Deficient interleukin-17 production in response to Mycobacterium abscessus in cystic fibrosis

To the Editor:

The respiratory tract of patients with cystic fibrosis (CF) is colonised with a high diversity of micro-organisms. Nontuberculous mycobacteria (NTM) show a high and increasing prevalence. 40% of these positive NTM cultures are caused by Mycobacterium abscessus [1], one of the rapidly growing NTMs present in the environment. Patients with M. abscessus infection are difficult to treat, due to natural and acquired antibiotic resistance [2, 3], and an infection with M. abscessus is controversially discussed as a contraindication for lung transplantation [4].

Immune-modulatory treatment strategies might contribute to overcome this problem. For their development, a better understanding of the defective immune response explaining the higher susceptibility of CF patients to M. abscessus is needed. Here we present three CF patients with M. abscessus infection, in whom we describe the pathogen-specific innate and adaptive cytokine production and compare this with non-CF patients with pulmonary infection caused by various NTMs: M. abscessus (n=1), M. avium (n=3), M. kansasii (n=2) and M. intracellulare (n=1).

Case 1 is a 24-year-old female patient with CF (dF508del/dF508del) with pancreatic insufficiency and Pseudomonas colonisation since 2003. In 2004, she presented with allergic bronchopulmonary aspergillosis (ABPA) which was successfully treated with corticosteroids. After years of infectious exacerbations she presented with an episode of haemoptysis in 2010. Shortly thereafter, M. abscessus was cultured from her sputum. In 2011, haemoptysis and clinical deterioration led to hospitalisation and several courses of antimycobacterial regimens (combinations of amikacin, clarithromycin, tigecycline, meropenem and clofazimine) were given without successful M. abscessus eradication.

Case 2 is a 23-year-old male patient with CF (dF508del/dF508del), pancreatic insufficiency and Staphylococcus aureus and Aspergillus colonisation who presented with ABPA. After a course of corticosteroids and itraconazole, he improved and serological markers for ABPA have remained at low levels ever since. After this episode, M. abscessus was consistently cultured and although he had no physical complaints, his pulmonary function deteriorated, and a computed tomography thorax scan showed several subpleural and intraparenchymatous nodular lesions compatible with mycobacterial disease. No clearance of M. abscessus was achieved, despite two courses of treatment with combination regimens of amikacin, meropenem, clarithromycin and clofazimine.

Case 3 is a 15-year-old male patient with CF (dF508del/G551D), pancreatic insufficiency and Pseudomonas colonisation. Since 2011 M. abscessus has been consistently cultured and he experienced several exacerbations in 2014, in which his pulmonary function deteriorated, despite several NTM regimens (including tigecycline, clofazimine, linezolid and azithromycin) and treatment with ivacaftor. However, he was noncompliant with the treatment and not able to complete several regimens due to side-effects and a complex psychosocial situation. No relevant clearance of M. abscessus was achieved.

Peripheral blood mononuclear cells (PBMCs) isolated from the three patients described above were stimulated with M. abscessus, isolated and cultured from the second case, Candida albicans, Escherichia coli lipopolysaccharide (LPS) and Aspergillus fumigatus. CF patients and pulmonary NTM patients did not demonstrate a distinct innate cytokine profile after PBMC stimulation with M. abscessus or C. albicans (figure 1a and b). Interestingly, all NTM patients showed higher tumour necrosis factor (TNF)-α and interleukin (IL)-1β production on LPS stimulation compared to the control group, while the A. fumigatus-specific innate immune response was selectively increased in the CF patients infected with M. abscessus (figure 1c and d).

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FIGURE 1

Innate and adaptive cytokine responses of three cystic fibrosis (CF) patients with Mycobacterium abscessus infection compared with non-CF patients with pulmonary nontuberculous mycobacteria (pNTM) infections. Peripheral blood mononuclear cells (2.5×10⁶ cells·mL^-1) of a healthy control group (n=11), non-CF patients with pNTM infections (n=7) and three CF patients with M. abscessus infection were stimulated with heat-killed M. abscessus (clinical isolate NLA001001395) (1×10⁵ cells·mL^-1), heat-killed Candida albicans (ATCC MYA-3573, UC820) (1×10⁵ cells·mL^-1), Escherichia coli lipopolysaccharide (E. coli serotype O55:B5; Sigma-Aldrich, St. Louis, MO, USA) (1 ng·mL⁻¹) or Aspergillus fumigatus conidia (clinical isolate V05-27) (1×10⁷ cells·mL^-1). The innate cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β were measured after 24 h and the adaptive cytokines interferon (IFN)-γ, IL-17 and IL-22 were measured using ELISA after 7 days in the cell culture supernatant. Innate cytokine response to a) M. abscessus; b) C. albicans; c) E. coli lipopolysaccharide; d) A. fumigatus. Adaptive cytokine response to e) M. abscessus; f) C. albicans; and g) A. fumigatus.

Cystic fibrosis transmembrane conductance regulator-deficient monocytes have been described as highly reactive to LPS stimulation due to a prolonged sensing period of Toll-like receptor-4 on the cell surface [5]. Furthermore, previous studies reported a hyperresponsive innate immune system in CF patients, with elevated TNF-α, IL-1β and IL-6 levels in the sputum [6], which is in line with the elevated innate immune profile described in the CF patients of our study. CF patients, who are often colonised with Aspergillus, are at high risk of developing ABPA (7.8%) [7], and two out of the three CF patients with NTM infection in this study fulfilled the criteria of ABPA. Whether and how the strongly increased innate cytokine response after Aspergillus stimulation, as seen in all the CF patients in this study contributes to the development of ABPA or is the result of a hypersensitivity reaction in an Aspergillus-colonised lung remains to be elucidated.

In contrast, the acquired immune responses revealed pathogen-specific differences. To investigate adaptive T-helper cell (Th) responses, PBMCs were stimulated with M. abscessus, C. albicans (which was used as a positive control for IL-17 production) and Aspergillus conidia. PBMCs from healthy subjects produced IL-17, IL-22 and interferon (IFN)-γ in response to M. abscessus (figure 1e). Although PBMCs isolated from both patient groups were capable of producing IL-17 in response to C. albicans (figure 1f), IL-17 in response to M. abscessus was very low in all CF patients, as well as in most of the patients with pulmonary NTM infection. In contrast, M. abscessus-induced IL-22 and IFN-γ was similar or even elevated in CF patients infected with M. abscessus, while the pulmonary NTM patients demonstrated low IFN-γ and IL-22 production upon M. abscessus stimulation. This suggests a selective pathogen-specific immunodeficiency in CF patients infected with M. abscessus shown by the total absence of M. abscessus-specific IL-17 production. Interestingly, the Aspergillus-specific Th-1 and -17 responses did not show differences between the groups.

CF is associated with a high prevalence of M. abscessus infections [1], but the underlying host defects leading to this increased susceptibility are not fully understood. Abnormalities in the IL-12/IFN-γ-signalling pathways are a known risk factor for mycobacterial infections [8], and decreased IL-17 production in response to M. avium has been described in PBMCs isolated from patients with NTM lung disease [9]. This is in line with the low Th cytokine deficiency of the pulmonary NTM patients in this study. A protective role in mucosal immunity against extracellular fungal and bacterial infections has been described for the Th-17 subpopulation, especially in the lung [10]. Although some of the healthy volunteers responded only poorly on M. abscessus stimulation, in most donors a robust Th-17 response was induced, as described previously [11]. The three CF patients in this study demonstrated a M. abscessus-specific IL-17 deficiency, while the cells were not intrinsically deficient in IL-17 production. Despite earlier reports that described significantly elevated IL-17 level in sputum and airway mucosa of CF patients, we propose that M. abscessus-specific IL-17 deficiency can promote the susceptibility to chronic NTM infections [12].

Treatment with biological drugs blocking innate cytokines that promote Th-17 differentiation is associated with an increased risk of NTM infections [13]. Therefore we propose the inverse correlation that patients with low IL-17-responses, such as the three CF patients in this study, might profit from an IL-17 supporting treatment regimen. Only a few studies have been performed to date. Next to treatment with IFN-γ, which had beneficial effects on the IL-17 and IL-22 production in patients with severe fungal infection [14], one might speculate about the supplementation of recombinant IL-17. Low-dose IL-17 has recently been shown to prevent diabetic nephropathy and organ fibrosis [15].

In conclusion, we describe pathogen-specific cytokine signatures in three CF patients with M. abscessus infection, two of them with ABPA, which might contribute to their higher susceptibility of NTM and Aspergillus-specific responses. The M. abscessus-specific IL-17 deficiency, rather than an IFN-γ defect might play a crucial role in promoting pulmonary NTM infections in CF. This might have direct implementation for different immune-modulatory treatment regimens in pulmonary NTM infection in patients, either with or without CF.