Absence Of Surfactant Protein D Research Articles

SP-D attenuates LPS-induced formation of human neutrophil extracellular traps (NETs), protecting pulmonary surfactant inactivation by NETs

An exacerbated amount of neutrophil extracellular traps (NETs) can cause dysfunction of systems during inflammation. However, host proteins and factors that suppress NET formation (NETosis) are not clearly identified. Here we show that an innate immune collectin, pulmonary surfactant protein-D (SP-D), attenuates lipopolysaccharide (LPS)-mediated NETosis in human neutrophils by binding to LPS. SP-D deficiency in mice (Sftpd−/−) leads to excess NET formation in the lungs during LPS-mediated inflammation. In the absence of SP-D, NETs inhibit the surface-active properties of lung surfactant, essential to prevent the collapse of alveoli, the air breathing structures of the lungs. SP-D reverses NET-mediated inhibition of surfactant and restores the biophysical properties of surfactant. To the best of our knowledge, this study establishes for the first time that (i) SP-D suppresses LPS-mediated NETosis, (ii) NETs inhibit pulmonary surfactant function in the absence of SP-D, and (iii) SP-D can restore NET-mediated inhibition of the surfactant system.

Lung Lining Interaction Determines the Fate of Multi‐Walled Carbon Nanotubes (MWCNTs) in vivo

MWCNTs are coated by lipid when incubated with dipalmitoylphosphatidylcholine (DPPC)—the primary component of lung lining fluid (LLF). This lipid coating allows MWCNTs to integrate into the LLF, and to be phagocytized by lung macrophages. We propose that in vivo, the presence of surfactant protein‐D (SP‐D) is needed for clearance of MWCNTs. MWCNTs, functionalized by carboxylation (C) or acid‐etched (A), (30‐50nm OD, 0.5‐2μm length) were instilled at 1.5μg/g body weight into C57Bl/6 or SP‐D‐/‐ mice. In C57Bl/6, C‐MWCNT induced neutrophilia (120.9 ± 55.9 v 0.2 ± 0.24 x103) and increased cell count (234 ± 57.6 v 100 ± 35.3 x103), 1 day post instillation (not seen with A‐MWCNTs). Lung mechanics were unchanged; but there was a decrease in phospholipid (PL) content and an increase in surfactant protein‐B (SP‐B) in the LLF (A‐ & C‐MWCNTs); consistent with lipid‐coating of MWCNTs and consequent phagocytosis. 3 days post instillation, neutrophilia, PL content and SP‐B levels resolved, but there was an increase in SP‐D; without significant change in lung mechanics (A‐ & C‐MWCNTs). In SP‐D‐/‐ mice, C‐MWCNTs elicited an acute neutrophilic response (63.6 ± 23.4 v 1.4 ± 1.44 x103). SP‐D‐/‐ mice displayed increased PL at baseline, but no reduction in response to MWCNT exposure, and no change to lung mechanics. Neutrophilia had resolved at 3 days in SP‐D‐/‐, but there was impairment of lung mechanics; especially with A‐MWCNTs. Though lipid coating allows for MWCNTs to be phagocytized, in the absence of SP‐D, MWCNTs are not cleared efficiently (as shown by maintenance of PL levels & the presence of MWCNTs in the LLF at 3 days). Failure to clear MWCNTs appears to increase lung collapse by surfactant dysfunction.

Radiation-induced lung injury and inflammation in mice: role of inducible nitric oxide synthase and surfactant protein D.

Reactive nitrogen species (RNS) generated after exposure to radiation have been implicated in lung injury. Surfactant protein D (SP-D) is a pulmonary collectin that suppresses inducible nitric oxide synthase (iNOS)-mediated RNS production. Herein, we analyzed the role of iNOS and SP-D in radiation-induced lung injury. Exposure of wild-type (WT) mice to γ-radiation (8 Gy) caused acute lung injury and inflammation, as measured by increases in bronchoalveolar lavage (BAL) protein and cell content at 24 h. Radiation also caused alterations in SP-D structure at 24 h and 4 weeks post exposure. These responses were blunted in iNOS(-/-) mice. Conversely, loss of iNOS had no effect on radiation-induced expression of phospho-H2A.X or tumor necrosis factor (TNF)-α. Additionally, at 24 h post radiation, cyclooxygenase expression and BAL lipocalin-2 levels were increased in iNOS(-/-) mice, and heme oxygenase (HO)-1(+) and Ym1(+) macrophages were evident. Loss of SP-D resulted in increased numbers of enlarged HO-1(+) macrophages in the lung following radiation, along with upregulation of TNF-α, CCL2, and CXCL2, whereas expression of phospho-H2A.X was diminished. To determine if RNS play a role in the altered sensitivity of SP-D(-/-) mice to radiation, iNOS(-/-)/SP-D(-/-) mice were used. Radiation-induced injury, oxidative stress, and tissue repair were generally similar in iNOS(-/-)/SP-D(-/-) and SP-D(-/-) mice. In contrast, TNF-α, CCL2, and CXCL2 expression was attenuated. These data indicate that although iNOS is involved in radiation-induced injury and altered SP-D structure, in the absence of SP-D, it functions to promote proinflammatory signaling. Thus, multiple inflammatory pathways contribute to the pathogenic response to radiation.

SP-D counteracts GM-CSF-mediated increase of granuloma formation by alveolar macrophages in lysinuric protein intolerance

BackgroundPulmonary alveolar proteinosis (PAP) is a syndrome with multiple etiologies and is often deadly in lysinuric protein intolerance (LPI). At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain. We hypothesized that GM-CSF and surfactant protein D (SP-D) would enhance the clearance of proteins and dying cells that are typically present in the airways of PAP lungs.MethodsCells and cell-free supernatant of therapeutic bronchoalveolar lavage fluid (BALF) of a two-year-old patient with LPI were isolated on multiple occasions. Diagnostic BALF samples from an age-matched patient with bronchitis or adult PAP patients were used as controls. SP-D and total protein content of the supernatants were determined by BCA assays and Western blots, respectively. Cholesterol content was determined by a calorimetic assay or Oil Red O staining of cytospin preparations. The cells and surfactant lipids were also analyzed by transmission electron microscopy. Uptake of Alexa-647 conjugated BSA and DiI-labelled apoptotic Jurkat T-cells by BAL cells were studied separately in the presence or absence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml), ex vivo. Specimens were analyzed by light and fluorescence microscopy.ResultsHere we show that large amounts of cholesterol, and large numbers of cholesterol crystals, dying cells, and lipid-laden foamy alveolar macrophages were present in the airways of the LPI patient. Although SP-D is present, its bioavailability is low in the airways. SP-D was partially degraded and entrapped in the unusual surfactant lipid tubules with circular lattice, in vivo. We also show that supplementing SP-D and GM-CSF increases the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo. Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.ConclusionsWe propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI. To improve the lung condition of LPI patients with PAP, it would be useful to explore alternative therapies for increasing dead cell clearance while decreasing cholesterol content in the airways.

Helicobacter Infection in the Surfactant Protein D-Deficient Mouse

Surfactant protein D (SP-D), a component of innate immunity, is expressed in the gastric mucosa and is up-regulated in the presence of Helicobacter infection. SP-D binds to Helicobacter in vitro, suggesting the involvement of SP-D in Helicobacter-induced immune responses. The aim of this study was to determine the role of SP-D in gastric epithelial defense in vivo. Specific pathogen-free SP-D-deficient mice (SP-D(-/-)) and C57BL/6 wild-type controls were challenged by gavage with different doses of Helicobacter felis, a mouse-adapted Helicobacter strain. Mice were assessed for colonization rates and density of infection. Inflammatory responses were measured by neutrophil counting and T-cell responses by proliferation assays on spleen cells stimulated with H. felis sonicate. The in vitro effect of SP-D on Helicobacter uptake by monocyte-derived dendritic cells was assessed by confocal microscopy and FACS analyses. SP-D(-/-) mice were more susceptible to low-dose infectious challenge than C57BL/6 controls (p = .02). The density of colonization was higher in the SP-D(-/-) infected mice. Neutrophil infiltrates were lower in the SP-D(-/-) mice, particularly in the acid-secreting regions of the stomach. T-cell proliferative responses to Helicobacter antigen were reduced in SP-D(-/-) mice (p = .001) after 12 weeks infection. In vitro uptake of Helicobacter by dendritic cells was significantly enhanced in the presence of SP-D (p = .001). In the absence of SP-D, Helicobacter uptake by dendritic cells is impaired. This provides an explanation for the diminished inflammation and immune responses in the SP-D(-/-) mice.

Surfactant protein D deficiency influences allergic immune responses

The collectin surfactant protein D (SP-D) confers protection against pulmonary infection and inflammation. Recent data suggest a role for SP-D in the modulation of allergic inflammation. The aim of this study is to characterize the immune responses of SP-D-deficient (SP-D(-/-)) mice in a kinetic model of allergic inflammation. We determined whether allergic parameters were enhanced in SP-D(-/-) mice in vivo. Further, we examined whether functional immune responses in vitro such as lymphocyte proliferation (LP) and cytokine production were modulated in the absence of SP-D. In vivo, wild-type (WT) and SP-D(-/-) mice were sensitized and challenged with the allergen ovalbumin (OVA) and assessed for allergic parameters (bronchoalveolar lavage (BAL) eosinophils, IL-13 production, pulmonary IFN-gamma, IL-10 expression) at early time points (1 and 3 days of challenge) in comparison with late time points (7 days of challenge). In vitro, spleen cells from WT and SP-D(-/-) mice were stimulated with the mitogen concanavalin A (ConA) and lipid A (LpA) and analysed for LP, IL-13 and IFN-gamma production. Toll-like receptor 4 (TLR4), ligand for LpA, was assessed by mRNA expression and immunohistochemistry in vivo. Following allergen exposure in vivo, SP-D(-/-) mice expressed higher BAL eosinophils and IL-13 concentrations and lower IFN-gamma expression at early time points compared with WT mice. IL-10 expression was increased at early time points in SP-D(-/-) compared with WT mice. Allergen-induced TLR4 expression was increased in WT, but not in SP-D(-/-) mice. After stimulation with LpA and ConA in vitro LP was increased and IFN-gamma concentration was decreased in SP-D(-/-) mice. SP-D may be critical for the modulation of early stages of allergic inflammation in vivo.

Proteolysis of surfactant protein D by cystic fibrosis relevant proteases.

Surfactant protein D (SP-D) is an important innate host defense molecule that has been shown to interact with cystic fibrosis (CF)-associated pathogens. Previous studies demonstrated that rat SP-D is highly resistant to degradation by a wide range of proteolytic enzymes. The aim of this study was to examine whether human SP-D can be degraded by CF relevant proteases ex vivo and in vitro. Bronchoalveolar lavage fluids (BALFs) of 11 patients with CF in a stable clinical condition were examined for SP-D by immunoblotting. In vitro, purified human SP-D was treated with human leukocyte elastase, proteinase 3, cathepsin G or Pseudomonas elastase followed by immunoblotting with specific antibodies to SP-D. In BALF of 8 of the 11 patients investigated, proteolytic fragments or absence of SP-D were detected. In vitro proteolysis of SP-D was observed in a time-dependent manner for each protease applied. The presence of Ca(++) at a physiologic concentration delayed, but did not prevent the degradation. We conclude that SP-D is an important target of numerous proteases present in the CF lung. Host defense is probably impaired due to proteolysis of SP-D and may contribute to the suppurative lung disease in CF.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.