Fig 2: Severe apoptosis and pathology in the orally infected heart. Experimental design is as outlined in Fig. 1B. (A) Heart sections from moribund mice were examined with H&E, and immunohistochemistry staining for pan-leukocyte infiltration using an anti-CD45 antibody. (B) The gap junction channel protein connexin 43 at the border of cardiomyocytes were lost in EV71-infected mice. (C) IFA TUNEL assay detected massive host DNA fragmentation. (D,E) Extensive apoptosis in ventricle and atrium was detected by IHC in orally infected (16 dpi), but not in i.p.-infected (8 dpi), moribund mice. See Table 2 for tissue distribution of VP1 in the i.p.-infected group. (F) Oral infection resulted in near 70% apoptotic cardiomyocytes by TUNEL assay (Merck). ***p < 0.0001. (G) Activated caspase 3 was detected by IHC.

Fig 3: VP1-positive infiltrating M2 macrophage and VP1-positive pneumocytes were detectable in the orally infected lung. Orally infected mice were sacrificed on day 16 post-infection (Fig. 1B). Adjacent lung sections were examined with (A) H&E and IHC staining for a pan-leukocyte marker CD45. Massive leukocyte infiltration was detected. (B) VP1 and CD163 (M2 macrophage), but not CD68 (M1 macrophage), were detected by IHC in adjacent lung sections. (C) Similarly, arginase 1 marker for M2 macrophages, but not iNOS marker for M1 macrophage, was visualized in the alveoli by IHC staining. (D) VP1-positive (brown) M2 macrophages (CD163, green) were observed in both panels by IHC double staining. (E,F) VP1-positive signal was detected in both type I (aquaporin 5) and type II pneumocytes (surfactant protein C). (G) Distribution of VP1 in heart, lung and muscle, as well as their respective infiltrating leukocytes.

Fig 4: CNBS patch modulated immune environment in vivo post MI. (a and b) Immunofluorescent staining images of CD45 (green) showing the leukocyte infiltration 4 weeks after transplantation and quantification of CD45-positive cells. (c and d) Representative IHC images of CD68 showing the infiltration of macrophages, and the quantification was conducted accordingly. (e and f) The reduction of pro-inflammatory IL-1ß (red) and elevation of anti-inflammatory CD163 (green) were detected by IHF staining. g) The relative expression of inflammation-related mRNA was measured in the three groups via RT-qPCR. (h) The serum concentrations of inflammatory cytokines IL-8, TNF-a and IL-10 were detected by ELISA. For (a and e), the nucleus was counterstained by DAPI (blue). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 according to one-way ANOVA followed by LSD post-hoc test (b, d, and f-h). All data were shown as mean ± SD. Values of independent experiments were: n = 3 (b, d, and f-h).