Heart transplantation is an important and potentially life-saving treatment for many patients with end-stage heart disease. Allograft rejection is a significant problem, despite the use and improvements that have been made with immunosuppressants. The majority of heart transplant patients experience rejection, who on average have 3.6 ± 2.7 episodes of rejection that require intervention with invasive procedures and toxic medications.1 Cardiac transplant rejection may occur as acute cellular rejection, antibody-mediated rejection, and allograft vasculopathy. Transplant recipients require the use of lifelong immunosuppressive therapy to inhibit the immune system response to the allograft. These medications are associated with a number of toxic side effects, opportunistic infections, and increased risk of cancer. The 10 year survival rate for transplanted organs is <50%2; and thus, a major goal in developing novel therapies for organ transplantation is to create new approaches for the development of allograft tolerance.
Studies have demonstrated the presence of immune cells with suppressive functions that can promote tolerance to transplanted allografts.3 The best characterized suppressor cells are CD4+, CD25+, FoxP3+ T-regulatory (Treg) cells, which play a significant function in the development and maintenance of allograft tolerance.4–6 T-regulatory cells in an animal model of liver transplant were increased in tolerant grafts and associated with the production of the regulatory cytokines CTLA4, interleukin (IL)-4, and transforming growth factor beta (TGF-beta); depletion of Tregs using an anti-CD25 monoclonal antibody resulted in allograft rejection and increased production of IL-2.7 Importantly, Tregs can promote allograft tolerance while maintaining the immune response to viral infections.8 Thus, mechanisms to promote the production of Tregs in organ transplant patients are expected to have a favorable impact on allograft rejection and survival.
Investigations of the FoxP3 promoter show hyperacetylation of H4 with gene expression,9 and acetylation of the FoxP3 protein by TIP60 is associated with Treg cell function.10 Thus, histone deacetylases and their inhibitors are expected to play a role in the regulation of FoxP3 expression and Treg function. Sirtuin 1 (silent mating type information regulation 2 homolog 1; Sirt1) is a NAD+ histone deacetylase that can deacetylate and render coexpressed FoxP3 relatively inactive and vulnerable to degradation via the ubiquitin proteasome 26S pathway.11 The use of a Sirt1 inhibitor may be a therapeutic option to expand the numbers and functionality of the FoxP3+ T-suppressor cell population and to reduce autoimmunity and theoretically graft rejection.12 The goal of this investigation was to perform a pilot study exploring the potential of Sirt1 as a therapeutic target by evaluating the Sirt1 expression in human cases of acute cellular rejection of cardiac allografts.
Thirteen endomyocardial biopsy specimens with acute cellular rejection (grade 2R or 3R) were selected. Ten cases of grade 0R or 1R were also collected to compare Sirt1 staining. The formalin-fixed, paraffin-embedded tissue blocks are archived specimens for which all testing needed for clinical care has already been performed and results given to the treating physicians. This study was approved by the Committee for the Protection of Human Subjects, which is the Institutional Review Board for the University of Texas Health Science Center at Houston (HSC-MS-13-0748).
Five micron tissue sections of the paraffin-embedded tissues were deparaffinized and underwent antigen retrieval. Ethylenediaminetetraacetic acid was used for antigen retrieval for CD8, and citrate was used for FoxP3, T-cell intracytoplasmic antigen (TIA-1), and Sirt1. The following primary antibodies were used: CD8 (Dako, Carpinteria, CA), FoxP3 (Abcam, Cambridge, MA), Sirt1 (Abcam), and TIA-1 (Abcam). The secondary antibodies used were the Starr Trek Universal HRP Detection System (Concord, CA) for CD8, VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA) for Sirt1, and PowerVision Poly-HRP anti-mouse/rabbit IgG (Leica Biosystems, Buffalo Grove, IL) for FoxP3 and TIA-1. Staining for CD3, CD4, CD20, and CD68 was performed as previously described.13
All images were taken using the Nuance Multispectral Imaging System (CRI, Woburn, MA), which allows enumeration of cellular phenotypes in defined areas of pathology. Images from at least four regions of each case were captured with the ×20 objective. Quantification of cells expressing specific markers was performed with the tissue and cell segmenting functions of inForm software (CRI) according to the manufacturer’s instructions. Specifically, the lymphocytic infiltrate was identified by the tissue segmenting function. The total number of mononuclear cells and the number of cells positive for a given marker within the lymphocytic infiltrate were enumerated with the cell segmenting function.
Data are reported as the median and interquartile range. The percentage of Sirt1 expression in mononuclear cells and the CD8/FoxP3 ratio was determined for each case and reported as the median value.
All 13 cases of acute cellular rejection had histological evidence of acute cellular rejection, including lymphocytic infiltrates, areas of myocyte loss, and edema. Two cases were classified as grade 3R acute cellular rejection and 11 cases were considered grade 2R acute cellular rejection. The cases used for comparison were 5 cases of grade 0R and 5 cases of grade 1R. All cases were negative for antibody-mediated rejection, including negative immunohistochemical staining for C4d.
Representative images of immunohistochemical performed for Sirt1 on cases with acute cellular rejection are shown in Figure 1. Sirt1 is expressed in the majority of mononuclear cells. Sirt1 staining was also noted in the nuclei of cardiomyocytes in cases of acute cellular rejection and those without rejection.
Representative images of staining for CD3, CD4, CD8, FoxP3, and TIA-1 and negative control for the 13 cases of acute cellular rejection are shown in Figure 2. The majority of the lymphocytic infiltrate is positive for CD3 with numerous cells positive for CD4. Abundant CD8+ cells are present in the lymphocyte infiltrate with corresponding positivity for TIA-1. Relatively few FoxP3+ cells are present. Few B-cells identified by CD20 staining were present (not shown). CD68-positive cells were not quantified because of very few positive cells present (not shown).
Cases without the evidence of acute cellular rejection (grade 0R) or mild rejection (grade 1R) were evaluated for Sirt1 expression. The five cases of grade 0R showed Sirt1 staining in the nuclei of the cardiomyocytes; no lymphocytic infiltrate was present in these cases. The number of lymphocytes in the five cases of grade 1R was variable between cases. Sirt1 expression in the lymphocytes of these cases of grade 1R rejection was also variable, ranging from little expression to the majority of lymphocytes.
The mean numbers ± standard deviation obtained by quantitative image analysis of the various markers in the 13 cases of acute cellular rejection are shown in Table 1. Sirt1 expression was noted in median percentage of 73.5 of mononuclear cells (interquartile range, 51.2–100%). Twelve of the 13 cases (92.3%) had an elevated CD8/FoxP3 ratio, coinciding with myocardial injury and favoring acute cellular rejection. The median CD8/FoxP3 was 10.9 (interquartile range, 4.5–16.6). The two cases of grade 3R acute cellular rejection had increased numbers of mononuclear cells compared with the cases of grade 2R, the majority of which were positive for CD3.
All 23 patients were on a maintenance regimen of prednisone, tacrolimus, and mycophenolate mofetil. The acute cellular rejection episode was treated with pulse doses of prednisolone with or without adjustment of the other maintenance medications. Subsequent biopsies were taken 1 week later for all patients. One patient showed a biopsy that was negative for acute cellular rejection, nine patients showed grade 1R, and three patients showed grade 2R.
Naive CD4+ T-helper cells may develop into T-helper (Th) type 1, type 2, type 17, or Treg phenotypes, depending on the antigen stimulus and cytokine environment (Figure 3).14 The shift of Th responses to Th1 and Th17 with a corresponding decrease in Treg function is hypothesized to contribute to transplant rejection.15 Thus, strategies to promote naive CD4+ cell differentiation toward a Treg phenotype are expected to have a favorable impact on cardiac transplant rejection.
Our data show expression of Sirt1 in the majority of the lymphocytic infiltrate in acute cellular rejection. Sirtuin 1 is a class III histone deacetylase that deacetylates a number of proteins including the histones H1, H3, and H4, as well as other cellular proteins.16 A number of biological processes are regulated by Sirt1, including apoptosis, DNA repair, inflammation, and insulin production.17 Importantly, Sirt1 plays a role in the regulation of FoxP3 expression. Kwon et al.18 reported negative regulation of Treg activity by Sirt1 because of deacetylation of three sites on FoxP3; treatment with a Sirt1 inhibitor resulted in enhanced induction and stabilization of FoxP3 expression. Other investigations have also found Sirt1 as a negative regulator of FoxP3, with inhibition resulting in increased FoxP3 expression and functional Treg cells.12,19
Strategies to inhibit Sirt1 may enhance Treg activities and provide a therapeutic approach to treat autoimmune diseases and allograft rejection. Sirtuin 1 deletion in mice resulted in enhanced FoxP3 expression, increased Treg suppressive activity, and longer survival of cardiac allografts with mismatched major histocompatibility complex (MHC); function of effector T-cells was not affected as determined by proliferation, activation, or production of IL-2, IL-4, IL-17, or interferon-gamma.12 Similar results have been achieved using agents that inhibit histone deacetylases. Rhesus monkey splenocytes treated with suberoylanilide hydroxamic (SAHA) resulted in FoxP3 expression and promoted Treg suppressive activities, suggesting a potential role of SAHA in preventing allograft rejection.20 An investigation of a murine MHC-mismatched cardiac transplant model treated with SAHA showed prolonged allograft survival time, with enhanced survival when low-dose SAHA was combined with low-dose tacrolimus.21 A different investigation found that SAHA treatment decreased B-cell proliferation and immunoglobulin production in vitro and reduced immunoglobulin and C4d deposition in the mouse cardiac transplant model, suggesting that SAHA treatment may be effective in preventing antibody-mediated transplant rejection.22 Other therapeutics such as sirolimus are known to expand the numbers and function of FoxP3+ cells while decreasing CD8 effector cell function through inhibition of the mammalian target of rapamycin23; one study found potential synergistic activity between SAHA and sirolimus in enhancement of FoxP3.24 Although additional investigations are needed, the use of histone deacetylase inhibitors show promise for the prevention and management of allograft rejection alone and as part of a combinatorial strategy to improve the functionality of Tregs.
Nuclear staining for Sirt1 was noted in the cardiac myocytes. A number of studies have suggested that Sirt1 may have several beneficial cardiac effects. Moderate overexpression of Sirt1 prevents cardiomyocyte apoptosis and has protective effects from oxidative stress; however, high expression of Sirt1 has a number of detrimental properties, including induction of apoptosis, cardiac hypertrophy, and reduction of cardiac function.25,26 Resveratrol activation of Sirt1 has a number of favorable cardiac effects including improved mitochondrial function, antioxidative properties, and attenuation of the age-associated decline in function.27 Thus, inhibition of Sirt1 as a therapeutic strategy for cardiac allograft rejection necessitates consideration of the potential cardioprotective functions of Sirt1. Suberoylanilide hydroxamic (vorinostat) is used to treat cutaneous T-cell lymphoma. Suberoylanilide hydroxamic was reported to be associated with nonspecific electrocardiogram changes in phase I clinical trials but without cardiac angina, arrhythmias, or other cardiotoxicity.28,29
In summary, we show expression of Sirt1 in the majority of lymphocytes and an elevated CD8/FoxP3 ratio in cases of acute cellular rejection. Our data are limited by the small sample size and retrospective design. Other studies have reported a CD4/CD8 ratio of 1.49 in cases of acute cellular rejection in comparison with our ratio of approximately one30; this may reflect the use of the objective, quantitative imaging used in our investigation or the small sample size. Nonetheless, our study highlights the potential of Sirt1 as a therapeutic target for cardiac allograft rejection and may serve as the basis for improved and less toxic immunosuppressant regimens.
The authors thank Pamela K. Johnston, HT (ASCP), for technical assistance and Ms. Bheravi Patel for graphic support.
1. Deuse T, Haddad F, Pham M, et al.: Twenty-year survivors of heart transplantation at Stanford University. Am J Transplant 2008.8: 17691774.
2. Hong JC, Kahan BD: Immunosuppressive agents in organ transplantation: Past, present, and future. Semin Nephrol 2000.20: 108125.
3. Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC, von Boehmer H: Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 2005.6: 12191227.
4. Steger U, Kingsley CI, Karim M, Bushell AR, Wood KJ: CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection
. Transplantation 2006.82: 12021209.
5. Veronese F, Rotman S, Smith RN, et al.: Pathological and clinical correlates of FOXP3
+ cells in renal allografts during acute rejection
. Am J Transplant 2007.7: 914922.
6. Wang Z, Shi B, Jin H, Xiao L, Chen Y, Qian Y: Low-dose of tacrolimus favors the induction of functional CD4(+)CD25(+)FoxP3
(+) regulatory T cells in solid-organ transplantation. Int Immunopharmacol 2009.9: 564569.
7. Li W, Kuhr CS, Zheng XX, et al.: New insights into mechanisms of spontaneous liver transplant tolerance: The role of Foxp3
-expressing CD25+CD4+ regulatory T cells. Am J Transplant 2008.8: 16391651.
8. Bushell A, Jones E, Gallimore A, Wood K: The generation of CD25+ CD4+ regulatory T cells that prevent allograft rejection
does not compromise immunity to a viral pathogen. J Immunol 2005.174: 32903297.
9. Mantel PY, Ouaked N, Rückert B, et al.: Molecular mechanisms underlying FOXP3
induction in human T cells. J Immunol 2006.176: 35933602.
10. Li B, Samanta A, Song X, et al.: FOXP3
interactions with histone acetyltransferase and class II histone deacetylases are required for repression. Proc Natl Acad Sci U S A 2007.104: 45714576.
11. Lucas JL, Mirshahpanah P, Haas-Stapleton E, Asadullah K, Zollner TM, Numerof RP: Induction of Foxp3
+ regulatory T cells with histone deacetylase inhibitors. Cell Immunol 2009.257: 97104.
12. Beier UH, Wang L, Bhatti TR, et al.: Sirtuin-1 targeting promotes Foxp3
+ T-regulatory cell function and prolongs allograft survival. Mol Cell Biol 2011.31: 10221029.
13. Welsh KJ, Risin SA, Actor JK, Hunter RL: Immunopathology of postprimary tuberculosis: Increased T-regulatory cells and DEC-205-positive foamy macrophages in cavitary lesions. Clin Dev Immunol 2011.2011: 307631.
14. Afzali B, Lombardi G, Lechler RI, Lord GM: The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007.148: 3246.
15. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM: Th17: An effector CD4 T cell lineage with regulatory T cell ties. Immunity 2006.24: 677688.
16. Vaquero A, Scher M, Lee D, Erdjument-Bromage H, Tempst P, Reinberg D: Human SirT1
interacts with histone H1 and promotes formation of facultative heterochromatin. Mol Cell 2004.16: 93105.
17. Hubbard BP, Gomes AP, Dai H, et al.: Evidence for a common mechanism of SIRT1
regulation by allosteric activators. Science 2013.339: 12161219.
18. Kwon HS, Lim HW, Wu J, Schnölzer M, Verdin E, Ott M: Three novel acetylation sites in the Foxp3
transcription factor regulate the suppressive activity of regulatory T cells. J Immunol 2012.188: 27122721.
19. van Loosdregt J, Vercoulen Y, Guichelaar T, et al.: Regulation of Treg functionality by acetylation-mediated Foxp3
protein stabilization. Blood 2010.115: 965974.
20. Johnson J, Pahuja A, Graham M, Hering B, Hancock WW, Bansal-Pakala P: Effects of histone deacetylase inhibitor SAHA on effector and FOXP3
+regulatory T cells in rhesus macaques. Transplant Proc 2008.40: 459461.
21. Zhang X, Han S, Kang Y, et al.: SAHA, an HDAC inhibitor, synergizes with tacrolimus to prevent murine cardiac allograft rejection
. Cell Mol Immunol 2012.9: 390398.
22. Zhang X, Guo M, Kang Y, et al.: SAHA, an HDAC inhibitor, attenuates antibody-mediated allograft rejection
. Transplantation 2013.96: 529537.
23. Sabbatini M, Ruggiero G, Palatucci AT, et al.: Oscillatory mTOR inhibition and Treg increase in kidney transplantation. Clin Exp Immunol 2015.182: 230240.
24. Subbiah V, Brown RE, McGuire MF, et al.: A novel immunomodulatory molecularly targeted strategy for refractory Hodgkin’s lymphoma. Oncotarget 2014.5: 95102.
25. Alcendor RR, Gao S, Zhai P, et al.: Sirt1
regulates aging and resistance to oxidative stress in the heart. Circ Res 2007.100: 15121521.
26. Sundaresan NR, Pillai VB, Gupta MP: Emerging roles of SIRT1
deacetylase in regulating cardiomyocyte survival and hypertrophy. J Mol Cell Cardiol 2011.51: 614618.
27. Turan B, Tuncay E, Vassort G: Resveratrol and diabetic cardiac function: Focus on recent in vitro
and in vivo
studies. J Bioenerg Biomembr 2012.44: 281296.
28. Kelly WK, O’Connor OA, Krug LM, et al.: Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol 2005.23: 39233931.
29. Kelly WK, Richon VM, O’Connor O, et al.: Phase I clinical trial of histone deacetylase inhibitor: Suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 2003.9 (10 pt 1): 35783588.
30. Frank R, Dean SA, Molina MR, Kamoun M, Lal P: Correlations of lymphocyte subset infiltrates with donor-specific antibodies and acute antibody-mediated rejection
in endomyocardial biopsies. Cardiovasc Pathol 2015.24: 168172.