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A recombinant fragment of human SP-D promotes phagocytosis of Haemophilus influenzae strains whose in vivo pathogenicity is inversely related to SP-D binding

Presented at the Neonatal Society 2005 Summer Meeting (programme).

Mackay R-M, Deadman M, Moxon ER, Clark H

MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

Premature infants have been shown to have low levels of innate immune collectins which could leave them susceptible to infectious disease. Native SP-D binds to LPS on the surface of gram negative bacteria and promotes their phagocytosis and killing. The aims of this study were to test the ability of a recombinant fragment to promote phagocytosis of a model gram negative pathogen, Haemophilus influenzae Eagan and by using mutant strains with well characterised surface LPS structures, characterise SPD-LPS interaction in vitro and assess the relevance of this interaction in vivo.

Methods: Wild-type Haemophilus Eagan and mutant Eagan strains with truncated LPS structure were generated by deletion of genes governing LPS biosynthesis in the laboratory of Professor ER Moxon. The binding of human surfactant protein D to purified LPS from wild type (Eagan) and mutant strains (Eagan 4A, Eagan CA7) with truncated LPS structure and variable exposed core oligosaccharides) were compared by ELISA. Binding to the whole wild-type and mutant bacteria was demonstrated by FACS analysis. Wild type Eagan and strain Eagan 4A with the greatest differences in SP-D binding were evaluated for their pathogenicity in wild type (c57BL/6) and SP-D knock-out mice. Clearance of the organism from the lungs was compared in WT and SP-D knock-out mice, 2.5h, 6h and 12 hours after intranasal challenge with 107 organisms in 50μl of PBS by schedule 1 sacrifice and counting colony forming units from plated lung homogenates. Cytokines ( MIP-2 and TNF alpha) were assayed in BAL and lung homogenates by ELISA at the same time points. Differential counts of alveolar macrophage and neutrophils after challenge were assessed on cytospins of BAL cells, stained with DiffQuick.

Results: There was a direct inverse correlation between SP-D binding and the number of branch chain heptoses in the LPS from wild-type and mutants strains. In line with our expectations, wild type Eagan was more pathogenic than the mutant strain with a truncated LPS- Eagan 4A. Eagan 4A was cleared more efficiently from the lungs of wild type mice than the parent wildtype strain Eagan. (n=4-6, p<0.05). Surprisingly it was found that SP-D knock-out mice were more efficient than wild-type mice in clearing both Eagan and Eagan 4AHaemophilus strains from mouse lungs, despite the lack of endogenous SP-D (n=6, p<0.01). However there was a more pronounced inflammatory reaction in SP-D knock–out mice compared to wild type mice even after infection with the non-pathogenic Eagan 4A strain at 2.5h (n=6, p<0.01) and 6h. The recombinant fragment of SP-D promoted phagocytosis of both wild type and mutant Haemophilus strains.

Conclusions: The results confirm that the in vitro interaction between SP-D and LPS on gram negative bacteria correlates with pathogenicity in vivo and that a recombinant fragment of SP-D without the collagenous tail is effective at promoting phagocytosis of this model gram negative organism.

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