Poractant alfa (Curosurf®) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

<p>Abstract</p> <p>Background</p> <p>Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.</p> <p>Methods</p> <p>Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.</p> <p>Results</p> <p>Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.</p> <p>Conclusions</p> <p>We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.</p

Ureaplasma-driven neonatal neuroinflammation: novel insights from an ovine model

Ureaplasma species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including meningitis. Data on mechanisms involved in Ureaplasma-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to Ureaplasma parvum (UP) in utero. 7 days after intra-amniotic injection of UP (n = 10) or saline (n = 11), lambs were surgically delivered at gestational day 128–129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using ureB-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C–X–C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in Ureaplasma-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood–brain barrier function. According to these data and previous in vitro findings from our group, we speculate that Ureaplasma-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation

Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells

Background: Expression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441. Methods: Different quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D. Results: SP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone. Conclusion: SP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research

Immunoblots against claudin-5 and claudin-8 from H441 cells at different densities.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) quantities and either left untreated or incubated with 1 μM dexamethasone for 48 h. Immunoblots against claudin-5 (<b>A</b>) and claudin-8 (<b>B</b>) were performed, protein levels were normalized to total blotted protein, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. * <i>p</i> < 0.05 compared to 1 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

Impact of cell density on mature SP-B expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone. After 48 h immunoblotting against SP-B and β-actin was performed. SP-B levels were normalized to β-actin and fold differences compared to 2.5 × 10⁴ dexamethasone-treated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. ** <i>p</i> < 0.01 compared to 5 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

SP-B mRNA expression in H441 cells co-cultured with A549 cells at different cell densities.

<p>Different quantities of H441 cells were seeded alone or co-cultured with different quantities of A549 cells as indicated for 48 h. (<b>A</b>) SP-B mRNA levels were normalized to β-actin and fold differences compared to 1 × 10⁴ H441 cells per cm² without A549 cells were calculated. Means +SD of n = 4 independent experiments are shown. (<b>B</b>, <b>C</b>) Representative bright field images of H441 cells alone (<b>B</b>) or co-cultured with A549 cells (<b>C</b>). The bar in the upper left image represents 500 μm. *** <i>p</i> < 0.001 compared to 1 × 10⁴ H441 cells per cm² without A549 cells, ### <i>p</i> < 0.001 compared to 2 × 10⁴ H441 cells per cm² without A549 cells, $<i>p</i> < 0.05 and$ $$ <i>p</i> < 0.001 compared to 7.5 × 10⁴ H441 cells per cm² without A549 cells, §§ <i>p</i> < 0.01 compared to 2 × 10⁴ H441 cells per cm² co-cultured with 1 × 10⁴ A549 cells per cm².</p

Impact of cell density on mature SP-B expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone. After 48 h immunoblotting against SP-B and β-actin was performed. SP-B levels were normalized to β-actin and fold differences compared to 2.5 × 10⁴ dexamethasone-treated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. ** <i>p</i> < 0.01 compared to 5 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

SP-B mRNA expression in H441 cells co-cultured with A549 cells at different cell densities.

<p>Different quantities of H441 cells were seeded alone or co-cultured with different quantities of A549 cells as indicated for 48 h. (<b>A</b>) SP-B mRNA levels were normalized to β-actin and fold differences compared to 1 × 10⁴ H441 cells per cm² without A549 cells were calculated. Means +SD of n = 4 independent experiments are shown. (<b>B</b>, <b>C</b>) Representative bright field images of H441 cells alone (<b>B</b>) or co-cultured with A549 cells (<b>C</b>). The bar in the upper left image represents 500 μm. *** <i>p</i> < 0.001 compared to 1 × 10⁴ H441 cells per cm² without A549 cells, ### <i>p</i> < 0.001 compared to 2 × 10⁴ H441 cells per cm² without A549 cells, $<i>p</i> < 0.05 and$ $$ <i>p</i> < 0.001 compared to 7.5 × 10⁴ H441 cells per cm² without A549 cells, §§ <i>p</i> < 0.01 compared to 2 × 10⁴ H441 cells per cm² co-cultured with 1 × 10⁴ A549 cells per cm².</p

Impact of cell density on the expression of SP-B transcription factor mRNAs in H441 cells.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) densities and either left untreated or incubated with 1 μM dexamethasone for 24 h. qPCR of TTF-1 (<b>A</b>), Sp1 (<b>B</b>), Sp3 (<b>C</b>), HNF-3α (<b>D</b>), and ErbB4 mRNA (<b>E</b>) was performed, mRNA levels were normalized to those of GAPDH, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 5 experiments are shown. *** <i>p</i> < 0.001 compared to 1 × 10⁴ untreated cells per cm², ### <i>p</i> < 0.001 compared to 1 × 10⁴ dexamethasone-treated cells per cm², $$ $ <i>p</i> < 0.001 compared to 7.5 × 10⁴ untreated cells per cm².</p

Primers for qPCR.

<p>Primers for qPCR.</p