Surfactant Protein D Expression Research Articles

Changes in surfactant protein A and D in ovine ovaries related to follicle development.

Surfactant protein A (SP-A) and Surfactant protein D (SP-D) glycoproteins play a crucial role in maintaining lung homeostasis and lung host defense. Interestingly, these proteins are also expressed in extra-pulmonary tissues, including the female genital tract. The ovarian tissue, where SP-A and SP-D expression increases with follicular development, may serve as the primary site of defense for this tissue. However, their functions in these tissues are not well understood and are currently an active area of research. Therefore, the objective of this study is to investigate the expression of SP-A and SP-D in the ovine ovary throughout the ovarian cycle using immunohistochemistry by semiquantitative intensity classification and Western blotting techniques. These findings revealed the presence of SP-A and SP-D in various compartments of the ovary, such as the follicular epithelium, granulosa cells, cumulus cells, theca cells, oocyte I, follicular fluid, and luteal cells of Graafian follicles, excluding the corpus albicans. SP-A and SP-D likely act as a first line of defense against potential pathogens that infiltrate the ovaries. Further investigation of the differential expression of SP-A and SP-D proteins in ovarian follicles will provide a basis for understanding their interactions with key proteins involved in oogenesis.

Identification of Crucial Genes and Signaling Pathways in Alectinib-Resistant Lung Adenocarcinoma Using Bioinformatic Analysis.

Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has been shown to be effective for patients with ALK-positive non-small cell lung cancer (NSCLC). However, alectinib resistance is a serious problem worldwide. To the best of our knowledge, little information is available on its molecular mechanisms using the Gene Expression Omnibus (GEO) database. In this study, the differentially expressed genes (DEGs) were selected from the gene expression profile GSE73167 between parental and alectinib-resistant human lung adenocarcinoma (LUAD) cell samples. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) annotation enrichment analyses were conducted using Database for Annotation, Visualization and Integrated Discovery (DAVID). The construction of protein-protein interaction (PPI) network was performed to visualize DEGs. The hub genes were extracted based on the analysis of the PPI network using plug-in cytoHubba of Cytoscape software. The functional roles of the key genes were investigated using Gene Expression Profiling Interactive Analysis (GEPIA), University of Alabama at Birmingham Cancer (UALCAN), Gene Set Enrichment Analysis (GSEA), and Tumor Immune Estimation Resource (TIMER) analysis. The networks of kinase, miRNA, and transcription-factor targets of SFTPD were explored using LinkedOmics. The drug sensitivity analysis of SFTPD was analyzed using the RNAactDrug database. Results showed a total of 144 DEGs were identified. Five hub genes were extracted, including mucin 5B (MUC5B), surfactant protein D (SFTPD), deleted in malignant brain tumors 1 (DMBT1), surfactant protein A2 (SFTPA2), and trefoil factor 3 (TFF3). The survival analysis using GEPIA displayed that low expression of SFTPD had a significantly negative effect on the prognosis of patients with LUAD. GSEA revealed that low expression of SFTPD was positively correlated with the pathways associated with drug resistance, such as DNA replication, cell cycle, drug metabolism, and DNA damage repair, including mismatch repair (MMR), base excision repair (BER), homologous recombination (HR), and nucleotide excision repair (NER). The SFTPD expression was negatively correlated with the drug sensitivity of alectinib according to RNAactDrug database. The expression of SFTPD was further validated in parental H3122 cells and alectinib-resistant H3122 cells by quantitative reverse transcription PCR (RT-qPCR). In conclusion, our study found that the five hub genes, especially low expression of SFTPD, are closely related to alectinib resistance in patients with LUAD.

Immunomodulatory Role of Surfactant Protein-D in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model.

Surfactant protein D (SP-D), a pattern recognition molecule, is emerging as a potent anti-tumoural innate immune defense molecule in a range of cancers. Previously, SP-D expression was found to be significantly downregulated at the malignant sites of human prostate adenocarcinoma and associated with an increasing Gleason score and severity. We recently reported selective induction of intrinsic apoptosis by a recombinant fragment of human SP-D (rfhSP-D) in the human Prostate cancer (PCa) biopsy explants and cells with glucose regulated protein of 78 (GRP78) as one of the key interacting partners. The present study evaluated the expression of SP-D in early and advanced stages of PCa using transgenic adenocarcinoma of mouse prostate (TRAMP) model. Both early and late stages of PCa showed significantly decreased SP-D mRNA expression and increased proteolytic degradation of SP-D protein. Systemic and tumoural immunophenotyping of TRAMP model revealed increased serine proteases producing granulocytes and polymorphonuclear myeloid-derived suppressor cells (PMN MDSCs) in the late stage; the serine proteases secreted by these cells could be involved in the degradation of SP-D. Susceptibility of rfhSP-D to elastase-mediated proteolysis provided the rationale to use an elastase-inhibitor to sustain intact rfhSP-D in the tumour microenvironment. The study revealed an immunomodulatory potential of rfhSP-D and elastase inhibitor, sivelestat, to induce macrophage polarization towards M1 with downregulation of PMN MDSCs in ex-vivo cultured TRAMP tumours. Furthermore, rfhSP-D induced immunogenic cell death in murine PCa cells and in TRAMP explants. The findings highlight that SP-D plays an anti-tumourigenic role in PCa by inducing immunogenic cell death and immunomodulation while the prostate tumour milieu adversely impacts SP-D by inhibiting its transcription, and enhancing its proteolytic degradation. Transformation of an immunologically “cold tumour” into a “hot tumour” implicates therapeutic potential of rfhSP-D in PCa.

Presence of surfactant proteins in the uteri and placentae of pregnant mares.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.

Role of Surfactant Protein D in Experimental Otitis Media

Surfactant protein D (SP-D) is a C-type collectin and plays an important role in innate immunity and homeostasis in the lung. This study studied SP-D role in the nontypeable Haemophilus influenzae (NTHi)-induced otitis media (OM) mouse model. Wild-type C57BL/6 (WT) and SP-D knockout (KO) mice were used in this study. Mice were injected in the middle ear (ME) with 5 μL of NTHi bacterial solution (3.5 × 10⁵ CFU/ear) or with the same volume of sterile saline (control). Mice were sacrificed at 3 time points, days 1, 3, and 7, after treatment. We found SP-D expression in the Eustachian tube (ET) and ME mucosa of WT mice but not in SP-D KO mice. After infection, SP-D KO mice showed more intense inflammatory changes evidenced by the increased mucosal thickness and inflammatory cell infiltration in the ME and ET compared to WT mice (p < 0.05). Increased bacterial colony-forming units and cytokine (IL-6 and IL-1β) levels in the ear washing fluid of infected SP-D KO mice were compared to infected WT mice. Molecular analysis revealed higher levels of NF-κB and NLRP3 activation in infected SP-D KO compared to WT mice (p < 0.05). In vitro studies demonstrated that SP-D significantly induced NTHi bacterial aggregation and enhanced bacterial phagocytosis by macrophages (p < 0.05). Furthermore, human ME epithelial cells showed a dose-dependent increased expression of NLRP3 and SP-D proteins after LPS treatment. We conclude that SP-D plays a critical role in innate immunity and disease resolution through enhancing host defense and regulating inflammatory NF-κB and NLRP3 activation in experimental OM mice.

Ozone Inhalation Attenuated the Effects of Budesonide on Aspergillus fumigatus-Induced Airway Inflammation and Hyperreactivity in Mice.

Inhaled glucocorticoids form the mainstay of asthma treatment because of their anti-inflammatory effects in the lung. Exposure to the air pollutant ozone (O3) exacerbates chronic airways disease. We and others showed that presence of the epithelial-derived surfactant protein-D (SP-D) is important in immunoprotection against inflammatory changes including those induced by O3 inhalation in the airways. SP-D synthesis requires glucocorticoids. We hypothesized here that O3 exposure impairs glucocorticoid responsiveness (including SP-D production) in allergic airway inflammation. The effects of O3 inhalation and glucocorticoid treatment were studied in a mouse model of allergic asthma induced by sensitization and challenge with Aspergillus fumigatus (Af) in vivo. The role of O3 and glucocorticoids in regulation of SP-D expression was investigated in A549 and primary human type II alveolar epithelial cells in vitro. Budesonide inhibited airway hyperreactivity, eosinophil counts in the lung and bronchoalveolar lavage (BAL) and CCL11, IL-13, and IL-23p19 release in the BAL of mice sensitized and challenged with Af (p < 0.05). The inhibitory effects of budesonide were attenuated on inflammatory changes and were completely abolished on airway hyperreactivity after O3 exposure of mice sensitized and challenged with Af. O3 stimulated release of pro-neutrophilic mediators including CCL20 and IL-6 into the airways and impaired the inhibitory effects of budesonide on CCL11, IL-13 and IL-23. O3 also prevented budesonide-induced release of the immunoprotective lung collectin SP-D into the airways of allergen-challenged mice. O3 had a bi-phasic direct effect with early (<12 h) inhibition and late (>48 h) activation of SP-D mRNA (sftpd) in vitro. Dexamethasone and budesonide induced sftpd transcription and translation in human type II alveolar epithelial cells in a glucocorticoid receptor and STAT3 (an IL-6 responsive transcription factor) dependent manner. Our study indicates that O3 exposure counteracts the effects of budesonide on airway inflammation, airway hyperreactivity, and SP-D production. We speculate that impairment of SP-D expression may contribute to the acute O3-induced airway inflammation. Asthmatics exposed to high ambient O3 levels may become less responsive to glucocorticoid treatment during acute exacerbations.

Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 −/−) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways.

Colonic Epithelial Surfactant Protein D Expression Correlates with Inflammation in Clinical Colonic Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract. Surfactant protein D (SP-D) is expressed in the intestinal epithelium and is essential for innate host defense and regulation of inflammatory responses. Genetic variations of SP-D are associated with IBD, but the effects of SP-D in clinical disease development have not been clarified. We hypothesized that colonic epithelial SP-D expression is increased in parallel with intestinal inflammation with the capacity to dampen deleterious effects. Surgical specimens from IBD patients including Crohn's disease (n = 9) and ulcerative colitis (n = 18) were scored for expression of SP-D and inflammatory activity. Cohoused Sftpd+/+ and Sftpd-/- mouse littermates were subjected to dextran sodium sulfate (DSS) for 7 days to induce colitis. Colonic tissue was scored for histologic damage and analyzed for inflammatory markers and expression of SP-D. Surgical specimens from IBD patients showed a strong positive correlation between immunoscore for SP-D and inflammatory activity (R2 = 0.78, P < 0.0001). In mice, colonic epithelial SP-D expression was very low, and DSS-induced colitis was unaffected by SP-D deficiency, although DSS induced transcription of colonic SP-D to a mild degree. A strong positive correlation between inflammatory activity and epithelial expression of SP-D was observed in surgical specimens from IBD patients supporting a role for SP-D in clinical disease. The in vivo study was inconclusive due to very low intestinal SP-D expression in the mouse. Further studies are warranted to support that increased SP-D expression in the human colonic epithelium is protective against intestinal inflammation.

Surfactant protein D attenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis, inflammation and NF-\u03baB signaling

Pneumonia and sepsis are major risk factors for acute kidney injury (AKI). Patients with pneumonia and AKI are at increased risk for morbidity and mortality. Surfactant protein D (SP-D) expressed in lung and kidney plays important roles in innate immunity. However, little is known about the role of organ-specific SP-D in the sepsis. The current study uses wild type (WT), SP-D knockout (KO), and humanized SP-D transgenic (hTG, lung-specific SP-D expression) mice to study organ-specific role of SP-D in pneumonia-induced sepsis. Analyses demonstrated differential lung and kidney injury among three-type mice infected with Pseudomonas aeruginosa. After infection, KO mice showed higher injurious scores in both lung and kidney, and decreased renal function than WT and hTG mice. hTG mice exhibited comparable lung injury but more severe kidney injury compared to WT mice. Increased renal tubular apoptosis, NF-κB activation and proinflammatory cytokines in the kidney of KO mice were found when compared with WT and hTG mice. Furthermore, in vitro primary proximal tubular epithelial cells from KO mice showed more apoptosis with higher level of activated caspase-3 than those from WT mice after LPS treatment. Collectively, SP-D attenuates AKI in the sepsis by modulating renal apoptosis, inflammation and NF-κB signaling.

Surfactant protein D attenuates nitric oxide-stimulated apoptosis in rat chondrocyte by suppressing p38 MAPK signaling

Innate immune molecule surfactant protein D (SP-D), a member of the C-type lectin protein family, plays an indispensable role in host defense and the regulation of inflammation in the lung and other tissues. Osteoarthritis (OA) is a degenerative disease of cartilage, with inflammation that causes pathologic changes and tissue damage. However, it is unknown whether there exist SP-D expression and its potential role in the pathogenesis of OA. In this study, we examined SP-D expression and explored its biological function in a sodium nitroprusside (SNP)-stimulated rat chondrocytes and surgically-induced rat OA model. We found SP-D expression in both human and rat articular chondrocytes, with higher level in normal chondrocytes compared to in OA chondrocytes. Furthermore, In vivo study demonstrated that recombinant human SP-D (rhSP-D) ameliorated cartilage degeneration in surgically-induced rat OA model. In vitro cell culture study showed that rhSP-D markedly inhibited the expression of caspase-3 as an apoptosis biomarker, and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which resulted in maintaining normal nuclear morphology and increasing mitochondrial membrane potential in SNP-stimulated rat chondrocytes. Collectively, these findings indicate that SP-D expresses in articular chondrocytes and suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via suppressing p38 MAPK activity.

Variable ventilation improves pulmonary function and reduces lung damage without increasing bacterial translocation in a rat model of experimental pneumonia.

BackgroundVariable ventilation has been shown to improve pulmonary function and reduce lung damage in different models of acute respiratory distress syndrome. Nevertheless, variable ventilation has not been tested during pneumonia. Theoretically, periodic increases in tidal volume (VT) and airway pressures might worsen the impairment of alveolar barrier function usually seen in pneumonia and could increase bacterial translocation into the bloodstream. We investigated the impact of variable ventilation on lung function and histologic damage, as well as markers of lung inflammation, epithelial and endothelial cell damage, and alveolar stress, and bacterial translocation in experimental pneumonia.MethodsThirty-two Wistar rats were randomly assigned to receive intratracheal of Pseudomonas aeruginosa (PA) or saline (SAL) (n = 16/group). After 24-h, animals were anesthetized and ventilated for 2 h with either conventional volume-controlled (VCV) or variable volume-controlled ventilation (VV), with mean VT = 6 mL/kg, PEEP = 5cmH2O, and FiO2 = 0.4. During VV, tidal volume varied randomly with a coefficient of variation of 30% and a Gaussian distribution. Additional animals assigned to receive either PA or SAL (n = 8/group) were not ventilated (NV) to serve as controls.ResultsIn both SAL and PA, VV improved oxygenation and lung elastance compared to VCV. In SAL, VV decreased interleukin (IL)-6 expression compared to VCV (median [interquartile range]: 1.3 [0.3–2.3] vs. 5.3 [3.6–7.0]; p = 0.02) and increased surfactant protein-D expression compared to NV (2.5 [1.9–3.5] vs. 1.2 [0.8–1.2]; p = 0.0005). In PA, compared to VCV, VV reduced perivascular edema (2.5 [2.0–3.75] vs. 6.0 [4.5–6.0]; p < 0.0001), septum neutrophils (2.0 [1.0–4.0] vs. 5.0 [3.3–6.0]; p = 0.0008), necrotizing vasculitis (3.0 [2.0–5.5] vs. 6.0 [6.0–6.0]; p = 0.0003), and ultrastructural lung damage scores (16 [14–17] vs. 24 [14–27], p < 0.0001). Blood colony-forming-unit (CFU) counts were comparable (7 [0–28] vs. 6 [0–26], p = 0.77). Compared to NV, VCV, but not VV, increased expression amphiregulin, IL-6, and cytokine-induced neutrophil chemoattractant (CINC)-1 (2.1 [1.6–2.5] vs. 0.9 [0.7–1.2], p = 0.025; 12.3 [7.9–22.0] vs. 0.8 [0.6–1.9], p = 0.006; and 4.4 [2.9–5.6] vs. 0.9 [0.8–1.4], p = 0.003, respectively). Angiopoietin-2 expression was lower in VV compared to NV animals (0.5 [0.3–0.8] vs. 1.3 [1.0–1.5], p = 0.01).ConclusionIn this rat model of pneumonia, VV improved pulmonary function and reduced lung damage as compared to VCV, without increasing bacterial translocation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0476-7) contains supplementary material, which is available to authorized users.

Potential inflammatory markers in obstructive sleep apnea-hypopnea syndrome.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a complex chronic inflammatory respiratory disease with multiple pathogenic factors and high morbidity and mortality. Serum levels of nuclear factor-κB (NF-κB), hypoxia-inducible factor-1 alpha (HIF-1α), and surfactant protein D (SPD) were investigated in OSAHS patients, to determine their clinical significance and correlation with the pathogenesis. Patients were classified into a mild and moderate OSAHS group (n = 25) and severe OSAHS group (n = 33). Twenty healthy patients served as a control group. Peripheral blood levels of NF-κB, HIF-1α, and SPD were determined by Western blot, and a correlation analysis was performed. Severe OSAHS patients received nasal continuous positive airway pressure (nCPAP) therapy and were followed up after 2 months. NF-κB p65, HIF-1α, and SPD expression levels were determined after valid nCPAP therapy. NF-κB p65 and HIF-1α expression was significantly higher in severe OSAHS group than in the other two groups (p < 0.01), and was positively correlated with the apnea-hypopnea index (AHI) (r = 0.696, p < 0.001; r = 0.634, p < 0.001). SPD expression was significantly lower in severe OSAHS group than in the control group (p < 0.01) and mild and moderate OSAHS group (p < 0.01), and was negatively correlated with AHI (r = -0.569, p < 0.001). OSAHS pathogenesis was associated with changes in NF-κB, HIF-1α, and SPD protein expression levels. nCPAP therapy could improve the clinical characteristics of the patients, lower serum NF-κB and HIF-1α levels, and increase serum SPD levels. We conclude that OSAHS is related to the expression of NF-κB, HIF-1, and SPD.

Innate Immune Molecule Surfactant Protein D Attenuates Sepsis-induced Acute Pancreatic Injury through Modulating Apoptosis and NF-κB-mediated Inflammation.

Sepsis causes multiple-organ dysfunction including pancreatic injury, thus resulting in high mortality. Innate immune molecule surfactant protein D (SP-D) plays a critical role in host defense and regulating inflammation of infectious diseases. In this study we investigated SP-D functions in the acute pancreatic injury (API) with C57BL/6 Wild-type (WT) and SP-D knockout (KO) mice in cecal ligation and puncture (CLP) model. Our results confirm SP-D expression in pancreatic islets and intercalated ducts and are the first to explore the role of pancreatic SP-D in sepsis. CLP decreased pancreatic SP-D levels and caused severe pancreatic injury with higher serum amylase 24 h after CLP. Apoptosis and neutrophil infiltration were increased in the pancreas of septic KO mice (p < 0.05, vs septic WT mice), with lower Bcl-2 and higher caspase-3 levels in septic KO mice (p < 0.05). Molecular analysis revealed increased NF-κB-p65 and phosphorylated IκB-α levels along with higher serum levels of TNF-α and IL-6 in septic KO mice compared to septic WT mice (p < 0.01). Furthermore, in vitro islet cultures stimulated with LPS produced higher TNF-α and IL-6 (p < 0.05) from KO mice compared to WT mice. Collectively, these results demonstrate SP-D plays protective roles by inhibiting apoptosis and modulating NF-κB-mediated inflammation in CLP-induced API.

Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested concentrations. Moreover, blocking FGFR, PDGFR, and VEGFR function did not affect nintedanib-induced SP-D expression, suggesting that nintedanib mediates its effects through a mechanism that is distinct from its known role as a tyrosine kinase inhibitor. Nintedanib is also reported to inhibit Src kinase although pre-treatment of cells with a Src kinase inhibitor had no effect on nintedanib-induced SP-D expression. Increased expression of SFTPD mRNA and SP-D protein in the lungs of nintedanib-treated mice was also observed. In this work, we demonstrated that nintedanib up-regulated SP-D expression in A549 cells via the JNK-AP-1 pathway and did not affect cell proliferation. This is the first report describing SP-D induction by nintedanib.

Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant protein D in human models of airway epithelium.

BackgroundSurfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in host defense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light of infection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-D make it relevant in the development and pathogenesis of asthma.MethodsExpression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC) grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic and non-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, and cytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epithelium and its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmatic and asthmatic donors.ResultsAirway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D as demonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures (fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 days to 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.ConclusionsSP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients. Future functional studies on the role of SP-D in asthma can provide better insight into defects in the structure and regulation of SP-D.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0177-7) contains supplementary material, which is available to authorized users.

N-acetylcysteine counteracts oxidative stress and protects alveolar epithelial cells from lung contusion-induced apoptosis in rats with blunt chest trauma

The aim of this study was to investigate the protective effects of N-acetylcysteine (NAC) on peroxidative and apoptotic changes in the contused lungs of rats following blunt chest trauma. The rats were randomly divided into three groups: control, contusion, and contusion+NAC. All the rats, apart from those in the control group, performed moderate lung contusion. A daily intramuscular NAC injection (150mg/kg) was given immediately following the blunt chest trauma and was continued for two additional days following cessation of the trauma. Samples of lung tissue were taken in order to evaluate the tissue malondialdehyde (MDA) level, histopathology, and epithelial cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and active caspase-3 immunostaining. In addition, we immunohistochemically evaluated the expression of surfactant protein D (SP-D) in the lung tissue. The blunt chest trauma-induced lung contusion resulted in severe histopathological injury, as well as an increase in the MDA level and in the number of cells identified on TUNEL assay together with active caspase-3 positive epithelial cells, but a decrease in the number of SP-D positive alveolar type 2 (AT-2) cells. NAC treatment effectively attenuated histopathologic, peroxidative, and apoptotic changes, as well as reducing alterations in SP-D expression in the lung tissue. These findings indicate that the beneficial effects of NAC administrated following blunt chest trauma is related to the regulation of oxidative stress and apoptosis.

Functional heterogeneity of pulmonary surfactant protein-D in cystic fibrosis

Pulmonary surfactant protein-D (SP-D) is a soluble collagenous C-type lectin with important anti-microbial and anti-inflammatory properties. Although it is subject to functionally relevant modification by common polymorphisms and unregulated inflammation, the functional status of SP-D in cystic fibrosis (CF) remains unclear. Given the importance of infection and inflammation in CF lung pathology we have undertaken the first systematic analysis of SP-D lectin activity in this population. By ELISA, we found that airway lavage fluid SP-D expression was greater in CF compared to control patients but was reduced in CF patients with infection and correlated negatively with markers of neutrophilic inflammation. In a functional assay, the percentage of SP-D capable of binding zymosan rarely exceeded 60% in CF or control patients and similarly restricted binding activity was observed towards maltose–agarose. SP-D lectin activity also correlated negatively with infection and neutrophilic inflammation but there was little evidence of major proteolytic degradation amongst the non-bound material. SP-D which failed to bind zymosan exhibited features of lower oligomeric form compared to bound material when tested by native gel electrophoresis. Furthermore, when separated by gel chromatography, high and low oligomeric populations of SP-D were observed in CF lavage fluid but only high oligomeric forms exhibited substantial lectin activity towards yeast derived mannan. Our data demonstrate that oligomeric heterogeneity underlies functional diversity amongst SP-D in health and disease and that dynamic regulation of oligomerisation is an important feature of SP-D biology.

THU0050 Circulating Lung Surfactant Protein D (SP-D) Differs Between Rheumatoid Arthritis (RA) Patients According to Anti-Citrullinated Protein Antibody (Anti-CCP) and IGM-Rheumafactor (IGM-RF) Status

BackgroundSubclinical lung inflammation has been proposed as a triggering mechanism for the generation of anti-CCP. This concept has recently been supported by imaging studies showing clinically silent parenchymal lung changes...

Differential modulation of surfactant protein D under acute and persistent hypoxia in acute lung injury

Hypoxia contributes to the development of fibrosis with epithelial-mesenchymal transition (EMT) via stimulation of hypoxia-inducible factor 1α (HIF-1α) and de novo twist expression. Although hypoxemia is associated with increasing levels of surfactant protein D (SP-D) in acute lung injury (ALI), the longitudinal effects of hypoxia on SP-D expression in lung tissue injury/fibrosis have not been fully evaluated. Here, the involvement of hypoxia and SP-D modulation was evaluated in a model of bleomycin-induced lung injury. We also investigated the molecular mechanisms by which hypoxia might modulate SP-D expression in alveolar cells, by using a doxycycline (Dox)-dependent HIF-1α expression system. Tissue hypoxia and altered SP-D levels were present in bleomycin-induced fibrotic lesions. Acute hypoxia induced SP-D expression, supported by the finding that Dox-induced expression of HIF-1α increased SP-D expression. In contrast, persistent hypoxia repressed SP-D expression coupled with an EMT phenotype and twist expression. Long-term expression of HIF-1α caused SP-D repression with twist expression. Ectopic twist expression repressed SP-D expression. The longitudinal observation of hypoxia and SP-D levels in ALI in vivo was supported by the finding that HIF-1α expression stabilized by acute hypoxia induced increasing SP-D expression in alveolar cells, whereas persistent hypoxia induced de novo twist expression in these cells, causing repression of SP-D and acquisition of an EMT phenotype. Thus this is the first study to demonstrate the molecular mechanisms, in which SP-D expression under acute and persistent hypoxia in acute lung injury might be differentially modulated by stabilized HIF-1α expression and de novo twist expression.

The presence and activity of SP-D in porcine coronary endothelial cells depend on Akt/PI3K, Erk and nitric oxide and decrease after multiple passaging

Surfactant protein D (SP-D) mediates clearance of microorganisms and modulates inflammation in response to cytotoxic stimulation. It is present in various epithelia, but also in vascular smooth muscle and endothelial cells. Experiments were designed to determine whether or not SP-D is present in porcine coronary arterial endothelial cells and if so, to investigate the molecular mechanisms underlying this presence. The expression of SP-D, NO synthase, Akt 1/2 and Erk 1/2 proteins was determined in cultures at passages 1 (#1) and 4 (#4). SP-D in primary cells existed in three isoforms (37–38kDa and 50kDa). The 37–38kDa SP-D forms were the dominant isoforms in the porcine endothelium and were prominent at #1 but partially lost at #4. Tumor necrosis factor-α (TNF-α) significantly augmented the level of SP-D expression at #1 but not at #4. The basal level of 37–38kDa SP-D isoforms at #1 was reduced by L-NAME, wortmannin and PD 98059. The low basal expression at #4 could be increased by DETA NONOate (donor of NO) or insulin (activator of PI3K/Akt). The presence of nitric oxide synthase was reduced while that of Akt 1/2 and Erk 1/2 was increased at #4. In cells both at passages 1 and 4, TNF-α downregulated NO synthase and up-regulated p-Erk 1/2 protein. The present findings demonstrate the presence of SP-D in endothelial cells which is NO-, PI3K/Akt- and Erk-dependent. They suggest a protective role of SP-D in these cells.