Lung immunoglobulin A immunity dysregulation in cystic fibrosis

Amandine M. Collin1, Marylène Lecocq1, Sabrina Noel2, Bruno Detry1, François M. Carlier1, Frank Aboubakar Nana1, Caroline Bouzin3, Teresinha Leal2, Marjorie Vermeersch4, Virginia De Rose5, Lucile Regard6,7, Clémence Martin6,7, Pierre-Régis Burgel6,7, Delphine Hoton8, Stijn Verleden9, Antoine Froidure1,10, Charles Pilette*1,10 and Sophie Gohy*1,10,11.*These authors contributed equally.

Supplementary figures

Figure S1. CF patients display peribronchial lymphoid follicles. Subepithelial area from one representative control and from one representative CF patient (lymphoid aggregates showed by arrows) and number of lymphoid aggregates per mm² of lung tissue from 33 CF patients, compared with 35 controls (***p<0·0001, Mann-Whitney test). Bars indicate median and interquartile ranges. CF, cystic fibrosis. Scale bar, 100 µm.

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Figure S2. Neutrophil elastase, Ig and Pa-specific Ig concentrations are increased in airways and serum from CF patients. (a) Neutrophil elastase concentration in sputum from 51 controls and 65 CF patients (***p<0·0001, unpaired Student’s t-test), distributed following their genotype (***p<0·001, Kruskal-Wallis test followed by Dunn’s test) or distributed following Leeds criteria (***p<0·001, *p<0·05, Kruskal-Wallis test followed by Dunn’s test). (b) IgM concentration in sputum from 51 controls and 65 CF patients (***p<0·0001, unpaired Student’s t-test), distributed following their genotype (***p<0·001, *p<0·05, Kruskal-Wallis test followed by Dunn’s test) or distributed following Leeds criteria (***p<0·001, Kruskal-Wallis test followed by Dunn’s test). (c) IgA, (d) S-IgA and (e) SC concentration in sputum from 51 controls and 65 CF patients, distributed following their genotype (***p<0·001, **p<0·01, *p<0·05, Kruskal-Wallis test followed by Dunn’s test). (f) IgA concentration in serum from 51 controls and 66 CF patients, distributed following their genotype (*p<0·05, Kruskal-Wallis test followed by Dunn’s test). (g) Pa-specific IgA concentration in sputum from 51 controls and 65 CF patients, distributed following their genotype (***p<0·001, **p<0·01, Kruskal-Wallis test followed by Dunn’s test). (h) Pa-specific IgA concentration in serum from 51 controls and 66 CF patients, distributed following their genotype (***p<0·001, **p<0·01, Kruskal-Wallis test followed by Dunn’s test). Bars indicate mean and standard deviation or median and interquartile ranges (genotype and Leeds distribution). NE, neutrophil elastase; Ig, immunoglobulin; Pa, Pseudomonas aeruginosa; CF, cystic fibrosis; S-IgA, secretory immunoglobulin A; SC, secretory component; ΔF, F508del mutation; C, chronically infected by Pa; I, intermittently infected by Pa; F, free of Pa infection; N, never infected with Pa.

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Figure S3. Regulation of pIgR expression and SC/S-IgA release in CF-derived HBEC following stimulation with Pa supernatant. (a) Representative western blot for pIgR in ALI-HBEC from CF patient treated with Pa SN or PBS as control condition and its pIgR-SC/GAPDH expression quantification in ALI-HBEC from five CF patients treated with Pa SN or PBS (p=0·8125, Wilcoxon test). (b) PIGR mRNA expression (corrected for housekeeping genes) in ALI-HBEC from five CF patients treated with Pa SN or PBS as control condition (p=0·0625, Wilcoxon test). (c) SC release in apical washes of ALI-HBEC from five CF patients treated with Pa SN or PBS (p=0·6250, Wilcoxon test). (d) After transcytosis of d-IgA, S-IgA concentration in apical washes from two CF ALI-HBEC treated with Pa SN or PBS (p=0·5000, Wilcoxon test). Bars indicate median and interquartile ranges. pIgR, polymeric immunoglobulin receptor; SC, secretory component; S-IgA, secretory immunoglobulin A; HBEC, human bronchial epithelial cells; CF, cystic fibrosis; Pa, Pseudomonas aeruginosa; SN, supernatant; ALI, air-liquid interface.

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Figure S4. IL-17-producing immune cells are increased in CF and IL-17 upregulates pIgR mRNA expression in Calu-3 cells. (a) RORγt staining (and CD3) in the total lung tissue from one representative control and one representative CF patient, and number of RORγt+ cells, including CD3+ RORγt+ cells, in the total lung tissue from 13 CF patients, as compared with 10 controls (*p=0·0147, *p=0·0147, Mann-Whitney test). (b) PIGR mRNA expression (corrected for housekeeping genes) in Calu-3 cells treated with increasing concentrations of IL-17 (n=5). (c) SC release by Calu-3 cells treated with increasing concentrations of IL-17 (n=5). (d) Representative western blot for pIgR in Calu-3 cells treated with increasing concentrations of IL-17 and pIgR/GAPDH expression in Calu-3 cells treated with increasing concentrations of IL-17 (n=5). (e) XBP1s/XBP1u mRNA ratio (corrected for housekeeping genes) in Calu-3 cells treated with increasing concentrations of IL-17 (n=5). Bars indicate median and interquartile ranges. IL-17, interleukin 17; pIgR, polymeric immunoglobulin receptor; CF, cystic fibrosis; ROR, RAR-related orphan receptor; CD, cluster of differentiation; SC, secretory component; XBP1s/u, spliced/unspliced X-box binding protein 1. Scale bar, 100 µm.

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Supplementary tables

Table S1. List of primers.HumanGene Forward primer sequence 5’ 3’ Reverse Primer sequence 5’ 3’PIGR CTC TCT GGA GGA CCA CCG T CAG CCG TGA CAT TCC CTGXBP-1s ATG GAT GCC CTG GTT GCT GAA GA TGC ACC TGC TGC GGA CTC AXBP-1u AGC ACT CAG ACT ACG TGC ACC TCT CCA GAA TGC CCA ACA GGA TAT CAGIL17 TGG AAT CTC CAC CGC AAT GA GCT GGA TGG GGA CAG AGT TCHPRT1 TGA CAC TGG CAA AAC AAT GCA GGT CCT TTT CAC CAG CAA GCTRPS13 TCG GCT TTA CCC TAT CGA CGC AG ACG TAC TTG TGC AAC ACC ATG TGARPS18 TGT GGG CCG AAG ATA TGC T TGA TCA CAC GTT CCA CCT CATRPL27 TGG TAG GGC CGG GTG GTT GC ACT TTG CGG GGG TAG CGG TCMouseGene Forward primer sequence 5’ 3’ Reverse primer sequence 5’ 3’Pigr CTG TGC CCG AAA CTG GAT CA GTG GAG ACC CCT GAA AAG ACAXbp-1s CTG AGT CCG AAT CAG GTG CAG GTC CAT GGG AAG ATG TTC TGGXbp-1u CAG CAC TCA GAC TAT GTG CA GTC CAT GGG AAG ATG TTC TGGIl17 AGT CCA GGG AGA GCT TCA TCT GGT CTT CAT TGC GGT GGA GAG TTbp CTA TCA CTC CTG CCA CAC C ATG ACT GCA GCA AAT CGC TTGHprt AGT CCC AGC GTC GTG ATT AG TGA TGG CCT CCC ATC TCC TTYwhaz TGC GTG ACA TCT GCA ACG AT GAC TGG TCC ACA ATT CCT TTC T

Definition of abbreviations: pIgR, polymeric immunoglobulin receptor; XBP-1s, spliced X-box binding protein 1; XBP-1u, unspliced X-box binding protein 1; IL17, interleukin 17 ; HPRT1, hypoxanthine-guanine phosphoribosyltransferase 1; RPS13, ribosomal protein S13; RPS18, ribosomal protein S18; RPL27 ribosomal protein L27; TBP, TATA-box binding protein; YWHAZ, tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein zeta.

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