Surfactant protein-A (SP-A) and Toll-like receptor-4 (TLR4) proteins are recognized as pathogen-recognition receptors. An exaggerated activation of TLR4 induces inflammatory response, whereas SP-A protein down-regulates inflammation. We hypothesized that SP-A-TLR4 interaction may lead to inhibition of inflammation. In this study, we investigated interaction between native baboon lung SP-A and baboon and human TLR4-MD2 proteins by coimmunoprecipitation/immunoblotting and microwell-based methods. The interaction between SP-A and TLR4-MD2 proteins was then analyzed using a bioinformatics approach. In the in silico model of SP-A-TLR4 -MD2 complex, we identified potential binding regions and amino acids at the interface of SP-A-TLR4. Using this information, we synthesized a library of human SP-A-derived peptides that contained interacting amino acids. Next, we tested whether the TLR4-interacting SP-A peptides would suppress inflammatory cytokines. The peptides were screened for any changes in the tumor necrosis factor-␣ (TNF-␣) response against lipopolysaccharide (LPS) stimuli in the mouse JAWS II dendritic cell line. Different approaches used in this study suggested binding between SP-A and TLR4-MD2 proteins. In cells pretreated with peptides, three of seven peptides increased TNF-␣ production against LPS. However, two of these peptides (SPA4: GDFRYSDGTPVNYTNWYRGE and SPA5: YVGLTEGPSPGDFRYSDFTP) decreased the TNF-␣ production in LPS-challenged JAWS II dendritic cells; SPA4 peptide showed more pronounced inhibitory effect than SPA5 peptide. In conclusion, we identify a human SP-A-derived peptide (SPA4 peptide) that interacts with TLR4-MD2 protein and inhibits the LPS-stimulated release of TNF-␣ in JAWS II dendritic cells.

Surfactant proteins A and D (SP-A and SP-D) are believed to participate in the pulmonary host defense and the response to lung injury. In order to understand the effects of prematurity and lung injury on these proteins, we measured the amounts of SP-A and SP-D and their mRNAs in three groups of animals: (1) nonventilated premature baboon fetuses; (2) neonatal baboons delivered prematurely at 140 d gestation age (ga) and ventilated with PRN O(2); (3) animals of the same age ventilated with 100% O(2) to induce chronic lung injury. In nonventilated fetuses, tissue and lavage SP-A were barely detectable in baboons of 125 and 140 d ga, but they equaled or exceeded adult SP-A concentrations (g/g lung dry wt) at 175 d (term gestation, 185 d). In contrast, SP-D was readily detectable in tissue and lavage at 125 and 140 d ga. When the baboons of 140 d ga were ventilated for 10 d with 100% oxygen to produce chronic lung injury, the tissue concentration of SP-A was five times greater than that of normal adults; SP-D 16-times greater. Despite the sizable tissue pools of SP-A and SP-D, however, lavage SP-A was only 7% of that of normal adults and lavage SP-D just equaled the amount in normal adults. Nevertheless, because SP-D is normally in much lower concentration than is SP-A, their total comprised less than 12% of the SP-A and SP-D found in the lavage of a healthy adult. The results indicate that in chronic lung injury, SP-A is significantly reduced in the alveolar space. SP-D concentration in lavage is about equal to that in normal adults, possibly because of the 16-fold excess in tissue, but the total collectin pool in lavage is still significantly reduced. Because these collectins may bind and opsonize bacteria and viruses, decrements in their amounts may present additional risk to those premature infants who require prolonged periods of ventilatory support.

We are developing liposome-encapsulated hemoglobin (LEH) as an artificial oxygen carrier for resuscitation in indications, such as acute blood loss and surgery. Earlier attempts to formulate a viable LEH met with constraints of scale up and limited hemoglobin content. In this work, we report an LEH formulation containing novel anionic non-phospholipid (CHHDA) that enhances the encapsulation efficiency of hemoglobin inside the liposome bilayer. CHHDA was synthesized from inexpensive ingredients in high yields. The formulation was evaluated in vitro to investigate the cytotoxic effects on RAW 264.7 macrophages and HUVEC endothelial cells in culture by LDH, MTT and hexosaminidase assays. Under optimal conditions of manufacturing, the presence of 28 mole % of CHHDA enhanced the hemoglobin content to over 4 g/dL. The LEH containing CHHDA shows some cytotoxicity in HUVEC and RAW cells in vitro, especially by LDH assay. MTT assay was negative for cytotoxicity in both cells lines. By hexosaminidase assay, the proliferation of RAW cells, but not HUVEC cells, was inhibited. When CHHDA-LEH was incubated with isolated human platelets in vitro, no platelet activation was observed. The LEH formulation with novel anionic lipid and high hemoglobin content reported in this article is an improvement from the past preparations.

The interaction between surfactant protein-A (SP-A) and TLR4 is important for host defense. We have recently identified an SPA4 peptide region from the interface of SP-A-TLR4 complex. Here, we studied the involvement of the SPA4 peptide region in SP-A-TLR4 interaction using a two-hybrid system, and biological effects of SPA4 peptide in cell systems and a mouse model. HEK293 cells were transfected with plasmid DNAs encoding SP-A or a SP-A-mutant lacking SPA4 peptide region and TLR4. Luciferase activity was measured as the end-point of SP-A-TLR4 interaction. NF-κB activity was also assessed simultaneously. Next, the dendritic cells or mice were challenged with Escherichia coli-derived LPS and treated with SPA4 peptide. Endotoxic shock-like symptoms and inflammatory parameters (TNF-α, NF-κB, leukocyte influx) were assessed. Our results reveal that the SPA4 peptide region contributes to the SP-A-TLR4 interaction and inhibits the LPS-induced NF-κB activity and TNF-α. We also observed that the SPA4 peptide inhibits LPS-induced expression of TNF-α, nuclear localization of NF-κB-p65 and cell influx, and alleviates the endotoxic shock-like symptoms in a mouse model. Our results suggest that the anti-inflammatory activity of the SPA4 peptide through its binding to TLR4 can be of therapeutic benefit.

No abstract