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. 2021 Jan;21(1):44-59.
doi: 10.1111/ajt.16149. Epub 2020 Jul 18.

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection

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Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection

Iris K A Jones et al. Am J Transplant. 2021 Jan.

Abstract

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1β maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.

Keywords: animal models; basic (laboratory) research/science; complication: infectious; heart (allograft) function/dysfunction; heart transplantation/cardiology; immune regulation; immunobiology; infection and infectious agents – viral: cytomegalovirus (CMV); infectious disease.

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Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

Figure 1.
Figure 1.
Study Design. Syngeneic donors (Lewis) were either mock infected or infected with RCMV at 1×105 PFU/animal 5 days prior to transplantation. One cohort of syngeneic cardiac grafts were soaked in UW solution containing 10μg/mL anakinra during the 4-hour cold ischemia time. At 1-hour post-transplantation, groups of recipients were treated by subcutaneous injection with either anakinra (100mg/kg) or parthenolide (5mg/kg) or their respective vehicles. Syngeneic cohorts were harvested at POD3. Allogeneic donors (F344) were infected with RCMV at 1×105 PFU/animal 5 days prior to transplantation. Allogeneic cohorts were treated subcutaneously with anakinra at 100mg/kg or vehicle by subcutaneous injection at 1 hour post-transplantation. Blood samples were taken at POD 14, 28, and 42. Animals were sacrificed at two time-points for analyses: POD14 and at the time of chronic rejection.
Figure 2.
Figure 2.
IRI causes myocardial tissue damage and PMN infiltration. Graft or control hearts were harvested at POD3 from isogeneic transplants, fixed in formalin, sectioned, and H&E stained. Tissue sections were then examined and graded for severity of myocardial injury, percent of examined area showing myocardial injury, and PMN infiltrate counts. (a) Representative images of control and I/R injured cardiac tissue at POD3 with or without prior RCMV infection. Control tissue was obtained from a non-transplanted animal. Scale bars represent 100μm. (b) Myocardial injury scores on a scale of 0 (no damage) to 4 (severe damage) as described in Supplemental Table 1, (c) Myocardial injury area determined as percent of examined area showing any degree of myocardial injury, (d) PMN infiltrate as measured by number of PMN cells per field of view at 400x magnification. n=4 for all groups. Error bars represent SEM.
Figure 3.
Figure 3.
RNAseq transcriptomic analysis of cardiac tissue and PBMC following IRI. Heart tissues were homogenized in Trizol. PBMC were isolated over lymphocyte separation media and resuspended in Trizol. RNA was extracted from heart and PBMC samples by Trizol preparation with isopropanol precipitation. One μg of total RNA was used for RNAseq. (a) Native heart from RCMV- transplant recipient at POD3 (cohort 2) compared to a non-transplanted control heart (cohort 1), (b) Graft heart from RCMV- transplant recipient at POD3 (cohort 2) compared to a non-transplanted control heart (cohort 1), (c) FDR-cutoffs and regulated gene counts for heart tissue comparisons. (d) PBMC isolated from whole blood of a RCMV- transplant recipient at POD3 (cohort 2) compared to PBMC isolated from whole blood of a non-transplanted control animal (cohort 1), (e) FDR-cutoffs and regulated gene counts for PBMC heart tissues. Genes showing fold-change greater than 2 (Heart) or 1.5 (PBMC) with an FDR adjusted p-value of 0.05 are shown in green in volcano plots (a, b, d) and were selected for further investigation.
Figure 4.
Figure 4.
IL-1R signaling molecules are up-regulated in graft and PBMC samples following transplantation leading to a pro-inflammatory response. Statistically significant alterations in gene expression from RNAseq analysis of graft tissues are represented by red (up-regulation) and blue (down-regulation). Molecules which did not meet statistical significance, or for which mapping was unavailable in the rat genome are shown in white. FDR-p<0.05. FDR-corrected p-values and Cohort 2 versus Cohort 1 ratios are listed in Table 3.
Figure 5.
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.
Figure 6.
Figure 6.
Anakinra reduces pro-inflammatory cytokines elevated during transplantation. Luminex cytokine profiling of serum and heart tissue homogenates was analyzed following IL-1R antagonist treatment of recipients compared to non-treated controls and non-transplanted controls. Treatment groups are as indicated with (Transplant-/Treatment-) Native heart from a non-transplanted, uninfected animal (n=5); (Transplant+/VA) Graft heart from RCMV- donor with anakinra vehicle injection of recipient animals 1-hour post-transplant (n=4); (Transplant+/AD) Graft heart from RCMV- donor with anakinra treatment of donor organ pre-transplantation (n=4); (Transplant+/AR) Graft heart from RCMV- donor with anakinra treatment of recipient 1-hour post-transplant (n=4). Error bars represent SEM. Statistical significance determined by one-way ANOVA with Dunnett’s correction for multiple comparisons, *P<0.05, ***P<0.001.
Figure 7.
Figure 7.
One dose of anakinra at 1-hour post-transplant significantly improves survival time and decreases viral loads at rejection. Donor animals were infected with RCMV 5 days prior to transplantation. Recipients were then treated with vehicle or with anakinra at 1-hour post-transplantation. (a) Time to rejection was significantly improved by recipient treatment with anakinra following transplantation. Graft heart rejection was monitored by palpation for heart beat. Animals were sacrificed and tissues were harvested at the time of rejection. Vehicle treated animals had a mean survival time of 52 days and Anakinra treated animals had a mean survival time of 65 days. Statistical significance determined by Mann-Whitney test, ***P<0.001 vs. Vehicle-control, n=10. PBS-treated RCMV- historical controls included for references (light green) (b,c) Neointimal index of coronary arteries in graft hearts at chronic rejection was decreased in Anakinra-treated cohorts. Tissue sections were taken from graft hearts at the time of rejection and fixed in formalin. Tissue sections were stained with H&E and Elastin staining and the neointimal index was determined as an average of 6 coronary artery sections per animal (b). Representative images are shown in (c). Statistical significance was determined by Mann-Whitney test, **P<0.01, n=10. PBS-treated RCMV- historical controls included for references (light green). (d,e) Neointimal index of coronary arteries in graft hearts at POD14 was decreased in Anakinra-treated cohorts. Tissue sections were stained and the neointimal index was scored as in (b/c). Neointimal index scores are summarized in (d). Representative images are shown in (e). Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons, *P<0.05, **P<0.01. n=6. (f-h) Anti-RCMV IgG antibody responses were reduced in Anakinra treated animals. Blood was taken from transplant recipients at 2, 4, and 6 weeks post-transplant until graft rejection. Enzyme-linked immunosorbent assays against anti-RCMV IgM (f) and IgG (g) antibodies and anti-F344 cardiac tissue IgG (h) antibodies were performed on serum samples and dilution titers were calculated for each sample. Averages for each cohort at 2, 4, and 6 weeks post-transplant are shown. n=10. Error bars represent SEM. Statistical significance determined by Mann-Whitney test, *P<0.05, **P<0.01, ***P<0.001 vs. Vehicle-control. (i)Viral loads in tissues at rejection showed a significant decrease in the number of animals with detectable viral loads in SMG following Anakinra treatment. Tissues were harvested at time of cardiac graft rejection in RNAlater. Tissues were then homogenized in DNAzol and DNA was extracted. qPCR for RCMV viral DNA polymerase was used to quantitate RCMV genome copies in tissues. Error bars represent SEM. Statistical significance determined by fisher’s exact test, *p=0.0325 (two-tailed). (j) Viral loads in tissues at POD14 did not yet show significant impairment in viral loads in Anakinra-treated cohorts. Tissues were harvested as in (i). n=6.

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