Upregulation of defensins in burn sheep small intestine

Abstract

Objective: The aim of this study was to visualize and localize the sheep antimicrobials, beta-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented.

Methods: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction.

Results: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased.

Conclusion: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine.

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Figure 1

Longitudinal image of villi to demonstrate the localization of the antimicrobial SBD-3 (red) in the epithelial cells of the small intestine. Blue (DAPI) indicates cell nuclei and green (Phallicidin) shows F-actin. Note the green microvillous, brush border on the apical side of the cells (magnification ×200; deconvolution via 5 iterations). Red arrow shows SBD-3 in the apex of the intestinal epithelium, the green arrow points to the lacteal, the blue arrow indicates the microvilli, and the white arrow points along the layer of intestinal epithelial cells. Scale bar = 50 microns. The smaller panels on the left are split channels images to show nuclei (top), F-actin (middle panel), and SBD-3 (bottom panel).

Figure 2

Longitudinal image of villi to demonstrate the localization of the antimicrobial SBD-3 (red) in the epithelial cells of the small intestine. Blue (DAPI) indicates cell nuclei and green (Phallicidin) shows F-actin. Note the green microvillous, brush border on the apical side of the cells (magnification ×200; deconvolution via 5 iterations). Panel A is magnification ×1200, Panel B is magnification ×500, and Panel C is magnification ×500. White arrows indicate SBD-3 in the epithelium and subepithelial capillaries in the panel A and are associated with the lacteal and surrounding capillaries in panel C. Scale bar in panel B = 90 microns.

Figure 3

Comparison of all the targeted peptides with sham samples on the top row, burn samples on the bottom. Magnification ×200. Red = peptide as indicated (orange/yellow = colocalized red and green probes), blue = nuclei, green = F-actin.

Figure 4

Image of a villus to show the smooth muscle actin of the vasculature (blue) located juxtapositioned to the lacteal. This particular image (magnification ×200) shows SBD-1 in red, F-actin is green, and smooth muscle actin is blue. Scale bar = 50 microns. White arrows indicate 1, intraepithelial SBD-1; 2, perilacteal capillary SBD-1; and 3, subepithelial capillary-associated SBD-1.

Figure 5

A low-power acquisition (magnification ×200) and a high-power (magnification ×500) outtake of cross sections of crypts to show the localization of SBD-3 to both the apical and basal aspects of the epithelial cells. Red = SBD-3; green = F-actin; and blue = nuclei. Scale bar = 50 microns in low-power image. White arrows indicate epithelial cells and capillaries in panel A and both the basal and apical aspects of cells in panel B.

Figure 6

Comparison of cross sections of crypts from small intestine of sham and burn animals (magnification ×300). Note the dramatic increases in content of SBD-3 (panels A and C) and LL-37 (panels B and D) in the intestinal epithelial cells in these burn tissues (panels C and D), compared with sham-operated sections (panels A and B). Red = peptide; green = F-actin; and blue = nuclei. Scale bar = 50 microns.

Figure 7

Three pairs of images, showing changes in content in the outer, externa, aspects of small intestine samples. Panel A shows 2 images to compare the dramatic increase of SNP-1 following burn injury, panel B shows the dramatic decrease in SBD-2, and panel C shows the dramatic decrease in LL-37 following burn injury in the region of the Auerbach's plexus. Magnification ×200; red = peptide, green = F-actin, and blue = nuclei.

Figure 8

Generated 3D models of a burn versus sham tissue acquisition of SBD-3 in enterocytes of the villi. Note the perinuclear distribution of the peptide (red) as well as throughout the cytoplasm.