Fig. 5.

Paneth cell apoptosis and exfoliation shown by anti-lysozyme Ab IHC and development of ileal pathology in the post-weaning DKO mice. Panel A shows the normal appearance of Paneth cells stained by anti-Lyz1 IHC in a non-DKO (GPx1^-/-GPx2^+/-) mouse (DAB substrate; hematoxylin counterstained). Panel B shows a putative apoptotic Paneth cell judging by shriveled Lys1+ appearance in the DKO crypt (pointed by a white arrow). Apoptotic Paneth cells, in situ, are also demonstrated in Supplementary Fig. 3 as double-stained Paneth cells with sequential TUNEL IHC (dark grey to black color stain) and anti-Lyz1 Ab IHC (red/brown stain). Panel C shows Lyz+ exfoliated cells (green arrows) in DKO crypt. Panel D shows that Paneth cell number begins to decline at 26 days and nearly depleted by 32-day-old. From 27 days on, the Paneth cell number is significantly lower in DKO compared to the control mice (*, one-way ANOVA test). The progression of the apoptosis pathology (Panel E), crypt exfoliation (Panel F), counts of MPO+ cells in the ileal submucosa (Panel G), appearance of ileum crypt abscesses (Panel H) and increase of Nox1 mRNA levels (Panel I) were analyzed from the ileum of 24- to 35-day-old DKO mice. Two to four mice were analyzed for each day of DKO mice and 35-day-old non-DKO controls. All panels are shown as mean±SD. Panel D, E, F and H were analyzed by histology. The numbers of non-DKO and DKO mice analyzed were 7, 8 of 24-day-old; 2, 7 of 25-day-old; 4, 12 of 26-day-old; 6 each of 27-day-old; 8 each of 28-day-old; 5, 7 of 29-day-old; 5 each of 30-day-old; 3, 5 of 31-day-old; and 14, 13 of 35-day-old, respectively. MPO IHC was done on 4 samples each and Nox1 qPCR on 2 to 4 samples at each time point. For Panel D the comparison is between the non-DKO and DKO at each age using t-test. In Panel E–H analysis is done by one-way ANOVA test using 24- to 35-day-old sets as reference sequentially against the remaining sets using Dunnett's multiple-correction test. Panel I was analyzed by pair-wise t-test.