Haptoglobin Enhances Cardiac Transplant RejectionNovelty and Significance
Rationale: Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated.
Objective: To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation.
Methods and Results: We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and ≤72 hours after transplantation revealed that 22 proteins including haptoglobin, a known antioxidant, are significantly upregulated in our grafts. Through the use of haptoglobin-deficient mice, we show that 80% of haptoglobin-deficient recipients treated with perioperative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited >100-day survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild-type recipients rejected their transplants by 21 days after transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine interleukin-6 and the chemokine MIP-2 (macrophage inflammatory protein 2) but impair levels of the immunosuppressive cytokine interleukin-10. Haptoglobin also enhances dendritic cell graft recruitment and augments antidonor T-cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection.
Conclusions: Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported antioxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions.
Sterile inflammation occurs in several medical conditions, including after organ transplantation.1–3 In the latter, the harvest and subsequent preservation of an organ induces ischemic injury to the transplant, which is exacerbated at organ implantation by restoration of blood flow, a condition known as ischemia reperfusion injury (IRI).4 IRI, in turn, induces intragraft inflammation, which can have detrimental effects on long-term allograft function.5
In organ transplantation, several putative inflammatory triggers including cellular chaperones, nuclear proteins, and components of the extracellular matrix have been associated with acute allograft rejection.6–8 These putative triggers may be released by the cellular necrosis that accompanies organ implantation and reperfusion. However, it is unclear what role these triggers play in inflammation induction after organ transplantation. Importantly, inhibition of some of these triggers only modestly extends cardiac allograft survival,8 indicating that other yet undiscovered factors may contribute to inflammation after cardiac transplantation.
As graft inflammation has detrimental effects on graft outcomes,9 the identification of novel inflammatory mediators is therefore key for identifying lead candidates for the development of novel therapies to reduce inflammation after organ transplantation. Such lead candidates are particularly relevant to organ transplantation as the harvest and preservation of an organ provides a therapeutic window of opportunity to treat the graft before implantation while avoiding the inhibition of host defense pathways in recipients, an important strategy given that organ transplant recipients are intentionally immune suppressed.
Here, we used an unbiased proteomic screen to identify novel inflammatory modulators in transplanted tissue after cardiac transplantation in a murine model. Our study revealed that by 72 hours after transplantation, 22 proteins are significantly upregulated within the graft including haptoglobin, a heme-binding protein with antioxidative properties. In our murine cardiac transplant model, haptoglobin remarkably prevents the effects of costimulatory blockade therapy to induce indefinite allograft survival. We also found the protein in human cardiac allograft specimens undergoing acute graft rejection. Haptoglobin therefore may be a potential candidate for the development of novel therapeutics to reduce inflammation within cardiac allografts and possibly other organ transplants.
Note that detailed experimental procedures and methods are reported in the Methods section in the Online Data Supplement.
Mice Heart Transplant Model
C57BL/6J (stock# 000664), BALB/cJ (stock# 000651) mice were purchased from Jackson Laboratories (Bar Habor ME). C57BL/6 haptoglobin deficient (Hp−/−), C57BL/6 MyD88−/− (each backcrossed ten times) and relevant wild type (WT) littermate controls 2 to 4 months of age, both males and females, were maintained in our colony. The Hp−/− mice were initially provided by Dr Maffei (Dulbecco Telethon Institute, University Hospital of Pisa, Pisa Italy). The use of vertebrate animals was approved by the Yale School of Medicine IACUC.
A murine heterotopic heart transplant model was used to assess intragraft inflammatory responses10 (detailed in the Online Data Supplement). Anesthesia was induced by isofluorane via a pressurized vaporizer and maintained with ketamine (0.1 mg/g body weight) and xylazine (0.1 mg/g body weight).
In Vitro Culture, Cytokine Measurement, and Flow Cytometry
To determine whether cardiac transplantation led to an increase in inflammatory modulators, we procured cardiac lysates before and after transplantation and cultured them with bone marrow–derived dendritic cells (DCs) as previously reported.11 Cardiac lysates were free of lipopolysaccharide (LPS) (Limulus assay) and did not contain any microbial peptides as assessed by mass spectrometry. Lysates were treated with pronase (0.5 U/mL; Calbiochem-Behring Corp, La Jolla, CA), DNase I (60 U/mL, Roche), or RNase (10 μg/mL, Thermo Scientific) at 37°C for 1 hour and then added to the DC cultures overnight as indicated. Target cytokines and chemokines analytes in the cardiac lysates were measured by ELISA for interleukin (IL) -6, IL-10, monocyte chemotactic protein 1 (MCP-1) (eBioscience), macrophage inflammatory protein 2 (MIP-2) (R&D Systems), hemeoxygenase-1 (Enzo Life Sciences, Farmingdale, NY), and haptoglobin (Immunology Consultants Laboratory, Tampa, FL). We enriched subpopulations of cells within cellular suspension from cardiac tissue with a serial magnetic enrichment protocol (magnetic beads and columns from Milenyi Biotec, Inc, CA) as detailed in the Online Data Supplement. Purified human haptoglobin was obtained from Sigma and found to contain <40 pg/mL of LPS. Mixed lymphocyte cultures consisted of magnetically enriched T cells (Stem Cell Technologies, Vancouver, British Columbia) cultured in a 1:5 ratio with irradiated donor spleen cells. Antidonor T-cell responses were assessed either by measuring cytokine production by ELISPOT (enzyme-linked immuno spot), ELISA or proliferation by thymidine incorporation. Cellular suspensions (ie, spleen cells and cells harvested from cardiac tissue) were stained with relevant fluorescent-tagged antibodies and data acquired on an LSR II (BD Bioscience) flow cytometer and analyzed with FlowJo software (Treestar, Ashland, OR).
To determine the presence of intragraft haptoglobin after human heart transplantation, we stained for haptoglobin in human, archived endomyocardial specimens from the Pathology Department of the Yale New Haven Hospital. The samples had been histologically scored as either no evidence of cellular graft rejection or moderate cellular graft rejection according to the diagnostic criteria of the International Society for Heart and Lung Transplantation.12 These specimens were paraffin embedded at the time of routine protocol biopsy after heart transplantation and were deparaffinized and counterstained with an antihuman haptoglobin antibody (AbCam, Cambridge, MA) or isotype control via immune histochemistry. Slides were read blindly. The use of clinical specimens and accessing patient data from de-identified medical records were approved by the Human Regulatory Board at Yale School of Medicine.
To assess histological inflammation after cardiac transplantation in mice, native and transplanted hearts were harvested and fixed by immersion in 10% neutral buffered formalin, bisected lengthwise, processed, sectioned, and stained for hematoxylin and eosin by routine methods. An antimurine haptoglobin antibody (Proteintech, Chicago, IL) was used to stain for haptoglobin in murine hearts. Murine histological data were read in a blinded fashion.
Statistical analysis is detailed in the Online Data Supplement.
Characterization of Early Intragraft Inflammation
After organ implantation, graft inflammation occurs within hours in mice and humans.13 To establish a model to assess early intragraft inflammation after cardiac transplantation, we subjected murine BALB/c hearts to increasing cold storage and then implanted these organs into C57BL/6 murine recipients. This model differs by a full major histocompatibility complex mismatch between the donor and the recipient.
When we measured inflammatory cytokines and chemokines within the transplant 24 hours after implantation, we found that regardless of the exposure of the organ to cold storage, the inflammatory mediators MCP-1, MIP-2, and IL-6 were increased 2- to 10-fold over those in nontransplanted hearts (Online Figure IA), indicating an early phase of intragraft inflammation after organ implantation.
Characterization of the intragraft levels of cytokines and chemokines demonstrated similar levels between syngeneic and allogeneic transplants at 24 hours after transplantation (Online Figure IB). At this time point the T-cell response to transplantation was not yet evident within the transplant (Online Figure II). Our observations also held true when the hearts were exposed to 4 hours of cold storage before implantation (Online Figure II), indicating that the early inflammatory phase within the graft after cardiac transplantation is antigen independent.
To determine the kinetics of the early, antigen-independent (ie, syngeneic transplants), inflammatory phase for cardiac transplants, we analyzed the levels of inflammatory mediators including IL-6, MCP-1, and MIP-2. These three mediators peaked within the graft between 6 and 48 hours after transplantation and then subsided by 72 hours after transplantation (Online Figure IC). In parallel, the levels of inflammatory mediators within the recipient’s native heart (denoted as native heart), which was exposed to perioperative stressors, but not to the effects of organ procurement and implantation, were comparable with those in the donor heart before implantation (Online Figure IB and IC), indicating that the early antigen-independent phase of graft inflammation is mainly driven by the effects of organ procurement and implantation.
Proteins Are Contributors to Inflammation After Cardiac Transplantation
To characterize intragraft inflammation further after cardiac transplantation, we adapted an in vitro assay in which DCs are cultured with tissue lysates.11 We then assessed the production of the proinflammatory cytokine IL-6 by DCs during the culture to determine the degree of DC activation induced by the lysates. We measured IL-6 in the assay, as there are prior reports that IL-6 contributes to the tempo of cardiac allograft rejection in mice.14–16 The IL-6 response by DCs was increased after the culture with the lysates obtained from cardiac transplants at 24 and 48 hours after transplantation but not with the lysates obtained from nontransplanted hearts or recipient native hearts (Online Figure IIIA). These findings indicate that cardiac transplantation increases the concentration of inflammatory mediators within the graft.
To evaluate the nature of the inflammatory mediators within cardiac grafts, we added RNase, DNase, or pronase separately to lysates from syngeneic cardiac grafts at 24 hours after transplantation to degrade RNA, DNA, and proteins, respectively. Our findings suggest that proteins are the major contributors to the early antigen-independent phase of inflammation after cardiac transplantation as digestion of proteins abrogated the induced IL-6 response of DCs (Online Figure IIIB). In contrast, digestion of RNA or DNA did not reduce the IL-6 response (concentrations of RNase and DNase used in our experiments effectively digested known RNA and DNA-based DC activators, and the concentration of pronase used in the assay did not impair DCs to respond to LPS; Online Figure IIIC and IIID).
Proteomic Screen of Cardiac Tissue Before and After Transplantation
Based on the results from our in vitro DC assay, we identified proteins that are differentially regulated after cardiac transplantation by comparing nontransplanted cardiac tissue with tissue from syngeneic cardiac transplants at various time points after transplantation via mass spectrometry. By 6 hours after transplantation, only 1 protein, NADP transhydrogenase was differentially (down) regulated in cardiac grafts, whereas by 24 to 72 hours after transplantation, several proteins were differentially down- and upregulated within the transplant (Online Figure IVA and IVB). The entire proteomic screen identified a total of 1318 up- and downregulated proteins (Online Table I). Among these proteins, bioinformatic analyses indicated enrichment for biological and molecular processes including immune response, phagocytosis, and cytoskeletal reorganization (Online Tables II and III) and cellular components including mitochondrial components and myofilaments (Online Table IV), reflective of the complex alterations that occur within the graft after cardiac transplantation.
Upregulation of Proteins After Cardiac Transplantation
Our proteomic screen identified 22 proteins that were significantly upregulated in the graft after cardiac transplantation (Online Table V). The only significantly upregulated proteins at both 24 and 72 hours after transplantation were calgranulin A, haptoglobin, chitinase-like 3 protein, and vimentin (Online Table V). We had previously used an unbiased proteomic screen of skin transplants and noted that haptoglobin, a protein with antioxidant and immune modulatory properties,17–19 was upregulated in the graft after skin transplantation.11 Our earlier findings indicated that haptoglobin increases the tempo of minor mismatched skin graft rejection.11 Given this result, we explored the role of haptoglobin in cardiac transplantation.
We confirmed the upregulation of intragraft haptoglobin in the syngeneic heart transplant model via an ELISA test (Figure 1A), and found that haptoglobin was highly induced in the serum 3 hours after cardiac transplantation in mice (Figure 1A). At 24 hours after transplantation, haptoglobin levels were 6-fold higher in the cardiac transplant than in the native heart (Figure 1B), indicating that haptoglobin preferentially enters sites of inflammation.
Haptoglobin Enhances Acute Cardiac Allograft Rejection in Mice
Although intragraft haptoglobin levels were similar between allografts and syngeneic grafts 1 day after transplantation, by 3 days after transplantation, haptoglobin levels were 2-fold higher in allografts than in syngeneic grafts (Figure 1C), although by day 7, after transplantation levels in allografts declined to the levels of syngeneic grafts (Figure 1C). We also observed that haptoglobin levels increased in the liver of murine heart transplant recipients and that recipients of allografts exhibited higher levels of haptoglobin in the liver than recipients of syngeneic grafts (Figure 1D). These results indicate that intragraft haptoglobin is highly induced in both allografts and syngeneic grafts but peaks at a higher level in allografts during the first week after transplantation. Furthermore, concentrations of haptoglobin in the liver, a known site of haptoglobin production, increase after cardiac transplantation.
Given these findings, we examined the effect of haptoglobin on the tempo of acute allograft rejection after cardiac transplantation. For this purpose, we transplanted WT or Hp−/− recipient mice with a WT BALB/c cardiac allograft with or without perioperative CTLA4 Ig. CTLA4 Ig is related to belatacept, a drug that is used currently in clinical organ transplantation and which inhibits costimulation between T cells and antigen-presenting cells, thus increasing transplant survival.20 Without CTLA4 Ig treatment, Hp−/− recipients exhibited a 3-day significant extension of allograft survival compared with WT recipients (Figure 2A). With CTLA4 Ig treatment, this difference was dramatically increased as Hp−/− recipients displayed significantly longer cardiac allograft survival (median survival time >100 days) than WT recipients (median survival <21 days; Figure 2B), indicating that recipient haptoglobin impairs the ability of CTLA4 Ig to induce prolonged allograft survival. Haptoglobin expression in the donor allograft did not affect the tempo of acute allograft rejection in our model (Online Figure V). At 21 days after transplantation, the majority (8/9) of Hp−/− recipients treated with CTLA4 Ig exhibited beating heart grafts and thus allograft survival, whereas all similarly treated WT recipients had rejected their allografts (Online Figure VI and Online Movies I and II). At this time point, Hp−/− recipients treated with CTLA4 Ig also exhibited reduced histological evidence of graft necrosis (Figure 2C). Immune histochemical staining for haptoglobin within the allografts of WT recipients showed that haptoglobin costained with some cardiomyocytes, macrophages and exhibited scant perivascular staining (Figure 2D).
Haptoglobin Alters the Intragraft Inflammatory Milieu After Cardiac Transplantation and Treatment With CTLA4 Ig
We previously demonstrated that haptoglobin activates bone marrow–derived DCs in vitro to induce the production of proinflammatory cytokines, specifically the production of IL-6.11 Therefore, we evaluated how recipient haptoglobin affected the intragraft inflammatory milieu after cardiac transplantation and perioperative treatment with CTLA4 Ig. During the first 3 weeks after transplantation, we noted lower intragraft levels of IL-6 and MIP-2 in Hp−/− than in WT recipients treated with CTLA4 Ig (Figure 3A and 3B). In addition, graft levels of the immunosuppressive cytokine IL-10 were higher in the allografts of Hp−/− recipients treated with CTLA4 Ig than in similarly treated WT recipients (Figure 3C).
This elevation of IL-10 in the grafts of Hp−/− recipients was surprising given that prior work found that haptoglobin–hemoglobin complexes are internalized in macrophages to induce hemeoxygenase-1 and IL-10 production.19,21 We determined that intragraft levels of hemeoxygenase-1 were not altered by haptoglobin after cardiac transplantation and treatment with CTLA4 Ig (Online Figure VII). Furthermore, Hp−/− recipients of WT and thus haptoglobin sufficient allografts treated with CTLA4 Ig exhibited abrogated intragraft levels of haptoglobin (Figure 3D), further supporting the idea that haptoglobin, which impairs the ability of CTLA4 Ig to enhance graft survival (Figure 2B), originates from the recipient. Finally, we found that haptoglobin levels were significantly increased in the livers of WT cardiac transplant recipients treated with CTLA4 Ig but not in nontransplanted recipients (Online Figure VIII), indicating that the liver is a contributing source to haptoglobin after cardiac transplantation.
We further characterized how haptoglobin altered the inflammatory response of specific cells within heart allografts by enriching immune cells, fibroblasts, and endothelial cells (ECs) from allografts at day 21 after transplantation from WT and Hp−/− recipients treated with CTLA4 Ig. The total numbers of these different cell types were obtained from the allografts of WT or Hp−/− recipients and the cells were then cultured ex vivo. As IL-6 and MIP-2 were reduced in the allografts of Hp−/− recipients (Figure 3A and 3B), we measured IL-6 and MIP2 in the supernatants after 12 hours of cell culture. Compared with cells obtained from nontransplanted WT BALB/c hearts, immune cells enriched from BALB/c allografts from WT recipients produced significantly elevated (ie, 5- to 10-fold) levels of both IL-6 and MIP-2 compared with immune cells enriched from Hp−/− recipients (Figure 3E and 3F). We also noted that there were significantly more immune cells (ie, 4-fold higher) in the allografts of WT recipients as compared with Hp−/− recipients at this time point after transplantation (Online Figure IX). These data suggest that infiltrating immune cells are a likely source of proinflammatory cytokines within the allograft and that haptoglobin enhances this response by increasing the recruitment of immune cells into the allografts.
Fibroblasts and ECs from allografts did not produce IL-6 and MIP-2 above levels from cells obtained from nontransplanted hearts (Online Figure X). Immune cells enriched from allografts also produced more IL-10 after culture than cells obtained from nontransplanted hearts (Online Figure XI). Furthermore, immune cells enriched from the allografts of Hp−/− recipients exhibited somewhat lower levels (ie, 50% reduced) of IL-10 than immune cells enriched from the allografts of WT recipients (Online Figure XI). ECs, but not fibroblasts, enriched from allografts had higher levels of IL-10 production than cells obtained from nontransplanted hearts, although we did not observe significant differences between ECs enriched from the allografts of Hp−/− recipients and WT recipients. These results suggest that in addition to immune cells, ECs contribute to the graft levels of IL-10 after cardiac transplantation and treatment with CTLA4 Ig.
Haptoglobin Increases Accumulation and Activation of Intragraft DCs After Cardiac Transplantation and Treatment With CTLA4 Ig
As DCs have been shown to be critical for activating CD4+ T cells to induce acute cardiac allograft rejection,22,23 we next assessed whether there were alterations in the activation phenotype and accumulation of intragraft DCs (defined as CD11c+ major histocompatibility complex class II+ cells) in the cardiac allografts of Hp−/− compared with WT recipients treated with CTLA4 Ig at day +7 after transplantation. We found that the upregulation of the costimulatory molecule CD80 was reduced 3-fold on the surface of intragraft DCs in the Hp−/− recipients as compared with WT recipients treated with CTLA4 Ig (Figure 4A and 4B). There was also a lower number of DCs within the allografts of Hp−/− recipients treated with CTLA4 Ig than in WT recipients treated with CTLA4 Ig (Figure 4C). Similar to what we observed at day +21 after transplantation, we found a 2-fold lower total number of immune cells within the allografts of Hp−/− recipients than in WT counterparts at day +7 after transplantation (Figure 4D). Analysis at day +7 after transplantation also found reduced numbers of neutrophils and T cells in the allografts of Hp−/− recipients as compared with their WT counterparts (Online Figure XII).
MyD88 Expression Within the Allograft Alters the Intragraft Inflammatory Milieu
We previously demonstrated that haptoglobin activates bone marrow–derived DCs to produce IL-6 in vitro via MyD88.11 Furthermore, we and others have shown that MyD88 is critical for IL-6 production by DCs and macrophages in response to microbial stimulation.24 We therefore examined the impact of MyD88 expression within the allograft on the development of graft inflammation. For this purpose, WT or MyD88−/− C57BL/6 hearts were transplanted into WT BALB/c recipients that were treated with CTLA4 Ig. At day 14 after transplantation, the allografts were obtained and graft levels of IL-6, MIP-2, and IL-10 were measured. We found that the graft levels of IL-6 and MIP-2 were significantly reduced 3- to 5-fold in MyD88−/− allografts as compared with WT allografts (Figure 5A and 5B). IL-10 levels were significantly elevated 3-fold in the MyD88−/− allografts as compared with WT allografts (Figure 5C). These results phenocopy the graft inflammatory alterations observed in Hp−/− recipients of WT allografts (Figure 3A–3C).
To determine the cellular targets of haptoglobin within resident heart cells, we isolated immune cells, fibroblasts, and ECs from WT and MyD88−/− nontransplanted hearts and stimulated the cells in vitro with haptoglobin. We found that only immune cells enriched from hearts produced IL-6 in response to haptoglobin and this response was mostly abrogated in immune cells obtained from MyD88−/− hearts (Figure 5D; immune cells, fibroblasts, and ECs produced IL-6 in response to in vitro stimulation with LPS, indicating that these populations of cells enriched from hearts were capable of producing IL-6; Online Figure XIII). Overall, these data show that donor expression of MyD88 enhances allograft inflammation after cardiac transplantation and treatment with CTLA4 Ig and that immune cells are the likely targets within the heart that respond to haptoglobin in a MyD88-dependent fashion.
Haptoglobin Enhances Antidonor T-Cell Responses Without Affecting Intrinsic T-Cell Function
As prior in vitro studies have indicated that haptoglobin may affect T-cell function to nominal antigens or nonspecific stimulation,18,25 we assessed whether haptoglobin alters intrinsic T-cell responses to allostimulation in a mixed lymphocyte culture. We found that purified WT and splenic Hp−/− polyclonal T cells stimulated in vitro with irradiated allogeneic spleen cells exhibited similar production of the Th1 cytokine, interferon-γ, and similar IL-2 levels as WT T cells (Figure 6A and 6B). The levels of proliferation measured in the mixed lymphocyte culture were also similar between WT and Hp−/− T cells (Figure 6C). However, when we assessed antidonor T-cell responses in T cells obtained from the spleens of WT and Hp−/− recipients treated with CTLA4 Ig and transplanted with cardiac allografts, Hp−/− recipients exhibited a 2- to 3-fold reduction in splenic antidonor T-cell interferon-γ and IL-2 responses (Figure 6D and 6E). Recipient haptoglobin had no impact on the numbers of splenic CD4+ FoxP3+ regulatory T cells either before or after transplantation and treatment with CTLA4 Ig (Figure 6F). Thus, haptoglobin seems to amplify intragraft inflammation (Figure 3A and 3B) and enhances antidonor Th1 T-cell alloimmunity to impair the graft-prolonging effects of costimulatory blockade.
Intragraft Expression of Haptoglobin Associates With Acute Allograft Rejection in Humans
To provide an initial determination of the presence of haptoglobin in human heart transplants, we performed a case-controlled study in which 10 endomyocardial biopsies that had no histological evidence of rejection on routine endomyocardial biopsy obtained at various time points after transplantation were compared with 9 biopsies that exhibited moderate histological evidence of rejection. We found that haptoglobin staining was present in 7 of 9 human cardiac transplant specimens that exhibited evidence of moderate allograft rejection but only in 2 of 10 cardiac transplant specimens that were free of acute rejection (P=0.02, Fisher exact test; Figure 7A and 7B). Examination of the 7 biopsies that exhibited moderate rejection and haptoglobin staining suggested that haptoglobin was present within some cardiomyocytes (Online Figure XIV) and within some scattered ECs (Figure 7B). In this small sample size, we did not observe haptoglobin staining with immune cells (the clinical characteristics of patients who had moderate rejection are compared with those of patients with no rejection in Online Table VI). These findings suggest that intragraft haptoglobin is also associated with acute allograft rejection in humans.
Our study identified haptoglobin as a novel enhancer of cardiac graft inflammation that prevents the allograft-prolonging effects of CTLA4 Ig. Although haptoglobin has important physiological functions, such as an antioxidant in the setting of hemolysis,17 our study has found that it also enhances the levels of inflammatory cytokines, such as IL-6, which have been shown to impair cardiac allograft survival.14
In contrast to a skin transplant model,11 our current study found that only recipient, but not donor, expression of haptoglobin accelerates the tempo of acute cardiac allograft rejection (Figure 2; Online Figure V). In previous studies, the skin has been shown to be a source of haptoglobin.26 Unlike the skin, however, which undergoes neovascularization after transplantation, cardiac transplants are immediately vascularized and subjected to a rapid IRI. Our study suggests that the liver is a source of haptoglobin in our cardiac transplantation model. Haptoglobin production from the liver may be induced by the initial inflammation and hypoxia that occurs after graft implantation. In turn, haptoglobin enters the graft within 24 hours after transplantation via the vasculature and amplifies inflammation.
Prior work has indicated that haptoglobin enhances the clearance of Salmonella and increases inflammation in delayed-type hypersensitivity reactions of the skin.18 There is also evidence that haptoglobin affects intrinsic T-cell function in particular Th1 responses, at least to nominal antigens or nonspecific stimulation in vitro.18,25 Furthermore, haptoglobin increases the cellularity of secondary lymphoid organs in mice,18 which could affect immune responses including the tempo of cardiac allograft rejection. Other studies have found that haptoglobin dampens inflammation induced by innate immune activation via lipopolysaccharide.27 In addition, haptoglobin reduces oxidative stress within the myocardium and reduces the rate of late ventricular rupture after acute myocardial infarction induced by coronary ligation in mice.28 Moreover, when haptoglobin binds to hemoglobin, this complex induces the production of the immune suppressive cytokine IL-10 by macrophages.19 Given that innate immune activation is implicated in IRI,29 we expected haptoglobin to reduce inflammation after cardiac transplantation. However, our study found that haptoglobin enhanced graft inflammation and impaired the graft prolonging properties of CTLA4 Ig, although it did not affect intrinsic T-cell function during in vitro stimulation with allogeneic spleen cells (Figure 6A–6C). Together, our current study and prior reports imply that haptoglobin exhibits either anti- or proinflammatory effects, and the dominant phenotype may depend on the disease context or the model used.
Our current study indicates that in the context of organ transplantation haptoglobin enhances the recruitment of immune cells into the allograft including DCs to amplify graft inflammation. We think that the initial IRI after cardiac transplantation induces graft inflammation. This leads to the production of haptoglobin in the recipient, which is released into the circulation and subsequently enters the graft to amplify inflammation, possibly through activating intragraft immune cells, such as DCs. In turn, activated immune cells within the transplant release cytokines and chemokines to enhance immune cell recruitment and promote inflammation. Future studies will be needed to determine how haptoglobin affects other possible cellular sources of pro- and anti-inflammatory molecules within the heart, such as cardiomyoctyes or vascular smooth muscle cells. Regardless of the source of inflammatory mediators, recipient haptoglobin enhances the intragraft inflammatory milieu and increases antidonor T-cell immunity after cardiac transplantation. Our study suggests that the inflammatory alterations mediated by haptoglobin renders T cells less susceptible to the effects of costimulatory blockade (Online Figure XV).
We previously demonstrated that haptoglobin activates bone marrow–derived DCs in vitro via MyD88, a signal adaptor downstream of TLR and IL-1 and 18 receptors, to induce inflammatory responses.11 In the current study, we found that the expression of MyD88 within the allograft enhances graft inflammation (ie, elevating levels of IL-6 and MIP-2 but impairing IL-10 levels) similar to the impact recipient haptoglobin has on graft inflammation. We also provide evidence that immune cells enriched from cardiac tissue respond to haptoglobin in vitro via MyD88. Together, these data imply that recipient-derived haptoglobin is sensed by immune cells in the graft to enhance alloimmunity. Future studies will be required to identify the MyD88 sensing cell within the allograft that is activated by haptoglobin in vivo to enhance intragraft inflammation after cardiac transplantation.
The cellular recognition mechanisms and pathways by which haptoglobin alters the intragraft inflammatory milieu after vascularized organ transplantation will require further investigations to discern detailed underlying molecular pathways. Moreover, haptoglobin was not the only upregulated protein in the graft after cardiac transplantation. Aside from haptoglobin, calgranulin A, chitinase-like 3 protein and vimentin were also significantly upregulated at both 24 and 72 hours after transplantation (Online Table V). Prior work has shown that calgranulin A suppresses DC priming and slows the tempo of major histocompatibility complex class II mismatched cardiac allograft rejection in mice.30 A recent study identified that chitinase like 1 protein, which is similar to the chitinase-like 3 protein, correlates with the degree of graft injury after kidney transplantation,31 although the role of either protein in cardiac transplantation is not known. In addition, there is evidence that vimentin is an important antigen in organ transplantation and antibodies to it are associated with cardiac allograft rejection.32 These other proteins therefore warrant further exploration as they may potentially also contribute to inflammation after heart transplantation.
Aside from the murine data that implicate haptoglobin as an amplifier of inflammation after cardiac transplantation, our study also showed that there is an association between intragraft haptoglobin expression and acute allograft rejection in archived human biopsies that were obtained at various time points after heart transplantation. Our murine data indicate that without immune suppression, haptoglobin is upregulated within the graft during the first week after transplantation (Figure 1C) and is present in cardiac transplants 3 weeks after transplantation in recipients treated with CTLA4 Ig (Figure 3D). Human heart transplant biopsies are not routinely available before 1 week after transplantation because of patient safety issues. We therefore could not determine whether haptoglobin is upregulated within the immediate perioperative period after human heart transplantation. As our small case-controlled study did not determine where haptoglobin localizes within human cardiac transplants, future clinical studies will be required to identify the time course and localization of haptoglobin within human hearts transplants, although it will be challenging to assess whether haptoglobin is induced in the perioperative period.
In summary, we show that haptoglobin is an important amplifier of inflammation after cardiac transplantation. Our results from the murine experimental data combined with our human archived samples suggest that haptoglobin enhances allograft inflammation after cardiac transplantation. The protein and its associated pathways may be key to identifying potential candidates for future anti-inflammatory treatments and therapies.
Sources of Funding
This study was supported by National Institutes of Health grants AI101918, AI098108, and AG028082 and an Established Investigator Award from the American Heart Association to D.R. Goldstein.
In February 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.9 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.305406/-/DC1.
- Nonstandard Abbreviations and Acronyms
- dendritic cells
- endothelial cells
- haptoglobin deficient
- ischemia reperfusion injury
- macrophage inflammatory protein 2
- wild type
- Received October 21, 2014.
- Revision received March 19, 2015.
- Accepted March 23, 2015.
- © 2015 American Heart Association, Inc.
- Shen H,
- Kreisel D,
- Goldstein DR.
- Chen GY,
- G. N.
- Huang Y,
- Yin H,
- Han J,
- Huang B,
- Xu J,
- Zheng F,
- Tan Z,
- Fang M,
- Rui L,
- Chen D,
- Wang S,
- Zheng X,
- Wang CY,
- Gong F.
- Quaye IK.
- Huntoon KM,
- Wang Y,
- Eppolito CA,
- Barbour KW,
- Berger FG,
- Shrikant PA,
- Baumann H.
- Philippidis P,
- Mason JC,
- Evans BJ,
- Nadra I,
- Taylor KM,
- Haskard DO,
- Landis RC.
- Vincenti F,
- Blancho G,
- Durrbach A,
- et al
- Schaer CA,
- Schoedon G,
- Imhof A,
- Kurrer MO,
- Schaer DJ.
- Kreisel D,
- Krasinskas AM,
- Krupnick AS,
- Gelman AE,
- Balsara KR,
- Popma SH,
- Riha M,
- Rosengard AM,
- Turka LA,
- Rosengard BR.
- Shen H,
- Tesar BM,
- Walker WE,
- Goldstein DR.
- Arslan F,
- Smeets MB,
- Buttari B,
- Profumo E,
- Riganò R,
- Akeroyd L,
- Kara E,
- Timmers L,
- Sluijter JP,
- van Middelaar B,
- den Ouden K,
- Pasterkamp G,
- Lim SK,
- de Kleijn DP.
- Shimizu K,
- Libby P,
- Rocha VZ,
- Folco EJ,
- Shubiki R,
- Grabie N,
- Jang S,
- Lichtman AH,
- Shimizu A,
- Hogg N,
- Simon DI,
- Mitchell RN,
- Croce K.
- Schmidt IM,
- Hall IE,
- Kale S,
- Lee S,
- He CH,
- Lee Y,
- Chupp GL,
- Moeckel GW,
- Lee CG,
- Elias JA,
- Parikh CR,
- Cantley LG.
Novelty and Significance
What Is Known?
Cardiac transplantation leads to ischemia reperfusion injury and inflammation after graft implantation.
Graft inflammation impairs the induction of transplantation tolerance and enhances acute and chronic transplant rejection.
What New Information Does This Article Contribute?
Haptoglobin levels increase in the serum and the graft after cardiac transplantation in mice.
Haptoglobin amplifies graft inflammation via activation of the innate immune system to impair indefinite cardiac transplant survival induced by costimulatory blockade.
Haptoglobin expression in human heart transplants associates with acute transplant rejection.
After organ transplantation, inflammation is induced by the innate immune system. However, the mechanisms that induce and maintain graft inflammation remain unclear. We performed a proteomic analysis of murine cardiac transplants and showed that the protein haptoglobin, which is known for its antioxidant properties, is upregulated in these grafts. Recipient, but not donor, expression of haptoglobin impairs the ability of immune modulators to induce indefinite cardiac allograft survival in mice. Furthermore, haptoglobin in cardiac allografts enhances the production of the proinflammatory cytokine interleukin-6, the chemokine MIP-2 but impairs the immune suppressive cytokine, interleukin-10. We also showed that haptoglobin activates resident immune cells in heart tissue via the innate immune adaptor, MyD88. Importantly, the presence of haptoglobin in human cardiac allograft biopsies correlates with acute allograft rejection. Together, these findings indicate a novel and surprising function for haptoglobin in cardiac allografts: activation of innate immunity to impair indefinite allograft survival. Our study reveals that the inflammatory pathway amplified by haptoglobin may be therapeutically targeted to reduced inflammation after organ transplantation and possibly in other sterile inflammatory conditions.