Low Th17 cells in patients with cystic fibrosis and allergic broncho-pulmonary aspergillosis

Abstract

Background: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity response to the allergens of Aspergillus fumigatus, which is the most frequently isolated fungus from the sputum of cystic fibrosis (CF) patients. Because a low number of Th17 lymphocytes is associated with the risk of fungal infections, we investigated inflammatory markers, Th17 cells, and T-cell polarization in CF patients with ABPA.

Methods: We analyzed the levels of inflammatory markers, blood counts, chemokines, cytokines, and T cell subsets in blood and sputum of CF subjects to elucidate the immunological factors associated with CF patients with Aspergillus fumigatus (AF) positive sputum (AFS⁺) or with ABPA.

Results: We observed that AFS+ patients have higher sputum and blood IL-6 levels than AF-negative sputum (AFS^-) patients. Analysis of blood memory T-helper subsets associated with Th1, Th2, and Th17 polarization among circulating CD45RA-/CD4+ memory T-cell subsets showed higher numbers of CCR4⁺/CCR6⁺/CXCR3^- and CCR4⁺/CCR6⁺/CXCR3⁺ memory CD4 cells in AFS+ compared to AFS^- subjects. Further analysis of Th17-related subsets and IL-17 secreting T cells in subjects with AFS+ showed that those with ABPA have statistically significantly lower levels of Th17 cells as compared to those without ABPA.

Conclusion: In CF, AF airway colonization is associated with increased blood counts of Th17-related subsets. However, CF patients with ABPA exhibit lower numbers of CCR4+/CCR6+/CXCR3+ memory CD4 cells and IL-17-secreting CD4 cells compared to control subjects and CF patients without AF sensitization.

Keywords: Th17 cells; allergic bronchopulmonary aspergillosis; cystic fibrosis; pediatrics.

FIGURE 1

(A) Analysis of PCR plasma values and (B) SAA plasma levels and (C) neutrophils count and (D) IL6 plasma values between AFS+ subjects versus AFS− group of patients. (E) CXCL8 plasma levels comparison and (F) CXCL8 sputum levels comparison between AFS+ subjects compared to AFS− group. (G) CXCL10 sputum values and h CXCL9. Sputum plasma levels, between AFS+ and AFS‐ subjects. p Values are shown when a significant difference was present between two groups of patients. AFSP, Aspergillus fumigatus positive sputum culture; AFSN, Aspergillus fumigatus negative sputum culture. p Value for figure (A) obtained by unpaired t‐test, for figures (B, C, D, E, F, G, and H) obtained by Mann–Whitney U‐test.

FIGURE 2

(A) Th17 cell analysis by cell surface markers in AFS+ subjects compared to the AFS− group. (B) CCR6DP cell comparison between AFS+ and AFS− subjects. (C) Th17 analysis with surface markers in ABPA+ versus ABPA−. (D) CCR6DP cell subset comparison between ABPA+ versus ABPA−. (E) Gating strategies for Th1, Th1/Th17, Th2, Th17, Th9, Th22, CCR6DP, and CCR6DN subsets analysis are shown. p Values are shown when a significant difference was present between two groups of patients. ABPA+, allergic broncopulmonary aspergillosis positive group; ABPA−, allergic broncopulmonary aspergillosis negative group; AFSN, Aspergillus fumigatus negative sputum culture; AFSP, Aspergillus fumigatus positive sputum culture. p Value obtained by Mann–Whitney U‐test.

FIGURE 3

(A) Th17 evaluation with intracellular staining between ABPA positive subject, ABPA negative group, Hyper‐IgE syndrome patients and a HD subject group. (B) Th1 level evaluation with intracellular staining between ABPA positive subject, ABPA negative group, Hyer‐IgE syndrome patients and a healthy donor subject group. (C) IL‐10 levels values of ABPA+ and ABPA− subjects. p Values are shown when a significant difference was present between two groups of patients. ABPA+, allergic broncopulmonary aspergillosis positive group; ABPA−, allergic broncopulmonary aspergillosis negative group; HD, healthy donor. Global p value obtained by Kruskal–Wallis test, pairwise p values obtained by Welch's t‐test.