Flow cytometry was used to evaluate the frequency and intensity of sub-populations of lymphocytes and macrophages of lamina propria cells from jejune and colon fragments. A quicker isolation, improves the quantity and viability of cells. The use of enzymes alters the expression of surface antigens so that the subsequence FACS analysis may not reflected in detail the real percentage of cells in the suspension. The incubation of tissue fragments (Fig. 1c) with collagenase II is more effective when it stays for 45 minutes. In less than this, the separation of cells is not effective and over this time, there is increased cell death with debris. The collagenase type I is not efficient in obtaining canine gut cells, as it is done in murine protocols. The analysis of the cells should focus on the composition of the subpopulation. As demonstrated in Fig. 2a, cells were readily identified by their high SSC/FSC properties, allowing contamination with epithelial cells and debris that are common in mucosal preparations to be excluded. The phenotypic aspects of the cells were expressed in two different forms: percentage of cells expressing a given phenotypic marker, using cells and isotype control cutoff, with a bimodal distribution, and geometric mean fluorescence intensity, as seen in Fig. 2b.
An analysis of these cells (Figs. 3a-j) gives an opportunity to compare various situations of the lamina propria in different groups. In our case, in control dogs subpopulations showed nearly 15,64% of the CD4+; 1,94% of the CD8+; 9,78% of the CD4+FOXP3+; 23,00% of the CD11c+; 23,49% of the CD11b+; 3,24% of the CD14+; 10,22% of the mannose+; 26,41% of the TLR2+ and 86,19% of the TLR9+ cells. In the colon samples originate of infected dogs, increased numbers of CD4+ to 33,90%; CD4FOXP3+ to 23,61%; CD11c+ to 51,78%; CD11b+ to 43,21%; CD14+ to 28,45%; mannose+ to 12,58% and TLR2+ to 34,50% and decreased numbers of CD8+ to 1,59% and TLR9+ to 20,93% (Figs. 4a,b).
The resulting numbers of lamina propria cells varied from about 1x105 – 1x108 cells mass of 40mg per fragment sample. Less than 40mg of biopsy specimens showed no sufficient cells for the experiment. The isolated cell of infected dogs does not differ from cells isolated from control dogs, on the number and viability of cells. When comparing male versus female, no difference in the amount of collected cells was observed. So far, these findings will support continuing comparative studies involving the infected dogs with L. infantum and health dogs where we will be able to determine the phenotypic properties of cells of all small and large intestine segments.
In this work we propose a method for to obtain lamina propria cells of canine gut and respective analysis using flow cytometer. We have also demonstrated a protocol that can be useful for studies of general cellular markers. For example, the technique utilized herein demonstrates that the presence of parasites in the gut of dogs infected with L. infantum is correlated with the alteration in immunological parameters and in immunoregulation, since these parasites survive and multiply in the micro-environment of the intestinal mucosa and do not appear to damage the host.