Figure 5.

Anakinra treatment of the recipient immediately post-transplantation improves myocardial injury score and PMN infiltrate into cardiac tissue. (a-d) Myocardial injury score (a), myocardial injury area (b), PMN infiltrate (c), and macrophage infiltrate (d). Error bars represent SEM. Statistical significance determined by one-way ANOVA with Tukey’s multiple comparison, *P<0.05, **P<0.01, ***P<0.001 versus vehicle transplant. Representative images are shown in Supplemental Figure 3. Fields counted for (c) and (d) were viewed at magnification equal to 400x. Groups are labeled (Transplant-/RCMV-/Treatment-) Native heart from a non-transplanted, uninfected animal (n=5); (Transplant+/RCMV-/VA) Graft heart from RCMV- donor with anakinra vehicle injection of recipient animals 1-hour post-transplant (n=4); (Transplant+/RCMV-/VP) Graft heart from RCMV- donor with parthenolide vehicle injection of recipient animals 1-hour post-transplant (n=5); (Transplant+/RCMV-/AD) Graft heart from RCMV- donor with anakinra treatment of donor organ pre-transplantation (n=4); (Transplant+/RCMV-/AR) Graft heart from RCMV- donor with anakinra treatment of recipient 1-hour post-transplant (n=4); (Transplant+/RCMV-/P) Graft heart from RCMV- donor with parthenolide treatment of recipient 1-hour post-transplant (n=5); (Transplant+/RCMV+/VA) Donor infected i.p. with 1×10⁵PFU RCMV 5 days prior to transplant, graft heart with anakinra vehicle injection of recipients 1-hour post-transplant (n=4); (Transplant+/RCMV+/VP) Donor infected i.p. with 1×10⁵ PFU RCMV 5 days prior to transplant, graft heart with parthenolide vehicle injection of recipients 1-hour post-transplant (n=4); (Transplant+/RCMV+/AR) Donor infected i.p. with 1×10⁵PFU RCMV 5 days prior to transplant followed by anakinra treatment of recipient 1-hour post-transplant (n=4); (Transplant+/RCMV+/P) Donor infected i.p. with 1×10⁵PFU RCMV 5 days prior to transplant with parthenolide treatment of recipient 1-hour post-transplant (n=5). (e,f) Native (e) and graft (f) heart tissues were harvested at 3 days post-transplant from RCMV- PBS treated (cohort 2), RCMV- Anakinra treated (cohort 4), RCMV+ PBS treated (cohort 5), and RCMV+ Anakinra treated (cohort 6) recipients and processed for flow cytometry staining. Antibodies were directed against cellular markers for T-cells (CD3+, CD4+/CD8+), B-cells (CD45ra+, CD3-, CD161a low), Neutrophils (CD43+, CD3-, CD161a low, CD45rA-), macrophages (CD68+, CD3-), and NK cells (CD161a high, CD3-). Gating strategy as in Supplemental Figure 4. (e) Native heart tissues from transplant recipients at POD3, cell percentages reported as percent of total live cells. (f) Graft heart tissues from transplant recipients at POD3, cell percentages reported as percent of total live cells. Statistical significance determined by two-way ANOVA with Tukey’s multiple comparison, *P<0.05, **P<0.01, ***P<0.001. n=4. Error bars represent SEM.