PMA synergistically enhances up-regulation of MUC5AC induced by activation of TLR signaling.

<p>(<b>A</b>) PMA synergistically enhanced MUC5AC expression induced only by NTHi, <i>S.p.</i> and PGN, but not by TNF-α and IL-6, as assessed by MUC5AC-dependent promoter Luciferase assay. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. NTHi, <i>S.p.</i>, or PGN alone; <i>^#p>0.05</i> vs. TNF-α or IL-6 alone. (<b>B</b>) PMA synergized with either NTHi or <i>S.p.</i>, but not with TNF-α to enhance MUC5AC expression in a dose-dependent manner. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. PMA alone; <i>^#p>0.05</i> vs. PMA alone. (<b>C</b>) PGN-induced MUC5AC transcription was synergistically enhanced by PMA in HEK293-TLR2 cells, but not in HEK293-pcDNA cells. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. PGN alone. (<b>D</b>) Synergistic enhancement of MUC5AC expression by PMA was also observed in the cells treated with TLR ligands, such as PGN, Zymosan, Poly(I:C) and LPS, respectively. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. PGN, Zymosan, Poly(I:C), or LPS alone. The data shown are representative of three independent experiments. −, absence of; +, presence of; <i>S.p.</i>, <i>Streptococcus pneumoniae</i>; NTHi, nontypeable <i>Haemophilus influenzae</i>.</p

CARMA1 mediates TLR-dependent synergistic MUC5AC induction by NTHi and PMA via cross-talk with TRAF6.

<p>(<b>A</b>) CARMA1 knockdown by CARMA1-siRNA efficiently reduced the endogenous CARMA1 expression at both the mRNA and protein level, as assessed by Q-PCR and WB, respectively. (<b>B</b>) CARMA1-siRNA inhibited the synergistic induction of MUC5AC transcription by NTHi and PMA. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. NTHi alone; <i>**p<0.05</i> vs. control siRNA transfeced cells. (<b>C</b>) Synergistic enhancement of NTHi-induced TRAF6 polyubiquitination by PMA was attenuated by CARMA1 knockdown. (<b>D</b>) Schematic representation depicting how CARMA1 mediates the synergistic enhancement of <i>MUC5AC</i> expression in human epithelial cells. The data shown are representative of three independent experiments. CON, control; −, absence of; +, presence of; NTHi, nontypeable <i>Haemophilus influenzae</i>; Ubn, ubiquitin; TLRs, toll-like receptors; TRAF6, TNF receptor associated factor 6; PKC, protein kinase C.</p

PMA synergistically enhances NTHi-induced MUC5AC expression via MKK3/6-p38 MAPK pathway.

<p>(<b>A</b>) PMA synergistically enhanced NTHi-induced phosphorylation of p38 MAPK and MKK3/6, but not ERK and MEK1. (<b>B</b>) The synergistic induction of MUC5AC transcription by NTHi and PMA was inhibited by overexpressing DN mutant forms of p38α and p38β in human epithelial cells, as assessed by MUC5AC-dependent promoter Luciferase assay. Cells were transfected with 0.8 µg of DN p38α, DN p38β, or control vector, and treated with NTHi with or without PMA. Relative luciferase activity of MUC5AC was measured from the cell lysate. (<b>C</b>) SB203580, a specific inhibitor for p38 MAPK signaling, attenuated the synergistic induction of MUC5AC expression by NTHi and PMA at the mRNA level as assessed by Q-PCR. Cells were pre-treated with 10 µM of SB203580 or vehicle control, and treated with NTHi with or without PMA. mRNA expression level of MUC5AC was measured by Q-PCR. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. NTHi alone; <i>***p<0.05</i> vs NTHi with PMA in control vector transfected (<b>B</b>) or vehicle treated (<b>C</b>) cells. The data shown are representative of three independent experiments. −, absence of; +, presence of; DN, dominant negative; NTHi, nontypeable <i>Haemophilus influenzae</i>.</p

CARMA1 acts downstream of PKCθ in mediating PMA-induced synergistic enhancement of TLR-TRAF6-dependent MUC5AC expression.

<p>(<b>A</b>) CARMA1 was expressed in a variety of epithelial cells, such as in human cervix HeLa, colon HM3, airway A549, middle ear HMEEC-1 and primary bronchial epithelial NHBE cells, as assessed by WB using antibody against CARMA1. (<b>B</b>) Overexpression of a DN mutant form of CARMA1 attenuated C/A-PKCθ-induced MUC5AC expression. Cells were transfeced with 0.3 µg of C/A PKCθ with or without 0.8 µg of DN-CARMA1. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. C/A PKCθ transfected cells. (<b>C</b>) The synergistic induction of MUC5AC transcription by NTHi and PMA was potently inhibited by overexpression of DN-CARMA1. Cells were transfeced with 0.8 µg of DN-CARMA1 or control vector, and treated with NTHi with or without PMA. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. NTHi alone; <i>**p<0.05</i> vs. control vector transfected cells. (<b>D</b>) Overexpression of a DN mutant form of CARMA1 greatly inhibited not only synergistic phosphorylation of p38 MAPK and MKK3/6 induced by NTHi and PMA, but also ERK and MEK phosphorylation. Cells were transfeced with 0.8 µg of DN-CARMA1 or control vector, and treated with NTHi with or without PMA. Cell lysate was blotted with antibodies indicated in the figure. The data shown are representative of three independent experiments. −, absence of; +, presence of; NTHi, nontypeable <i>Haemophilus influenzae</i>; DN, dominant negative; C/A, constitutively active form.</p

TLR-TRAF6-dependent synergistic MUC5AC induction by NTHi and PMA is mediated by PKCθ.

<p>(<b>A</b>) Rottlerin, a specific PKC™/θ inhibitor, blocked the synergistic induction of MUC5AC transcription by NTHi and PMA in human epithelial cells, as assessed by MUC5AC-dependent promoter Luciferase assay. Cells were pre-treated with 20 µM of Rottlerin or vehicle control, and treated with NTHi with or without PMA. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. NTHi alone; ***<i>p<0.05</i> vs. NTHi with PMA in vehicle treated cells. (<b>B</b>) The synergistic induction of MUC5AC expression was also attenuated by Rottlerin at the mRNA level, as assessed by performing Q-PCR. Cells were pre-treated with 20 µM of Rottlerin or vehicle control, and treated with NTHi with or without PMA. mRNA expression level of MUC5AC was measured by Q-PCR. (<b>C</b>) Co-expressing WT-PKCθ enhanced, whereas DN-PKCθ inhibited, the synergistic induction of MUC5AC transcription by NTHi and PMA. Cells were transfected with 0.3 µg of WT- PKCθ, 0.6 µg of DN PKCθ, or control vector, and treated with NTHi with or without PMA. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. NTHi alone; ***<i>p<0.05</i> vs. NTHi with PMA in control vector transfected cells. (<b>D</b>) C/A-PKC-induced MUC5AC expression was synergistically enhanced by NTHi in human epithelial cells. Cells were transfeced with 0.3 µg of C/A PKCθ or control vecttor, and treated with NTHi. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. NTHi alone. (<b>E</b>) C/A-PKCθ synergized with WT-TRAF6 to induce MUC5AC expression in a dose-dependent manner. Cells were transfected with 0.1, 0.3, or 0.6 µg of C/A PKCθ with or without 0.3 µg of WT-TRAF6. mRNA expression level of MUC5AC was measured by Q-PCR. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. C/A PKCθ transfected cells. The data shown are representative of three independent experiments. −, absence of; +, presence of; NTHi, nontypeable <i>Haemophilus influenzae</i>; WT, wild-type; DN, dominant negative; C/A, constitutively active form.</p

PMA synergizes with NTHi to induce MUC5AC expression in human epithelial cell.

<p>(<b>A</b>) PMA synergistically enhanced NTHi-induced MUC5AC expression at the mRNA level in human epithelial HM3 cell, as assessed by performing RT-PCR (left panel) and real-time quantitative PCR (Q-PCR) analysis (right panel). Cyclophilin was used as a control for amount of RNA used in each reaction. (<b>B</b>) PMA synergized with NTHi to induce MUC5AC expression at the transcriptional level in human epithelial cells, as assessed by MUC5AC-dependent promoter Luciferase assay. (<b>C</b>) Synergistic induction of MUC5AC expression by NTHi and PMA was also observed in HM3 cells stably transfected with pMUC5AC 3.7 kb-luc. (<b>D</b>) PMA synergizes with NTHi to induce MUC5AC transcription in a dose-dependent manner and vice versa. (<b>E</b>) PMA synergistically enhanced NTHi-induced MUC5AC expression at the transcriptional level in human airway A549, middle ear HMEEC-1 and primary bronchial epithelial NHBE cells, as assessed by MUC5AC-dependent promoter assays. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. NTHi alone. The data shown are representative of three independent experiments. −, absence of; +, presence of; NTHi, nontypeable <i>Haemophilus influenzae</i>.</p

Schematic model illustrating NTHi-induced MyD88s is regulated by IKKβ, CREB and ERK1/2 signaling pathways.

<p>As indicated NTHi-induced MyD88s expression is positively regulated via IKKβ and CREB and negatively regulated via ERK1/2 creating a negative feedback loop to maintain immune homeostasis.</p

IKKβ is required for NTHi-induced MyD88 short expression in airway epithelial cells.

<p><b>(A)</b> BEAS-2B cells were stimulated with NTHi at various intervals of time as indicated. Phospho-IKK, total IKK and β-actin were visualized via western blot analysis. <b>(B)</b> Cells were treated with IKKβ inhibitor (1 μM) prior to NTHi stimulation for 6 hours and relative quantity of human MyD88s mRNA was measured by real-time Q-PCR analysis. <b>(C-E)</b> Cells were transfected with (<b>C</b>) IKKα-DN, (<b>D</b>) IKKβ-DN or (<b>E</b>) cotransfected with IκBα-DN and IKKβ-CA. Following NTHi stimulation for 6 hours, the relative quantities of MyD88s mRNA, human IL-6 mRNA and human IL-1β were measured via real-time Q-PCR analysis. Data are mean ± SD (<i>n</i> = 3). *<i>p<0</i>.<i>05</i>. Statistical analysis was performed using Student’s <i>t-</i>test. n.s., nonsignificant. Data are representative of three or more independent experiments.</p

<i>In Vitro</i> Interaction of <i>Pseudomonas aeruginosa</i> with Human Middle Ear Epithelial Cells

<div><p>Background</p><p>Otitis media (OM) is an inflammation of the middle ear which can be acute or chronic. Acute OM is caused by <i>Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis</i> whereas <i>Pseudomonas aeruginosa</i> is a leading cause of chronic suppurative otitis media (CSOM). CSOM is a chronic inflammatory disorder of the middle ear characterized by infection and discharge. The survivors often suffer from hearing loss and neurological sequelae. However, no information is available regarding the interaction of <i>P. aeruginosa</i> with human middle ear epithelial cells (HMEECs).</p><p>Methodology and Findings</p><p>In the present investigation, we demonstrate that <i>P. aeruginosa</i> is able to enter and survive inside HMEECs via an uptake mechanism that is dependent on microtubule and actin microfilaments. The actin microfilament disrupting agent as well as microtubule inhibitors exhibited significant decrease in invasion of HMEECs by <i>P. aeruginosa</i>. Confocal microscopy demonstrated F-actin condensation associated with bacterial entry. This recruitment of F-actin was transient and returned to normal distribution after bacterial internalization. Scanning electron microscopy demonstrated the presence of bacteria on the surface of HMEECs, and transmission electron microscopy confirmed the internalization of <i>P. aeruginosa</i> located in the plasma membrane-bound vacuoles. We observed a significant decrease in cell invasion of <i>OprF</i> mutant compared to the wild-type strain. <i>P. aeruginosa</i> induced cytotoxicity, as demonstrated by the determination of lactate dehydrogenase levels in culture supernatants of infected HMEECs and by a fluorescent dye-based assay. Interestingly, <i>OprF</i> mutant showed little cell damage compared to wild-type <i>P. aeruginosa</i>.</p><p>Conclusions and Significance</p><p>This study deciphered the key events in the interaction of <i>P. aeruginosa</i> with HMEECs <i>in vitro</i> and highlighted the role of bacterial outer membrane protein, OprF, in this process. Understanding the molecular mechanisms in the pathogenesis of CSOM will help in identifying novel targets to design effective therapeutic strategies and to prevent hearing loss.</p></div

Scanning electron micrographs demonstrating interaction of <i>P. aeruginosa</i> with HMEECs.

<p>Epithelial cells were infected with <i>P. aeruginosa</i> for 30 min (A), 1h (B), 1.5h (C), 2h (D), 4h (E) and 8h (F) and then subjected to SEM. Large number of bacteria were seen on the surface of HMEECs at 8h post-infection. Results are representative of four independent experiments carried out in triplicate. Scale bars 2 μm.</p