In situ hybridization as a technique for the immunological investigation of canine intestine: jejunal expression of IFNγ and IL10 in Irish setters and beagles

In situ hybridization (ISH) has found numerous applications in biology and medicine. However, its use to demonstrate expression of cytokines within the canine small intestine has not been described. Digoxigenin-labelled riboprobes complementary to mRNA encoding canine IFNγ and IL10 were used to demonstrate expression of these cytokines within formalin-fixed, paraffin-embedded sections of jejunum obtained from healthy control Irish setter (IS) dogs (n=4), gluten-sensitive IS in remission (n=7), and beagles with high enteric bacterial populations (n=5). Proportional areas of cells within the lamina propria showing one of three mutually exclusive staining intensities were measured, as well as the total stained area. Intensity categories were chosen arbitrarily to represent cells showing weak, moderate or dense staining (grades 1–3 respectively), reflecting increasing expression of mRNA. Control and gluten-sensitive IS showed similar total and grade-by-grade areas of expression of IFNγ and IL10 in the lamina propria (p>0.05), in contrast to beagles, which showed greater total and grade 1 areas of expression of IFNγ, and greater total, grade 1 and grade 2 areas of expression of IL10, than both groups of IS (p<0.05). Epithelial expression of both cytokines was demonstrated in beagles and IS, but differences between groups for each cytokine were not apparent (p>0.05). This study has validated the use of in situ hybridization for the detection of IFNγ and IL10 mRNA within canine intestinal biopsies, and has shown heightened jejunal expression of both cytokines in beagles with high enteric bacterial populations.