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 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

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 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