung. The presence of follicles is well-established in other chronic inflammatory diseases such as COPD [17]. B cell follicles are the result of lymphoid neogenesis leading to the development of tertiary lymphoid organs in tissues that are under a chronic and constant influence of certain triggers [17]. In end-stage CF, it seems logic that this trigger has an infectious nature as the lungs have endured years of microbial assaults and colonization with different pathogens. Due to the nature of this disease, these pathogens become very difficult to eradicate which results in a sustained immune response with infiltration into the lung parenchyma of macrophages, dendritic cells, T cells and B cells. These cells frequently organize themselves at an anatomical and functional level into tertiary lymphoid organs [17]. However, as CF is characterized by airway destruction, the B cell follicles could also reflect an adaptive immune response against degradation products of the extracellular matrix, becoming so-called neo-self antigens, and may in fact represent an autoimmune process that occurs after breaking the immune tolerance. In order to determine the aetiology of the lymphoid follicles found in CF, future research might focus on lung tissue derived from non-CF bronchiectasis patients who are often colonized with P.aeruginosa, S.aureus and H.influenza as well. If follicles are equally numerous in this disease, their development might have an infectious nature. In COPD, lymphoid follicles were described as aggregates of B cells that are surrounded by lower numbers of predominantly CD4 and to lesser extent CD8 T cells [17, 18]. However, in CF we observed a composition shift of the lymphoid follicles from B cell to T-cell predominance, suggesting that the cellular adaptive immune response is specifically affected in CF.

We also found that the increased inflammation in end-stage CF lung disease appeared to be gender-specific. Given the lack of apparent differences in patient characteristics between male and female CF patients in our population which may account for the observed female predominance in airway inflammation, there is some room for speculation. It has been shown that oestrogen has a complex immunomodulatory effect on inflammation and mucoid P. aeruginosa density [19]. Human CF patients whose lungs are actively infected with P. aeruginosa have elevated sputum levels of IL-23 and IL-17 which decrease dramatically following antibiotic treatment [20, 21]. Experiments with adult male mice demonstrated a critical role for IL-23 (and the Th17 products it induces) in the pathogenesis of murine lung inflammation upon infection with P. aeruginosa. Administration of the exogenous female sex hormone 17β-estradiol (circulating oestrogen) to male CF knock-out mice resulted in a more pronounced inflammatory reaction to infection with P. aeruginosa, which was reflected by increased lung tissue mRNA levels of IL-23 and IL-17 [22]. Sex hormones also modulate autoimmunity. Autoimmune diseases are a class of illnesses associated with increased Th17 activity and showing distinct gender-based differences in prevalence [19]. The gender-based difference in inflammation in our CF population may be the result of overstimulation of Th17 activity by oestrogens similar to what is seen in autoimmune diseases. Oestrogen can also cause a reduced production of IL-8 in CF bronchial epithelium through inhibition of NF-κB and IL-8 gene expression. This may result in a hyporesponsive innate immune response in the CF lung during times of high oestrogen exposure and predispose to infection and bacterial colonization [23].

The distinct inflammatory signature in females could have clinical implications. As there is a marked eosinophilic component, a more liberal policy with oral steroids (both during and independent of exacerbations) might be advocated in females. Future clinical trials with anti-inflammatory therapies using monoclonal antibodies may also want to analyse the effect in female CF patients separately.

Although the comprehensive pathological analysis of end-stage CF lung tissue provided a valuable insight in the inflammatory diversity of this disease, our study has some limitations. The origin of our samples only allowed us to study end-stage disease. To visualize disease progression, earlier stages of the disease will need to be studied in a similar way. The study of Regamey et al. who used endobronchial biopsies of children with a median age of 7.3 years to investigate the inflammatory pattern in the bronchial mucosa, already suggested that the infiltrate predominantly consists of macrophages and lymphocytes [24]. Although we did not specifically study the airway compartment when quantifying the lymphoid cells, it is clear that also in our study the CD4 and CD8 T cells outnumber the B cells, both as single dispersed cells as in the follicles, where in addition a composition shift to a larger proportion of CD4 T cells in CF compared to controls was noted. However, it seems that the lymphocytic infiltrate in the subepithelial tissue of children with CF has a larger portion of B cells (18% in CF compared to 13% in controls) which may indicate that the humoral adaptive response plays a bigger role in the earlier stages of CF lung disease. Moreover, Regamey et al. only found a limited number of neutrophils infiltrating the bronchial mucosa. The numerous presence of neutrophils around the airways in our study suggests that they infiltrate the tissue surrounding the airways in later stages of the disease, eventually leading to tissue destruction. A second limitation is that the number of CF and control tissue included is limited. As a result, we cannot exclude that the observed differences between male and female patients may be partly due to the small sample size. Thirdly, due to the large surface area of the biopsies and the fragile nature of CF lung tissue, we chose to cut nine μm thick sections. This could however confound our results in two ways: it may increase all cell type counts in CF tissue compared with controls, and it may also increase the number of large cell types like macrophages and neutrophils compared with small cells. Therefore, we described our findings in a qualitative way, not elaborating on the extent of the quantitative difference between CF and controls. Moreover, we have not compared cell types to each other but only looked at the differences between groups. A last limitation of our study is the age discrepancy between CF patients and control subjects, due to the fact that transplanted CF patients at our centre are inevitably younger than patients undergoing resection of bronchial carcinoma or in whom lung tissue was obtained after autopsy.

By quantifying and localizing distinct myeloid and lymphoid subsets in CF and control lung tissue, the present study clearly showed a diverse inflammatory pattern in end-stage CF lung disease with an increased presence of neutrophils, mast cells, CD1a and CD207 dendritic cells, macrophages, CD8 and CD4 T cells. We also found a large amount of lymphoid follicles showing a composition shift from B cell to T cell predominance. Together with the elevated presence of single T cells and macrophages in CF tissue, this may suggest a role for the cellular adaptive immune system in the pathophysiology of CF. Furthermore, considering the specific increase of eosinophils, mast cells and CD4 T cell counts in the lung tissue of female CF patients, inflammation in the CF lung seemed to be gender-specific in our population. This may be the result of the complex immunomodulatory effects of oestrogen on inflammation.