Additional file 1: of Personalized objects can optimize the diagnosis of EMCS in the assessment of functional object use in the CRS-R: a double blind, randomized clinical trial

CONSORT 2010 checklist of information to include when reporting a randomised trial. (DOC 217 kb

IL-1β increases wound closure via a TGF-β1-dependent mechanism in primary human ATII cell monolayers.

<p>(<b>A</b>) IL-1β increases the rate of wound closure in primary human ATII cell monolayers. IL-1β (10 ng/ml) and/or TGF-βscRII or their respective vehicles were added to the monolayers after the scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 36 h (right panels t = 36 h). Scale bar: 100 µm. (<b>B</b>) TGF-β1 soluble receptor (TGF-βscRII) prevents IL-1β-dependent increase in rate of wound closure of primary human ATII cell monolayers. IL-1β (10 ng/ml) and/or TGF-βscRII or their respective vehicles were added to the monolayers after the scratch. Rate of wound closure was expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to IL-1β vehicle. **<i>p</i><0.05 from monolayers exposed to IL-1β.</p

List of class I PI3K isoform inhibitors and their respective in vitro IC₅₀.

<p>Assays were conducted side by side with 10 µM ATP using the method described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#pone.0063907-Knight1" target="_blank">[22]</a>.</p

Endogenous HMGB1 released by primary rat ATII cell monolayers after scratch wounds increases alveolar epithelial wound closure via an IL-1β-dependent mechanism.

<p>(<b>A</b>) Supernatant from primary rat ATII monolayers collected 6 hours after multiple scratches (MS Cell Sup) induces an increase in the secretion of IL-1β by primary rat ATII cell monolayers via a TLR4-dependent pathway. MS Cell Sup, a blocking TLR4 antibody or its isotype control IgG were added to the monolayers after the scratch. HMGB1 was depleted from MS Cell Sup by immunoprecipitation using 30 µg/ml of HMGB1 specific Ab (MS Cell Sup IP w/HMGB1 Ab). Controls were MS Cell Sup immunoprecipitated with a control IgG (MS Cell Sup IP w/Cont Ab). IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. (<b>B</b>) IL-1β receptor antagonist (IL-1RA) prevented the MS Cell Sup-dependent increase in the rate of wound closure of primary rat ATII cell monolayers. MS Cell Sup, and IL-1β receptor antagonist (IL-1RA, 1 µg/ml) or its vehicle were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to condition media. **<i>p</i><0.05 from monolayers exposed to MS Cell Sup.</p

Human recombinant HMGB1 increases alveolar epithelial wound closure via an IL-1β-dependent signaling pathway.

<p>(<b>A</b>) Human recombinant HMGB1 increases the rate of wound closure of primary rat ATII cell monolayers. HMGB1 (10 ng/ml) or its vehicle was added to the monolayers after scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 16 h (right panels t = 16 h). Scale bar: 100 µm. In some experiments, glycyrrhizin or its vehicle was added to the monolayers after the scratch. (<b>B</b>) IL-1 receptor antagonist (IL-1RA) prevents HMGB1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. HMGB1 (10 ng/ml) and/or IL-1 receptor antagonist (IL-1RA, 20 µg/ml) or their respective vehicles were added to the monolayers after the scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 16 h (right panels t = 16 h). Scale bar: 100 µm. (<b>C</b>) HMGB1 increases the secretion of IL-1β by primary rat ATII cell monolayers. HMGB1 (10, 50 and 250 ng/ml, 6 h) or fully oxidized HMGB1 (ox HMGB1, 250 ng/ml) or their respective vehicles was added to the cell monolayers, and IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. In some experiments, zVAD or its vehicle was added to the cell medium 30 min prior to HMGB1. (<b>D</b>) Glycyrrhizin prevents HMGB1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. (<b>E</b>) High dose of Human recombinant HMGB1 (1 µg/ml) is required to increase the rate of wound closure of 3T3 fibroblasts. HMGB1 (50, 250, 1000 ng/ml) or its vehicle was added to the monolayers after scratch. (<b>F</b>) HMGB1 does not increase the secretion of IL-1β by 3T3 fibroblasts. HMGB1 (50, 250 and 1000 ng/ml, 6 h) or its vehicle was added to the cell monolayers, and IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. IL-1β (10 ng/ml) and glycyrrhizin (20 µg/ml) or their respective vehicles were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to HMGB1 or IL-1β vehicles. **<i>p</i><0,005 from monolayers exposed to HMGB1.</p

TGF-β1 increases alveolar epithelial repair via PI3Kα in primary rat ATII cell monolayer.

<p>(<b>A</b>) A β6 blocking antibody (3G9) prevents IL-1β-dependent phosphorylation of Akt in primary rat ATII cell monolayers. IL-1β (10 ng/ml, 30 min) or its vehicle was added to the monolayers 30 minutes after pretreatment with a β6 blocking antibodies or its isotype control antibody (3G9, Cont IgG, 1 µg/ml). (<b>B</b>) TGF-β1 soluble receptor (TGF-βscRII) prevents IL-1β-dependent phosphorylation of Akt in primary rat ATII cell monolayers. IL-1β (10 ng/ml, 30 min) or its vehicle was added to the monolayers 30 minutes after pretreatment with a TGF-β1 soluble receptor (TGF-βscRII, 20 µg/ml) or its vehicle. (<b>C</b>) PI3 kinase inhibition prevents TGF-β1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. TGF-β1 (10 ng/ml) and a broad inhibitor of PI3K (PIK90, 1 µM) or their respective vehicles were added to the monolayers after the scratch. (<b>D</b>) Inhibition of PI3Kα prevents TGF-β1-dependent increase in rate of wound closure of primary rat ATII cell monolayers. TGF-β1 (10 ng/ml) and isoform-specific inhibitors of PI3K (PW12, TGX220, SW14, 0.5 µM) or their respective vehicles were added to the monolayers after the scratch. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#pone-0063907-t001" target="_blank"><b>Table 1</b></a> for PI3K isoform-specific inhibitors IC₅₀. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to TGF-β1 vehicle. For western blot experiments, one representative experiment is shown, three additional experiments gave comparable results; *<i>p</i><0.05 from monolayers exposed to IL-1β vehicle. For immunofluorescence experiments, one representative experiment is shown; four additional experiments gave comparable results.</p

IL-1β increases wound closure via αvβ6 integrin and TGF-β1 in primary rat ATII cell monolayers.

<p>(<b>A</b>) IL-1β increases the rate of wound closure of primary rat ATII cell monolayers. IL-1β (10 ng/ml) or its vehicle was added to the monolayers after the scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 16 h (right panels t = 16 h). Scale bar: 100 µm. (<b>B</b>) A β6 blocking antibody (3G9) prevents IL-1β-dependent increase in rate of wound closure of a primary rat ATII cell monolayer. IL-1β (10 ng/ml). A β6 blocking antibody or its isotype control antibody was added to the monolayers after the scratch. (<b>C</b>) RGD peptides prevent IL-1β-dependent increase in rate of wound closure of a primary rat ATII cell monolayers. IL-1β (10 ng/ml) and RGE or RGD peptide were added to the monolayers after the scratch. (<b>D</b>) A TGF-β1 soluble receptor (TGF-βscRII) prevents IL-1β-dependent increase in rate of wound closure of primary rat ATII cell monolayers. IL-1β (10 ng/ml) and/or TGF-βscRII or their respective vehicles were added to the monolayers after the scratch. Degree of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to IL-1β vehicle. **<i>p</i><0.05 from monolayers exposed to IL-1β.</p

Endogenous HMGB1 released by damaged primary rat ATII cells increases alveolar epithelial wound closure.

<p>(<b>A</b>) HMGB1 was elevated in cell supernatant from rat ATII monolayers that underwent scratch wounds (MS Cell Sup) compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds (condition media). (<b>B</b>) MS Cell Sup increases the rate of wound closure of primary rat ATII cell monolayers compared to cell supernatant from rat ATII monolayers that did not undergo scratch wounds. HMGB1 was depleted from MS Cell Sup by immunoprecipitation using 30 µg/ml of HMGB1 specific Ab (MS Cell Sup IP w/HMGB1 Ab). Controls were MS Cell Sup immunoprecipitated with a control IgG (MS Cell Sup IP w/Cont Ab). (<b>C</b>) HMGB1 is secreted by primary rat ATII cell monolayers after scratch wounds. Multiple scratches (MS) were performed on primary rat ATII cell monolayers. Fresh cell media were added for 6 hours to the monolayers after extensive washes. Cell supernatants were then centrifuged to remove dead cells and cell debris, then analyzed by western blot (40 µl loaded per lanes from a 1 ml MS Cell Sup sample). (<b>D</b>) MS Cell Sup increases the rate of wound closure of a primary rat ATII cell monolayers via RAGE- and TLR4-dependent pathways, but not via a CXCR4-dependent mechanism. MS Cell Sup, and either 30 µg/ml of blocking RAGE or TLR4 antibodies or their isotype control IgG, or 1 µM of AMD3100, a CXCR4 inhibitor, were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to control cell media; **<i>p</i><0.05 from monolayers exposed to MS Cell Sup. For western blot experiments, one representative experiment is shown, three additional experiments gave comparable results; *<i>p</i><0.05 from monolayers exposed to condition media.</p

Chromatin changes during AEC differentiation.

<p>A) Manhattan plot of differential chromatin changes. X-axis = chromosomal location, Y-axis = number of cell type-specific chromatin changes within 2 MB region. Upper panel = H3K9/14^Ac changes, blue = AT2 cell-specific acetylation, purple = AT1 cell-specific acetylation. Lower panel = H3K27^me3 changes, orange = AT2 cell-specific methylation, grey = AT1 cell-specific methylation. B) 135 TFBS enrichment in domains of chromatin change from HOMER. X-axis = H3K9/14^Ac, Y-axis = H3K27^me3 enrichment. AT2 enrichment is shown as the log₁₀ TFBS p-value, AT1 enrichment is shown as the −log₁₀ TFBS p-value. C) Example of chromatin changes at an upregulated gene, <i>FZD2</i>, using IGV to visualize chromatin tracks. Blue = H3K9/14^Ac raw reads and SICER peaks called, green = predicted RXR binding site from HOMER analysis. D) Example of downregulated gene expression at the <i>PGC</i> gene locus. Lavender = predicted FOXA1 binding sites from HOMER analysis. AT2 = AEC chromatin signature (D0), AT1 = AEC chromatin signature (D8).</p

Functional validation of a transcription factor signaling pathway predicted from bioinformatics analysis.

<p>A) Western blots examining AT2 and AT1 cell markers during differentiation in the presence or absence of RXR antagonist UVI-3003. LAMIN A/C is the loading control. B) Transepithelial resistance as measured in kΩ-cm² over the course of differentiation. Error bars represent technical duplicates for each plating. C) Rat <i>Aqp5</i>-luciferase 4.3 kb promoter construct. Grey lines = 34 putative PPARA:RXR binding sites (Explain3.0). No sites were predicted from −900 to +6 bp due to lack of rat sequence information in the Explain v3.0 database. The asterisk marks the approximate location in the promoter of the ChIPed RXR site in E, below. The average number of PPARA:RXR sites per kilobase in the listed human/rat/mouse promoters is given in the table, with consensus site listed at the top. D) MLE-15 cells were transiently transfected with the <i>Aqp5</i>-luciferase construct and treated for 48 hours with vehicle (DMSO) or 7.5 µM UVI-3003. UV1-3003 treatment reduced <i>Aqp5</i>-luc activity by 48%±0.06. Values were normalized to vehicle control and represent the mean, error bars represent SEM, N = 3. All experiments represent 3 biological replicates. E) ChIP was performed on primary cultured rat AEC at day 0 (AT2, D0, n = 2) and day 8 (AT1-like, D8, n = 3). A region ∼4 kb upstream of the transcription start site specifically precipitated with RXR in day 8 samples. ChIP of GAPDH with RXR was performed as a control, and POL2 (POLR2A) binding to the GAPDH promoter was included as a positive control for the quality of day 0 DNA.</p