Enhanced Antiviral Activity of Human Surfactant Protein D by Site-Specific Engineering of the Carbohydrate Recognition Domain.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as "improved SP-D" (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry.

Surfactant proteins A and D are pattern recognition molecules that play a role in pulmonary host defence. In this paper, we describe for the first time the expression and localisation of both collectins in various porcine tissues using a combination of in situ hybridisation (ISH), RT-PCR and immunohistochemistry (IHC). SP-D was expressed in several tissues including lung, tongue, intestinal tract, thymus, skin, gall bladder and lacrimal gland. Focal SP-D expression was detected in oesophagus, stomach, kidney, liver, prostate and spleen with both histological techniques. These tissues tested negative with RT-PCR. In contrast, SP-A expression was limited to the lung as measured by ISH and IHC. Interestingly, analysis by RT-PCR showed that thymus, trachea, jejunum and duodenum are positive for the presence of SP-A mRNA. We conclude that the combination of different methods can be advantageous if tissue-specific expression is studied. The importance of SP-D in innate immune defence of the pig is underlined by its expression at the potential ports of entry of pathogens.

Expression of beta-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig: lack of upregulation in vivo upon Salmonella typhimurium infection.

Defensins are antimicrobial peptides that play an important role in the innate immune response in the intestine. Up to date, only one beta-defensin (pBD-1), has been described in pig, which was found to be expressed at low levels in the intestine. We set-up a quantitative PCR method to detect the gene expression of pBD-1 and a newly discovered porcine beta-defensin, pBD-2. Expression of pBD-1 mRNA increased from the proximal to the distal part of the intestine whereas pBD-2 expression decreased. The main gene expression sites for pBD-2 were kidney and liver, whereas pBD-1 was mainly expressed in tongue. The porcine small intestinal segment perfusion (SISP) technique was used to investigate effects of Salmonella typhimurium DT104 on intestinal morphology and pBD-1 and pBD-2 mRNA levels in vivo. The early responses were studied 2, 4 and 8 h post-infection in four separate jejunal and ileal segments. Immunohistochemistry showed invasion of the mucosa by Salmonella and changes in intestinal morphology. However, no concomitant changes in expression of either pBD-1 or pBD-2 were observed. We conclude that at least two defensins are differentially expressed in the intestine of pigs, and that expression of both defensins is not altered by S. typhimurium under these conditions.

The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar typhimurium DT104.

Salmonella enterica serovar typhimurium (S. typhimurium) species are a leading cause of human invasive gastroenteritis. There is increasing in vitro evidence about Salmonella interaction with isolated cells or cell lines (macrophages, and enterocytes) on the molecular level, however, very little is known about in vivo interactions during actual invasion. We investigated the early interaction of S. typhimurium with intact small intestinal mucosa, in a pig model. Intestinal segments were infected with or without S. typhimurium DT104, and perfused. Whole mucosal gene expression was analyzed by cDNA array on 0, 2, 4, and 8h post-infection. Invasion resulted in the upregulation of only eight transcripts in jejunal mucosa, among those the proinflammatory IL-8 (at 4h only), and the antiinflammatory STAT3 (at 4 and 8h). The limited number of differentially expressed genes found here in vivo compared to in vitro is most likely due to the presence of multiple, heterogenous cell interactions in intact mucosa. Furthermore, it is concluded that S. typhimurium evades strong host responses by downregulating the local inflammatory response.