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Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S -nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2 , which is reduced 10-fold by SP-D OE-BAL. S -nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20 , Cxcl1 , and Vcam1 , that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a , Nos2 , Tnf , and Ptgs2 , which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Nitrogen mustard (NM) is a vesicant known to cause acute pulmonary injury which progresses to fibrosis. Macrophages contribute to both of these pathologies. Surfactant protein (SP)-D is a pulmonary collectin that suppresses lung macrophage activity. Herein, we analyzed the effects of loss of SP-D on NM-induced macrophage activation and lung toxicity. Wild-type (WT) and SP-D-/- mice were treated intratracheally with PBS or NM (0.08 mg/kg). Bronchoalveolar lavage (BAL) fluid and tissue were collected 14 days later. In WT mice, NM caused an increase in total SP-D levels in BAL; multiple lower molecular weight forms of SP-D were also identified, consistent with lung injury and oxidative stress. Flow cytometric analysis of BAL cells from NM treated WT mice revealed the presence of proinflammatory and anti-inflammatory macrophages. Whereas loss of SP-D had no effect on numbers of these cells, their activation state, as measured by proinflammatory (iNOS, MMP-9), and anti-inflammatory (MR-1, Ym-1) protein expression, was amplified. Loss of SP-D also exacerbated NM-induced oxidative stress and alveolar epithelial injury, as reflected by increases in heme oxygenase-1 expression, and BAL cell and protein content. This was correlated with alterations in pulmonary mechanics. In NM-treated SP-D-/-, but not WT mice, there was evidence of edema, epithelial hypertrophy and hyperplasia, bronchiectasis, and fibrosis, as well as increases in BAL phospholipid content. These data demonstrate that activated lung macrophages play a role in NM-induced lung injury and oxidative stress. Elucidating mechanisms regulating macrophage activity may be important in developing therapeutics to treat mustard-induced lung injury.

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces., (Copyright © 2015 the American Physiological Society.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rationale: Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis., Objective: In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS)., Methods: Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd(-/-)) and iNOS single knockout mice (NOS2(-/-)) as well as wild type (WT) littermates., Measurements and Results: DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd(-/-) demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS., Conclusion: iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.

Rationale: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators., Objectives: To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1., Methods: Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation., Measurements and Main Results: EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1., Conclusions: Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.

Rationale: Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells., Objectives: We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation., Methods: Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis., Measurements and Main Results: The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol-SP-D compared with patients without cross-linked SP-D., Conclusions: We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.

This work was designed to study the role of surfactant protein D in the regulation of NO synthesis by "non-alveolar" microphages. We evaluated whether the effects of surfactant protein D depend on the phenotype of macrophages. In the absence of surfactant protein D, the LPS-induced iNOS response was shown to decrease in macrophages of native and proinflammatory phenotypes by 30%, and in macrophages of the antiinflammatory phenotype (by 63%). Under the influence of lipopolysaccharide in high doses (500 ng/ml), NO(2)*- production by mouse macrophages without surfactant protein D was reduced in native cells (by 25%), but increased in proinflammatory (by 40%) and antiinflammatory phenotypes (by 12% compared to mouse macrophages with surfactant protein D). Our results suggest that surfactant protein D is involved in the immune response in the whole organism, but not only in the lungs. The effect of surfactant protein D depends on the phenotype of macrophages.

Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4(+) T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4(+) cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional "bunch of flowers" arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.

Surfactant protein D (SP-D)-deficient (SP-D-/-) mice exhibit early development of emphysema. Previously we have shown that SP-D deficiency results in increased production and activity of inducible NO synthase (iNOS). In this study, we examined whether treatment with the iNOS inhibitor 1400W could inhibit the inflammatory phenotype. Mice were treated with 1400W systemically for 7 wk from 3 wk of age. Treatment reduced total lung NO synthase activity to 14.7+/-6.1% of saline-treated 10-wk-old SP-D-/- littermates. Long-term administration of 1400W reduced lung inflammation and cellular infiltration; and significantly attenuated the increased levels of matrix metalloproteinases 2 and 9, chemokines (KC, TARC), and cytokines (IFN-gamma) seen in bronchoalveolar lavage (BAL) of SP-D-/- mice. Abrogation of these levels was associated with decreasing BAL chemotactic activity for RAW cells. Two weeks of treatment with 1400W reduced total lung NO synthase (NOS) activity to 12.7+/-6.3% of saline-treated SP-D-/- mice. Short-term iNOS inhibition resulted in attenuation of pulmonary inflammation within SP-D-/- mice as shown by decreases in total BAL cell count (63+/-6% of SP-D-/- control), macrophage size (>25 microm) within the BAL (62+/-10% of SP-D-/- control), and a percentage of BAL macrophages producing oxidants (76+/-9% of SP-D-/- control). These studies showed that s.c. delivery of 1400W can be achieved in vivo and can attenuate the inflammatory processes within SP-D deficiency. Our results represent the first report linking defects in the innate immune system in the lung with alterations in NO homeostasis.

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D-/-) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D-/- mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D-/- mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 +/- 1.4 x 10(5) in SP-D-/- vs. 4.04 +/- 0.25 x 10(5) in SP-D+/+ mice) and neutrophils (1.2 +/- 0.4 x 10(5) vs. 0.03 +/- 0.02 x 10(5) in SP-D-/- and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D-/- mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D-/- mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D-/- phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.

Rationale: Surfactant protein D (SP-D) is a collectin family member with demonstrated immunomodulatory properties in vitro. We hypothesized that SP-D modulates inflammation during noninfectious lung injury in vivo., Objectives: To investigate the association of alveolar SP-D and injury, we studied the responses of transgenic mice expressing varying levels of SP-D to intratracheal bleomycin (ITB)., Methods: Eight-week old C57/BL6 SP-D-deficient (-/-) mice and syngeneic wild-type (WT) controls or Swiss Black SP-D-overexpressing (SP-D OE) mice and littermate controls received either ITB or saline and were followed for up to 21 d., Measurements and Results: Kaplan-Meier analysis demonstrated a dose-dependent decrease in survival in ITB SP-D (-/-) mice receiving 2 U/kg bleomycin, with a 14-d mortality of 100% versus 0% mortality for WT receiving 2 U/kg ITB or SP-D (-/-) mice given saline (p < 0.05). At 8 d, ITB SP-D (-/-) mice had greater respiratory distress (frequency/tidal volume) and weight loss than ITB WT mice. Furthermore, bronchoalveolar lavage cellularity, pulmonary parenchymal inflammation, and tissue 3-nitrotyrosine (NO2 Y) were increased to a greater extent in ITB SP-D (-/-) mice. By 21 d, compared with all groups, ITB SP-D (-/-) survivors had increased Trichrome staining and tissue hydroxyproline levels. As proof of principle, SP-D OE mice were highly resistant to bleomycin-induced morbidity and mortality at doses up to 3 U/kg., Conclusions: These data provide new in vivo evidence for an antiinflammatory role for SP-D in response to noninfectious, subacute lung injury via modulation of oxidative-nitrative stress.

Surfactant protein-D (SP-D), a member of the "collectin" family, has been shown to play a role in innate immunity through modulation of inflammation and clearance of organisms. The role of SP-D in host defense against Pneumocystis carinii pneumonia was assessed using SP-D knockout (KO) mice. When inoculated with P. carinii, both wild-type (wt) and SP-D KO mice required CD4 cell depletion to develop infection. In CD4 cell-depleted models, 2 weeks after infection with P. carinii, SP-D KO mice developed increased intensity of infection, compared with wt mice, despite higher lung-inflammation scores and increased amounts of alveolar inflammatory cells. The increased inflammation seen in SP-D KO mice was accompanied by increases in lung weight, expression of inducible nitric oxide (NO) synthase, total NO levels, and 3-nitrotyrosine levels in lung tissue. These results indicate that SP-D plays a role in host defense against P. carinii in vivo by modulating clearance of organisms, lung inflammation, and metabolism of NO.

Surfactant protein (SP)-D, a 43-kD multifunctional collagen-like lectin, is synthesized and secreted by the airway epithelium. SP-D knockout (SP-D [-/-]) mice exhibit an increase in the number and size of airway macrophages, peribronchiolar inflammation, increases in metalloproteinase activity, and development of emphysema. Nitric oxide (NO) is involved in a variety of signaling processes, and because altered NO metabolism has been observed in inflammation, we hypothesized that alterations in its metabolism would underlie the proinflammatory state observed in SP-D deficiency. Examination of the bronchial alveolar lavage (BAL) from SP-D (-/-) mice reveals a significant increase in protein and phospholipid content and total cell count. NO production and inducible NO synthase expression were increased in the BAL; however, there was a decline in S-nitrosothiol (SNO) content in the BAL and a loss of SNO immunoreactivity within the tissue. This decline in SNO was accompanied by an increase in nitrotyrosine staining. We conclude that inflammation that occurs in SP-D deficiency results in an increase in NO production and a shift in the chemistry and targets of NO. We speculate that the proinflammatory response due to SP-D deficiency results, in part, from a disruption of NO-mediated signaling within the innate immune system.