Effect of Lipopolysaccharide on Glucocorticoid Receptor Function in Control Nasal Mucosa Fibroblasts and in Fibroblasts from Patients with Chronic Rhinosinusitis with Nasal Polyps and Asthma

<div><p>Background</p><p>Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment.</p><p>Objective</p><p>We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma.</p><p>Methods</p><p>NP (n = 12) and NM fibroblasts (n = 10) were <i>in vitro</i> pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing.</p><p>Results</p><p>Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/β MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone’s capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRβ expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone’s capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone’s capacity to suppress FBS-induced CXCL8 release.</p><p>Conclusion</p><p>The bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.</p></div

Effect of LPS on cytokine production.

<p>(A) ELISA quantification of IL-6 production in cell supernatants of NM and NP fibroblasts (n = 8 each) pre-incubated with 10% csFBS-supplemented medium with/without 10 μg/ml LPS (Pre-LPS, 24 hours) and then incubated with LPS or 10% FBS-supplemented medium (10% FBS) for 24 hours. *<i>P</i><.05. (B) Effect of increasing LPS concentrations on 10% FBS-induced IL-6 production (NM and NP, n = 4–5). (C) Effect of LPS (10 μg/ml) on 10% FBS-induced cytokine/chemokine production (NM and NP, n = 5–8). *<i>P</i><.05, ** <i>P</i><.01, and ***<i>P</i><.001 <i>versus</i> medium-treated cells.</p

Effect of LPS on dexamethasone induction of <i>MKP-1</i> and <i>GILZ</i> gene expression.

<p>RT-PCR quantification of <i>MKP-1</i> (A) and <i>GILZ</i> (B) mRNAs in NM (n = 9–10) and NP (n = 12) fibroblasts pre-incubated with 10% csFBS-supplemented medium with/without LPS (10 μg/ml, 24 hours) prior to dexamethasone (DEX, 10^–7 M) addition for the indicated times. *<i>P</i><.05, **<i>P</i><.01, and ***<i>P</i><.001 <i>versus</i> untreated cells at each time point.</p

Effect of kinase inhibitors on LPS-induced IL-6 and CXCL8 secretion.

<p>ELISA quantification of IL-6 (A) and CXCL8 (B) production in cell supernatants of NM and NP fibroblasts (n = 4–6) incubated with 10% csFBS-supplemented medium with/without 10 μM SB203580 (p38α/β MAPK inhibitor), 20 μM SP600125 (JNK inhibitor) or 2 μM BMS-345541 (IκB kinase/NF-κB inhibitor) for 1 hour prior to LPS (10 μg/ml) addition for 24 hours. *<i>P</i><.05 and **<i>P</i><.01 <i>versus</i> LPS alone (100%).</p

Role of MKP-1 and GILZ on dexamethasone inhibition of CXCL8 production.

<p>NM fibroblasts were transfected with MKP-1/GILZ/negative control siRNAs, as indicated in Methods. Twenty-four hours later, cells were pre-incubated with/without LPS (10 μg/ml, 24 hours) prior to incubation with 10% FBS-supplemented medium with/without dexamethasone (DEX, 10^–6 M) for one (MKP-1 protein), six (GILZ protein) or twenty-four (CXCL8 release) hours. (<b>A</b>) MKP-1 and GILZ protein analysis (n = 3–4). ***<i>P</i><.001 <i>versus</i> negative control siRNA. (<b>B</b>) FBS-induced CXCL8 production (n = 6). **<i>P</i><.01 <i>versus</i> no LPS. (<b>C</b>) Dexamethasone inhibition of FBS-induced CXCL8 in LPS-pre-incubated cells (n = 6). *<i>P</i><.05, **<i>P</i><.01, and ***<i>P</i><.001 <i>versus</i> LPS alone.</p

Effect of LPS on dexamethasone induction of GRα nuclear translocation.

<p>(A) GR immunofluorescence images of nasal fibroblasts pre-incubated with 10% csFBS-supplemented medium with/without LPS (10 μg/ml, 24 hours) prior to dexamethasone addition (10^–7 M). (B) Quantification of GRα nuclear translocation in NM and NP fibroblasts (n = 12 each). *<i>P</i><.05, **<i>P</i><.01, and ***<i>P</i><.001 <i>versus</i> 0 hours. (C) Ratio of GRα nuclear translocation induced by dexamethasone over time to the respective (medium or LPS) baseline (0 hours) values.</p

Effect of MAPK specific inhibitors on COX-2 protein expression.

<p>Fibroblasts from nasal mucosa (NM, N = 5, black spots) and nasal polyps from AIA patients (NP-AIA, N = 5, white spots) were pre-treated with p38 (A) MAPK (SB203580), (B) JNK (SP600125), and (C) ERK (PD98059) specific inhibitors at different concentrations (0.1–10 µM) for 1 h before addition of IL-1β (1 ng/ml) for 24 h. COX-2 and β-actin protein expression was analyzed by Western blot. Only the p38 MAPK inhibitor significantly blocked IL-1β-induced COX-2 expression in NM fibroblasts. Results are presented as COX-2/β-actin ratio. Graph shows individual experimental results and lines indicate the medians values. * p<0.05, † p = 0.06 compared to IL-1β treatment by Wilcoxon test.</p

Time-course and dose-response of IL-1β in COX-1 and COX-2 protein expression.

<p>Fibroblasts from nasal mucosa (NM) and nasal polyps from AIA patients (NP-AIA) were incubated at different times and concentrations with IL-1β. COX-1 expression was not altered by IL-1β treatment in either NM or NP-AIA fibroblasts. In NM fibroblasts, COX-2 expression was induced in a time-dependent and dose-response manner. In NP-AIA, COX-2 protein expression was not significantly increased, compared to baseline level. The image that is shown is representative of NM and NP-AIA Western blot.</p

Epidemiological characteristics of control subjects and patients with NP and AIA.

<p>SD, standard deviation.</p

Time-course of p65 and p50 nuclear translocation induced by IL-1β in nasal fibroblast cultures.

<p>Fibroblasts from nasal mucosa (NM, N = 3, black spots) and nasal polyps from AIA patients (NP-AIA, N = 3, white spots) were incubated with IL-1β (10 ng/ml) for 5 to 60 min. p65 (A) and p50 (B) nuclear translocation were measured by TransAM® and Western blot. Graphs show the fold change increase from individual experimental results and lines indicate the medians values. Insets show representative Western blot images of p65 (A) and p50 (B) subunits from both NM and NP-AIA fibroblasts and the loading control β-actin. No significant differences (NS by Mann-Whitney U-test) were found at any time between NM and NP-AIA fibroblasts.</p