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

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.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.NP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GR?, GR?, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GR? nuclear translocation by immunocytochemistry, and GR? localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing.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 p38?/? MAPK and IKK/NF-?B pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone's capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GR? nuclear translocation (only in NM fibroblasts), 3) did not alter GR?/GR? 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.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

Impact of the COVID-19 pandemic on diagnoses of common mental health disorders in adults in Catalonia, Spain : a population-based cohort study

To investigate how trends in incidence of anxiety and depressive disorders have been affected by the COVID-19 pandemic. Population-based cohort study. Retrospective cohort study from 2018 to 2021 using the Information System for Research in Primary Care (SIDIAP) database in Catalonia, Spain. 3 640 204 individuals aged 18 or older in SIDIAP on 1 March 2018 with no history of anxiety and depressive disorders. The incidence of anxiety and depressive disorders during the prelockdown period (March 2018-February 2020), lockdown period (March-June 2020) and postlockdown period (July 2020-March 2021) was calculated. Forecasted rates over the COVID-19 periods were estimated using negative binomial regression models based on prelockdown data. The percentage of reduction was estimated by comparing forecasted versus observed events, overall and by sex, age and socioeconomic status. The incidence rates per 100 000 person-months of anxiety and depressive disorders were 151.1 (95% CI 150.3 to 152.0) and 32.3 (31.9 to 32.6), respectively, during the prelockdown period. We observed an increase of 37.1% (95% prediction interval 25.5 to 50.2) in incident anxiety diagnoses compared with the expected in March 2020, followed by a reduction of 15.8% (7.3 to 23.5) during the postlockdown period. A reduction in incident depressive disorders occurred during the lockdown and postlockdown periods (45.6% (39.2 to 51.0) and 22.0% (12.6 to 30.1), respectively). Reductions were higher among women during the lockdown period, adults aged 18-34 years and individuals living in the most deprived areas. The COVID-19 pandemic in Catalonia was associated with an initial increase in anxiety disorders diagnosed in primary care but a reduction in cases as the pandemic continued. Diagnoses of depressive disorders were lower than expected throughout the pandemic

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis

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

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 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

Study of the antimicrobial activity of cationic carbosilane dendrimers against clinical strains of multidrug-resistant bacteria and their biofilms

IntroductionAntimicrobial Resistance is a serious public health problem, which is aggravated by the ability of the microorganisms to form biofilms. Therefore, new therapeutic strategies need to be found, one of them being the use of cationic dendritic systems (dendrimers and dendrons).MethodsThe aim of this study is to analyze the in vitro antimicrobial efficacy of six cationic carbosilane (CBS) dendrimers and one dendron with peripheral ammonium groups against multidrug-resistant bacteria, some of them isolated hospital strains, and their biofilms. For this purpose, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum eradication biofilm concentration (MBEC) studies were carried out. In addition, the cytotoxicity on Hela cells of those compounds that proved to be the most effective was analyzed.ResultsAll the tested compounds showed in vitro activity against the planktonic forms of methicillin-resistant Staphylococcus aureus and only the dendrimers BDSQ017, BDAC-001 and BDLS-001 and the dendron BDEF-130 against their biofilms. On the other hand, only the dendrimers BDAC 001, BDLS-001 and BDJS-049 and the dendron BDEF-130 were antibacterial in vitro against the planktonic forms of multidrug-resistant Pseudomonas aeruginosa, but they lacked activity against their preformed biofilms. In addition, the dendrimers BDAC-001, BDLS-001 and BDSQ-017 and the dendron BDEF-130 exhibited a good profile of cytotoxicity in vitro.DiscussionOur study demonstrates the possibility of using the four compounds mentioned above as possible topical antimicrobials against the clinical and reference strains of multidrug-resistant bacteria

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

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