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

Timing during translation matters: synonymous mutations in human pathologies influence protein folding and function.

Ribosomes translate mRNAs with non-uniform speed. Translation velocity patterns are a conserved feature of mRNA and have evolved to fine-tune protein folding, expression and function. Synonymous single-nucleotide polymorphisms (sSNPs) that alter programmed translational speed affect expression and function of the encoded protein. Synergistic advances in next-generation sequencing have led to the identification of sSNPs associated with disease penetrance. Here, we draw on studies with disease-related proteins to enhance our understanding of mechanistic contributions of sSNPs to functional alterations of the encoded protein. We emphasize the importance of identification of sSNPs along with disease-causing mutations to understand genotype-phenotype relationships.

Retinal degeneration mutation in Sftpa1tm1Kor/J and Sftpd -/- targeted mice.

Surfactant proteins are important collectin immune molecules with a wide distribution throughout the body, including the ocular system. Mice with gene deletions for the surfactant protein genes Sftpa1 and Sftpd were observed to have visual impairment and thinning of the outer nuclear layers of the retina. We hypothesized that gene deletion of Sftpa1 and Sftpd (Sftpa1tm1Kor/J and Sftpd-/-) results in early retinal degeneration in these mice. Sftpa1tm1Kor/J and Sftpd-/- retinas were evaluated by histopathology and optical coherence tomography (OCT). Retinas from Sftpa1tm1Kor/J and Sftpd -/- mice showed early retinal degeneration with loss of the outer nuclear layer. After screening of mice for known retinal degeneration mutations, the mice were found to carry a previously unrecognized Pde6brd1 genotype which resulted from earlier breeding of the strain with Black Swiss mice during their generation. The mutation was outbred and the genotype of Sftpa1tm1Kor/J and Sftpd-/- was confirmed. Outbreeding of the Pde6brd1 mutation resulted in restoration of normal retinal architecture confirmed by in vivo and in vitro examination. We can therefore conclude that loss of Sftpa1 and Sftpd do not result in retinal degeneration. We have now generated retinal Sftpa1 and Sftpd targeted mice that exhibit normal retinal histology.

Localization of surfactant protein-D in the rheumatoid synovial membrane.

Surfactant protein-D (SP-D) is a collectin, which plays an important role in airway protection and inflammation. The molecule has both pro- and anti-inflammatory capacities depending on its molecular size. Its involvement in joint diseases is largely unknown and the aim of this investigation was to study SP-D occurrence and distribution in the synovial membrane of patients with long-standing rheumatoid arthritis (RA) and osteoarthritis (OA). Six RA patients and six OA patients, who underwent total hip arthroplasty, were included in the study. Synovial tissue biopsies were obtained during surgery and subsequently prepared for immunohistochemistry. In this first, small-scale comparative study on the occurrence of SP-D in the synovial membrane of RA and OA, we report that SP-D was only present in the microvascular endothelium in subsynovial and pannus tissue and that the immunostaining was much stronger than in OA. This distribution pattern suggests that SP-D modulates RA inflammatory activities.

Surfactant protein D in chornic obstructive pulmonary disease (COPD).

In the recent years, a large number of potential biomarkers for Chronic Obstructive Pulmonary Disease (COPD) have been described. One of the important biomarkers is Surfactant Protein D (SPD) since serum SPD levels have been associated with lung function or health status in patients with severe COPD. Several interesting evidences of the protein and gene polymorphisms have been described. The present review highlights the current literature, recent patents and, future prospects of this important collection.

Linking surfactant protein SP-D and IL-13: implications in asthma and allergy.

Surfactant protein D (SP-D) is an innate immune molecule that plays a protective role against lung infection, allergy, asthma and inflammation. In vivo experiments with murine models have shown that SP-D can protect against allergic challenge via a range of mechanisms including inhibition of allergen-IgE interaction, histamine release by sensitised mast cells, downregulation of specific IgE production, suppression of pulmonary and peripheral eosinophilia, inhibition of mechanisms that cause airway remodelling, and induction of apoptosis in sensitised eosinophils. SP-D can also shift helper T cell polarisation following in vivo allergenic challenge, from pathogenic Th2 to a protective Th1 cytokine response. Interestingly, SP-D gene deficient (-/-) mice show an IL-13 over-expressing phenotype. IL-13 has been shown to be involved in the development of asthma. Transgenic mice over-expressing IL-13 in the lung develop several characteristics of asthma such as pulmonary eosinophilia, airway epithelial hyperplasia, mucus cell metaplasia, sub-epithelial fibrosis, charcot-Leyden-Like crystals, airways obstruction, and non-specific airways hyper-responsiveness to cholinergic stimulation. Although both IL-4 and IL-13 are capable of inducing asthma like phenotype, the effector activity of IL-13 appears to be greater than that of IL-4. SP-D -/- mice seem to express considerably higher levels of IL-13, which is consistent with increased sensitivity and exaggerated immune response of the mice to allergenic challenge. Allergenic exposure also induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which prevents further activation of sensitised T cells. This negative feedback loop seems essential in protecting the airways from inflammatory damage after allergen inhalation. Here, we examine this link between IL-13 and SP-D, and its implications in the progression/regulation of asthma and allergy.

MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro.

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype.

SP-A and SP-D in host defense against fungal infections and allergies.

Innate immunity mediated by pattern recognition proteins is relevant in the host defense against fungi. SP-A and SP-D are two such proteins belonging to the class of collagen domain containing C-type lectins, or collectins. They bind to the sugar moieties present on the cell walls of various fungi in a dose dependent manner via their carbohydrate recognition domain (CRD). SP-A and SP-D directly interact with alveolar macrophages, neutrophils, lymphocytes. We review these roles of SP-A and SP-D against various clinically relevant fungal pathogens and fungal allergens. SP-A and SP-D gene deficient mice showed increased susceptibility/ resistance to various fungal infections. Patients of fungal infections and allergies are reported with alterations in the serum or lung lavage levels of SP-A and SP-D. There are studies associating the gene polymorphisms in SP-A and SP-D with alterations in their levels or functions or susceptibility of the host to fungal diseases. In view of the protective role of SP-D in murine models of Aspergillus fumigatus infections and allergies, therapeutic use of SP-D could be explored further.

Surfactant protein-D regulates effector cell function and fibrotic lung remodeling in response to bleomycin injury.

RATIONALE: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known. OBJECTIVES: The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM). METHODS: Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps. MEASUREMENTS AND MAIN RESULTS: BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-ß1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-ß1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-ß1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice. CONCLUSIONS: These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.

Factors impacting corneal epithelial barrier function against Pseudomonas aeruginosa traversal.

PURPOSE: Mechanisms determining epithelial resistance versus susceptibility to microbial traversal in vivo remain poorly understood. Here, a novel murine model was used to explore factors influencing the corneal epithelial barrier to Pseudomonas aeruginosa penetration. METHODS: Murine corneas were blotted with tissue paper before inoculation with green fluorescent protein-expressing P. aeruginosa. The impact of blotting on epithelial integrity was evaluated by susceptibility to fluorescein staining and histology. Using fluorescence imaging, blotted corneas were compared to nonblotted corneas for susceptibility to bacterial binding and epithelial penetration after 5 hours or were monitored for disease development. In some experiments, inoculation was performed ex vivo to exclude tear fluid or corneas were pretreated with EGTA to disrupt Ca(2+)-dependent factors. The role of surfactant protein D (SP-D), which inhibits P. aeruginosa cell invasion in vitro, was examined using knockout mice. RESULTS: Blotting enabled fluorescein penetration through the epithelium into the underlying stroma without obvious disruption to corneal morphology. Although blotting enabled bacterial binding to the otherwise adhesion-resistant epithelial surface, adherent bacteria did not penetrate the surface or initiate pathology. In contrast, bacteria penetrated blotted corneas after EGTA treatment and in SP-D knockouts. Visible disease occurred and progressed only in aged, blotted, and EGTA-treated, SP-D knockout mice. CONCLUSIONS: Neither fluorescein staining nor bacterial adhesion necessarily predict or enable corneal susceptibility to bacterial penetration or disease. Corneal epithelial defenses limiting traversal by adherent bacteria include EGTA-sensitive factors and SP-D. Understanding mechanisms modulating epithelial traversal by microbes could improve our understanding of susceptibility to infection and may indicate new strategies for preventing disease.

Therapeutic effects of recombinant forms of full-length and truncated human surfactant protein D in a murine model of invasive pulmonary aspergillosis.

Aspergillus fumigatus (Afu) is an opportunistic fungal pathogen that can cause fatal invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. Previously, surfactant protein D (SP-D), a surfactant-associated innate immune molecule, has been shown to enhance phagocytosis and killing of Afu conidia by phagocytic cells in vitro. An intranasal treatment of SP-D significantly increased survival in a murine model of IPA. Here we have examined mechanisms via which recombinant forms of full-length (hSP-D) or truncated human SP-D (rhSP-D) offer protection in a murine model of IPA that were immunosuppressed with hydrocortisone and challenged intranasally with Afu conidia prior to the treatment. SP-D or rhSP-D treatment increased the survival rate to 70% and 80%, respectively (100% mortality on day 7 in IPA mice), with concomitant reduction in the growth of fungal hyphae in the lungs, and increased levels of TNF-alpha and IFN-gamma in the lung suspension supernatants, as compared to untreated IPA mice. The level of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lung cell suspension was also raised considerably following treatment with SP-D or rhSP-D. Our results appear to reaffirm the notion that under immunocompromised conditions, human SP-D or its truncated form can offer therapeutic protection against fatal challenge with Afu conidia challenge. Taken together, the SP-D-mediated protective mechanisms include enhanced phagocytosis by recruited macrophages and neutrophils and fungistatic properties, suppression of the levels of pathogenic Th2 cytokines (IL-4 and IL-5), enhanced local production of protective Th1 cytokines, TNF-alpha and IFN-gamma, and that of protective C-C chemokine, MIP-1 alpha.

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice.

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.

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 (37-38 kDa and 50 kDa). The 37-38 kDa SP-D forms were the dominant isoforms in the porcine endothelium and were prominent at #1 but partially lost at #4. Tumor necrosis factor-alpha (TNF-alpha) significantly augmented the level of SP-D expression at #1 but not at #4. The basal level of 37-38 kDa 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 PI(3)K/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-alpha 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-, PI(3)K/Akt- and Erk-dependent. They suggest a protective role of SP-D in these cells.

Roles of serum clara cell protein 16 and surfactant protein-D in the early diagnosis and progression of silicosis.

OBJECTIVE: To study roles of Clara cell protein 16 (CC16) and surfactant protein-D (SP-D) as serum biomarkers in the early diagnosis and the pathogenesis of silicosis. METHODS: Thirty healthy volunteers, 30 silica-exposed workers, and 30 workers with suspected silicosis and phase I silicosis were included. Serum CC16 and SP-D concentrations were determined using enzyme-linked immunosorbent assay. RESULTS: Serum CC16 concentrations decreased in silica-exposed workers when compared with in controls, but serum SP-D levels increased, and this trend was obvious in phase 0 and I groups. Discriminant analysis showed that the accuracies of classifying group membership into control, phase 0, phase 0, and phase I were 86.7%, 46.7%, 66.7%, and 70%, respectively, and the total classification accuracy rate was 67.5%. CONCLUSION: Serum CC16 and SP-D may be useful biomarkers for early diagnosis, and serum SP-D concentration may associate with the pathogenesis of silicosis.

Hydrophobic surfactant proteins and their analogues.

Lung surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). Its major function in the lung alveolus is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film enriched in surfactant lipids, hence preventing cellular collapse during respiration. Surfactant therapy using bovine or porcine lung surfactant extracts, which contain only polar lipids and native SP-B and SP-C, has dramatically improved the therapeutic outcomes of preterm infants with respiratory distress syndrome (RDS). One important goal of surfactant researchers is to replace animal-derived therapies with fully synthetic preparations based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. Here, we review recent research developments with peptide analogues of SP-B and SP-C, designed using either the known primary sequence and three-dimensional (3D) structure of the native proteins or, alternatively, the known 3D structures of closely homologous proteins. Such SP-B and SP-C mimics offer the possibility of studying the mechanisms of action of the respective native proteins, and may allow the design of optimized surfactant formulations for specific pulmonary diseases (e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS)). These synthetic surfactant preparations may also be a cost-saving therapeutic approach, with better quality control than may be obtained with animal-based treatments.

[Immunity tests for respiratory diseases--SP-A, SP-D, KL-6].

We present the measurement methods, physiological means and clinical utilities of surfactant protein-A (SP-A), surfactant protein-D and KL-6 of current immunity tests for pulmonary diseases in Japan. The measurement methods of SP-A and SP-D are Enzyme Immunoassays(EIA), and SP-D cannot be measured automatically. KL-6 can be measured directly by the Sandwich method, developed in 1985 by our co-researcher Professor Kohno. SP-A and SP-D, proteins in pulmonary surfactants (phosphoric lipids), can kill many causative bacteria of respiratory infectious diseases by the activation of natural immune defective functions of macrophages. KL-6 is MUC-1, which responded to sixth antibodies extracted from lung cancer cells by Professor Kohno's monoclonal antibody method. SP-A, SP D and KL-6 are very used in the clinical field as injury markers of the lungs, because they have been proved to increase abnormity in difficult pulmonary fibrotic diseases such as idiopathic interstitial pulmonitis (IIP), etc. In particular, we could prove that KL-6 increased the movement and volume of pulmonary fibroblastic cells and decreased the apoptosis of pulmonary fibroblastic cells. In the near future, we are planning to develop new therapeutic drugs for pulmonary fibrotic diseases, as the target the fibrotic mechanism of the lungs by KL-6.

The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages.

The severe acute respiratory syndrome coronavirus (SARS-CoV) infects host cells with its surface glycosylated spike-protein (S-protein). Here we expressed the SARS-CoV S-protein to investigate its interactions with innate immune mechanisms in the lung. The purified S-protein was detected as a 210 kDa glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required Ca(2+) and was inhibited by maltose. The serum collectin, mannan-binding lectin (MBL), exhibited no detectable binding to the purified S-protein. S-protein binds and activates macrophages but not dendritic cells (DCs). It suggests that SARS-CoV interacts with innate immune mechanisms in the lung through its S-protein and regulates pulmonary inflammation.