Anti-IL-1β antibody treatment has no significant effect on lung ultrastructure, nor on IL-1β, IL-6 and CCL2 mRNA production in lungs.

<p>Wild type (WT) and F508del CFTR mutation homozygote (d/d) mice (14-week-old females and males) were treated intra-peritoneally, once a week for 8 weeks, an anti-IL-1β antibody (10 mg/kg), or PBS. Mice were euthanatized 1 week after the last treatment and lung structure was observed on H&E stained slides observed at ×20 magnification (A). Cellular infiltration (B) was quantified on these H&E slides and mucus production (C) was analyzed on CAB stained lung slides. Lung injury score was recorded in anti-IL-1β antibody treated mice compared with PBS treated mice. (D and E) mRNA production of IL-1β, IL-6 and CCL2 was measured, and normalized with 2 housekeeping gene expression (<i>Hprt1</i> and <i>Gapdh</i>). The effect of anti-IL-1β antibody treatment compared to PBS control in WT and d/d animals is shown in (D), and the effect of F508del CFTR mutation compared to WT is shown in (E). (n = 6–7)</p

IL-1β participates in <i>P. aeruginosa</i>-induced inflammation at 20 h in WT mice.

<p>WT mice received 10⁶ CFU of <i>P. aeruginosa</i> PA011 intranasally, and WT mice treated with NaCl 0.9% were used as control. A group of WT mice was also treated intraperitoneally with anti-IL-1β antibody, 200 µg/mice, 15 h and 1 h before infection. Body weight variation 20 h after infection is shown in (A). Bacterial load was determined in BALF and in lung homogenate (B), myeloperoxidase activity was quantified (C) and absolute numbers of cells, (macrophages and neutrophils) were measured in BALF (D). Protein concentration was evaluated in BALF (E) and IL-1β (F), KC (G), TNF-α (H) and IL-6 (I), were measured in BALF and in lung homogenate. Cell infiltration was observed on H&E stained slide (J) and scored (K). (n = 5–6) Values are in mean +/− SEM; * for p<0.05; ** for p<0.01 and *** for p<0.001. ns for non-significant.</p

Adoptive transfer of iTreg cells significantly diminished Th1/Th2/Th17 cell frequencies in draining lymph nodes in asthmatic mice.

<p>Intracellular expression of IFN-γ, IL-5, and IL-17A in CD3⁺ T cells were determined by FACS. (A) A representative of 9 mice in each group. Cells were gated on CD3⁺ cells. (B) Results were mean ± SEM of values of 9 mice in each group. *<i>P<0.05</i>, **<i>P<0.01</i>, ***<i>P<0.001</i>. (C) Significant reductions of T cell differentiation markers, including T-bet1 and Gata-3, but not RORγT, were detected in iTreg-treated OVA mice compared to iTreg-untreated OVA control.</p

<i>P. aeruginosa</i> infection induces an increased inflammation in d/d mice.

<p>WT and d/d mice received 2×10⁵ CFU of <i>P. aeruginosa</i> PA011 intranasally, and WT mice treated with NaCl 0.9% were used as control. Body weight variation 20 h after infection is shown in (A). Myeloperoxidase activity was quantified (B) and absolute numbers of cells; (macrophages and neutrophils) were measured in BALF (C). Bacterial load (total CFU) was determined in BALF and in lung homogenate (D). Total protein concentration was evaluated in BALF (E) and KC (F), IL-1β (G) and TNF-α (H) were measured in lung homogenate. Cell infiltration was observed on H&E stained slide (I) and scored (J). (n = 5–6) Values are in mean +/− SEM; * for p<0.05; ** for p<0.01 and *** for p<0.001. ns for non-significant.</p

IL-1β participates in pathologic inflammation in F508del CFTR mutants, in response to <i>P. aeruginosa</i> LPS.

<p>d/d and double KO d/d x IL-1R1^−/− mice were treated intranasally with 80 µg of <i>P. aeruginosa</i> endotoxins/LPS in 40 µL PBS, once a week for 5 weeks. Untreated WT mice (UT) were used as control. Survival is presented in (A) and body weight variation 24 h after the last LPS challenge in (B). Absolute numbers of cells, (macrophages, lymphocytes and neutrophils) were measured in BALF 24 h after the last LPS challenge (C). CCL2 (D), IL-6 (E) and IL-1β (F) were measured in lung homogenate. Cell infiltration was observed on H&E staining (G), mucus production on PAS staining (H) and collagen deposition on CAB staining (I). Histopathological scores are shown in (J). n = 6–7 initially, n = 3 for d/d+LPS mice 24 h after last challenge instillation. Values are in mean +/− SEM; * for p<0.05 and ns for non-significant.</p

<i>In vitro</i> induction of regulatory T (iTreg) cells by TGF-β.

<p>Naive CD4⁺CD25⁻ cells were stimulated with anti-CD3/CD28 coated beads with IL-2 in the presence (CD4_TGF-β) and absence (CD4_med) of TGF-β for 5–6 days. nTreg cells were splenic CD4⁺CD25⁺ cells that were sorted and expanded with anti-CD3/CD28 coated beads with IL-2 for 6–7 days. (A). FoxP3 expression was determined by flow cytometry with anti-Foxp3 antibody. cIgG, control IgG. (B). T cells labeling with CFSE were stimulated with anti-CD3 with or without CD4 condition cells (ratio of CD4 condition to T responder = 1∶2) for three days and CFSE dilution was identified on the CD4⁺ cell gate. (C). T cell proliferation was determined by ³H-thymidine incorporation assay. (D). The T cell proliferation was determined in the different ratios of CD4 conditioned cells and T responder cells. Data was representative or mean ± SEM of three independent experiments.</p

Abnormal airway wall remodeling and AHR were subsided with iTreg cell treatment.

<p>(A) Excessive mucin expression in small airway epithelial cells was detected by PAS staining (red color). (B) The numbers of airways with PAS-positive epithelial cells per lung tissue section were quantified in different experimental groups (n = 5). (C) Clara cells in small airway epithelia were stained by CCSP immunofluorescence staining (green) and the surrounding airway smooth muscle cells were immunostained by SMA (red). (D) Airway resistance was measured upon Mch (40mg/ml) aerosol delivery. Although the airway resistance was still significantly higher in iTreg cell-treated group than that in normal control group, significant reduction of airway resistance was achieved in iTreg cell-treated group compared to non-treated (OVA) or control T cell-treated group. *P<0.05, **P<0.01.</p

Alteration of dentritic cells and related cytokine IL-23.

<p>Significant reduction of both CD11c⁺CD86⁺ and CD11c⁺CD80⁺ subsets of DCs in mediastinal lymph nodes were detected in iTreg-treated OVA mice compared to untreated OVA mice. (B) IL-23 expression at the mRNA level in draining lymph nodes was also significantly reduced by iTreg treatment compared to untreated OVA mice (Fig. 8B).</p

Adoptive transfer of iTreg cells altered cytokine production.

<p>(A) iTreg cells inhibited OVA-induced increase of Th2 cytokines. IL-5 and IL-13 in mouse serum were quantified by specific ELISA. Significant reduction of IL-5 and IL-13 in the group receiving iTreg treatment was detected compared to OVA challenge only control group. **P<0.01. (B) Expression of IRF-4 and IL-10 at the mRNA level of lung lymphocytes was significantly increased in iTreg-treated OVA mice compared to untreated OVA mice. *P<0.05.</p