Fig 2: ER stress and UPR activation in immune cells. mRNA relative expression of ER stress and UPR markers BiP, IRE1α, PERK, XBP1s, ERdj4, ATF4, CHOP, GADD34, TNF, and IL-6. (A) Primary PBMCs from HC volunteers (n = 8) and patients with CF (n = 14) at basal conditions and stimulated with LPS (10 ng/ml) for 4 h. (B) Isolated lymphocytes from HC volunteers (n = 6) and patients with CF (n = 6) at basal conditions and stimulated with LPS (10 ng/ml) for 4 h. (C) Freshly isolated Neutrophils from HC volunteers (n = 6) and patients with CF (n = 6) at basal conditions and stimulated with LPS (10 ng/ml) for 4 h. (D) Isolated monocytes from HC volunteers (n = 6) and patients with CF (n = 6) at basal conditions and stimulated with LPS (10 ng/ml) for 4 h. The 14 CF patients and 8 HC volunteers in (A) are independent from the other panels. The 6 CF patients and 6 HC volunteers in (B–D), are the same. All data are represented by logarithmic scale base 10 and presented as mean ± SEM. All n values represent biological independent samples. Statistical comparisons were performed by unpaired independent student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001; ND, Not detected.

Fig 3: The IRE1α-XBP1 pathway regulates inflammatory cytokine secretion in CF M1 macrophages. IRE1 inhibitors 4 μ8c (50 μM) and MKC-3946 (10 μM) were administrated 30 min before M1 macrophages activation. (A) Polarized M1 macrophages from HC volunteers (n = 9) and patients with CF (n = 9) are presented as percentage M1 of total macrophages measured by flow cytometry. (B) mRNA relative expression of ER stress and UPR markers BiP, XBP1s, ERdj4,TNF, and IL-6 in M1 macrophages from HC volunteers (n = 6) and patients with CF (n = 6). (C,D) IL-6 and TNF cytokine levels from M1 macrophages measured by ELISA, HC volunteers (n = 6) and patients with CF (n = 6). The same CF patients and HC volunteers were used in all panels. All data is presented as mean ± SEM. Data in panel B are represented by logarithmic scale base 10. Statistical comparisons were performed by paired student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Polarization of M1/M2 Macrophages and XBP1s expression. (A) Monocytes were differentiated into macrophages for 6 days with GM-CSF 20 ng/ml or M-CSF 20 ng/ml, then M1-type macrophage activation was achieved by supplementing growth media with 100 ng/ml human IFNγ, and 50 ng/ml LPS. M2-type macrophage activation was achieved by supplementing growth media with 20 ng/ml IL-13 and 20 ng/ml IL-4. Macrophages were characterized as M1-type (markers- CD14+, HLA-DR+, CD274+, CD86+ TNFHI) or M2-type (markers- CD14+, HLA-DR−, CD206+ and IL-10HI). (B) Polarized M2 macrophages (M-CSF, IL-4, and IL-13) from HC volunteers (n = 7) and patients with CF (n = 7) are presented as percentage M1 or M2 of total macrophages measured by flow cytometry. (C) IL-10 cytokine levels from M1 and M2 polarized macrophages measured by ELISA. (D) Polarized M1 macrophages (GM-CSF, LPS, and IFNγ) from HC volunteers (n = 7) and patients with CF (n = 7) are presented as percentage M1 or M2 of total macrophages measured by flow cytometry. (E) IL-6 cytokine levels from M1 and M2 polarized macrophages measured by ELISA. (F) mRNA relative expression of ER stress and UPR markers BiP, IRE1α, PERK, XBP1s, ERdj4, ATF4, CHOP, GADD34, TNF, and IL-6 in M1 macrophages from HC volunteers (n = 9) and patients with CF (n = 9) at basal conditions and stimulated with LPS (100 ngml) for 4 h. (G) XBP1s by RT-PCR in M1 macrophages from HC volunteers (n = 7) and CF patients (n = 9). (H,I) XBP1s levels in HC volunteers (n = 4) and CF (n = 5) M1 macrophages, cells were lysed and immunoblotted for XBP1s and HPRT (H), XBP1s was normalized to HPRT and the ratios were compared to the levels of HC (I). (J) XBP1s levels in HC volunteers (n = 4) and CF (n = 5) M2 macrophages, cells were lysed and immunoblotted for XBP1s and HPRT. All data is presented as mean ± SEM. Data in panel F are represented by logarithmic scale base 10. All n values represent biological independent samples. Statistical comparisons were performed by Mann-Whitney non-parametric test, *p < 0.05, **p < 0.01, ***p < 0.001 (B–E); unpaired independent student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001 (F,I).

Fig 5: Cytokine secretion in NLRP3-stimulated CF immune cells isolated from patients with CF (homozygous Phe508del), following treatment with IVA/LUM or IVA/TEZ.PBMCs isolated at baseline, one month and three months from patients homozygous for Phe508del CFTR mutations receiving compassionate use IVA/LUM (n = 12/13) or IVA/TEZ (n = 8) therapy. Following isolation, PBMCs were immediately stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect levels of A, IL-18; B, IL-1β; C, TNF; D, IL-6 and E, IL-10 secretion from PBMCs in the IVA/LUM treated group. ELISA assays were used to detect levels of F, IL-18; G, IL-1β; H,TNF; I, IL-6; J, IL-10 secretion from PBMCs in the IVA/TEZ treated group. For IVA/LUM: IL-18 (n = 13); IL-1β (n = 13); TNF (n = 12); IL-6 (n = 13); IL-10 (n = 12). A two-way ANOVA statistical test with Tukey’s multiple comparison was performed. P value for baseline to three months is shown on each graph. For IL-6 (IVA/LUM), a non-parametric Kruskal-Wallis statistical test with Dunn’s multiple comparison was performed. Baseline ranges were established for each cytokine from HC and clinically stable drug-naïve CF patients (Figure 2—figure supplement 3). Upper 95% confidence interval for baseline HC (solid blue line) or CF (solid pink line) with block colour shading (HC, blue; CF, pink) to lower 5% confidence interval is displayed for each cytokine.Figure 3—source data 1.Cytokine secretion in NLRP3-stimulated (LPS/ATP) CF immune cells isolated from patients with CF (homozygous Phe508del), following treatment with IVA/LUM or IVA/TEZ.The baseline (pre-therapy, zero month) values for each patient were calculated as a percentage of the average baseline within each patient group (IVA/LUM or IVA/TEZ). The one month and three month samples were calculated as a percentage of the baseline average. ELISA assays were used to detect IL-18, IL-1β, TNF, IL-6 or IL-10 secretion (n = 13 IVA/LUM; n = 8 IVA/TEZ).