Cardiac transplantation is the last resort for patients with end-stage heart failure. Ischemia-reperfusion (IR) injury is a major issue in cardiac transplantation. IR injury is associated with increased primary organ dysfunction and subsequent delayed organ function after cardiac transplantation. In the long term, this correlates with increased episodes of acute and chronic rejections (1).
Interleukin (IL)-17A is a member of the IL-17 family, which includes six structurally related isoforms: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (2). Th17 cells are recognized as the primary source of IL-17A (3). However, additional innate immune cell populations have been shown to secrete IL-17A, including γδT cells, natural killer (NK) cells, NKT cells, and neutrophils (4). IL-17A is a critical mediator of neutrophil recruitment and migration through induction of granulopoiesis and the production of neutrophil chemokines, including lipopolysaccharide-induced CXC chemokine (LIX), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein-2 (MIP-2) (5). Previous articles have indicated that IL-17A contribute to brain, kidney, intestine, and cardiac IR injury, but the mechanisms involved are still largely unknown (6–9).
High-mobility group box 1 (Hmgb1), a nonchromosomal nuclear protein, has been implicated in several disease states, including sepsis, arthritis, IR injury, and cancer (10–12). Toll-like receptor 4 (TLR4), one of the pattern recognition receptors, plays a crucial role in the induction of the inflammatory response, and its activation is linked to the activation of downstream signaling in several cell types (13–15). However, the cellular and molecular immune mechanisms of Hmgb1 in cardiac IR injury remain elusive. How the specific innate immune receptor for Hmgb1 activates macrophages and induces proinflammatory cytokines and how these cytokines prime the subsequent innate immune response and mediate inflammation are completely unclear.
In this study, we demonstrated that Hmgb1 stimulates IL-23 production by macrophages in a TLR4-dependent manner, and IL-23 aids in the generation of IL-17A–producing γδT cells in the heart. IL-17A secreted by γδT cells then induces neutrophil recruitment. Thus, the Hmgb1-TLR4-IL-23-IL-17A axis contributes to the accumulation of neutrophils and IR injury in cardiac transplantation.
RESULTS
IL-17A Increases After Myocardial IR Injury
We first investigated IL-17A levels in the myocardium at different reperfusion time points after IR injury. Both IL-17A mRNA and protein levels were significantly increased on day 1 after reperfusion and then began to decrease, although it remain at a high level compared with the sham group until day 7 after IR injury (Fig. 1A, B).
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FIGURE 1: Levels of IL-17A in cardiac isografts increased after myocardial IR injury. Levels of IL-17A were measured by real-time PCR (A) and Western blotting (B) in cardiac isografts from sham and cardium IR injury group for different times (n=6). *P<0.05 versus sham group. C, infiltrated IL-17A+ leukocytes in cardiac isografts day 1 after transplantation were analyzed by flow cytometry. CD45+ cells were isolated and restimulated. IL-17A+CD45+ cells were further analyzed for γδTCR, CD4, CD8, NK1.1, and Gr-1 expression to detect the cellular source of IL-17A. The proportion of different IL-17A–secreting cells in the IL-17A+ CD45+ cells was quantitative analysis (n=6). *P<0.05.
To identify the cellular source of IL-17A production in cardiac isografts, we prepared graft-infiltrating lymphocytes from cardiac isografts. IL-17A production by distinct subsets of lymphocytes was analyzed by intracellular cytokine staining combined with staining for various surface markers. We found that IL-17A was predominantly produced by γδT cells rather than CD4+ or CD8+ T cells infiltrated into the cardiac isografts (Fig. 1C).
IL-17A Neutralization Ameliorates Myocardial IR Injury
To evaluate the functional status of hearts exposed to ischemia, hearts were excised after 24 hr of reperfusion and mounted on a Langendorff apparatus to assess the pressure–volume ratio. For comparison, pressure–volume ratio was also performed in healthy hearts. The results showed that ischemic hearts were functionally impaired compared with healthy hearts, and either neutralization of IL-17A or γδTCR resulted in a significant improvement of hemodynamic performance compared with control hearts. Cardiac output at 80 mm Hg of after-load pressure was significantly increased in hearts with IL-17A or γδTCR neutralization using anti–IL-17A monoclonal antibody (mAb) or anti-γδTCR mAb (anti–IL-17A: 53.2±7.9 mL/min, anti-γδTCR: 48.4±4.4 mL/min, and control: 12.9±1.5 mL/min; P<0.005) (Fig. 2A). Then, we detected cardiac troponin T (TnT) production and found that, on day 1 after transplantation, the production of TnT was reduced by neutralization of IL-17A or γδTCR in comparison with controls (anti–IL-17A: 1.84±0.26 ng/mL, anti-γδTCR: 1.38±0.15 ng/mL, and control: 6.90±0.51; P<0.05) (Fig. 2B).
FIGURE 2: Anti–IL-17A mAb or anti-γδTCR mAb treatment increased hemodynamic performance of cardiac isografts and decreased serum TnT after transplantation. A, hearts were evaluated for hemodynamic function in a pressure–volume relationship (Frank-Starling) day 1 after transplantation. B, serum TnT was measured in sham, control IgG, anti–IL-17A mAb, and anti-γδTCR mAb-treated recipients day 1 after transplantation (n=6). *P<0.05 versus sham or control group.
IL-17A Neutralization Reduces Cardiomyocyte Apoptosis and Neutrophil Recruitment
Apoptosis contributes significantly to myocardial IR injury (16). We carried out terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) of cardiac isografts from different experiment groups at day 1 after transplantation. We found that anti–IL-17A mAb or anti-γδTCR mAb treatment remarkably decreased the number of TUNEL-positive cardiomyocytes compared with control group (Fig. 3A,B). Caspase-3 activity determined by a caspase colorimetric assay from cardiac isografts was concomitantly down-regulated by anti–IL-17A mAb or anti-γδTCR mAb (Fig. 3C).
FIGURE 3: IL-17A–mediated cardiomyocyte apoptosis and cardiac neutrophil recruitment. A, representative photographs of TUNEL-stained cardiac isograft sections day 1 after transplantation. Total nuclei were identified by DAPI staining (blue) and apoptotic nuclei were identified by TUNEL staining (red). Arrows indicate apoptotic cardiomyocytes. B, percentages of TUNEL-positive nuclei over total number of nuclei (n=6). C, caspase-3 activity in cardiac isograft was assessed day 1 after transplantation, and values were normalized to sham (n=6). D, cardiac MPO activity in tissue samples (n=6). E, Number of CD11b+Gr-1+ neutrophils infiltrated in myocardium was analyzed by flow cytometry (n=6). F, LIX, KC, and MIP-2 mRNA levels were analyzed by real-time PCR (n=6). *P<0.05 versus sham or control group. DAPI, 4′,6-diamidino-2-phenylindole; KC, cytokine-induced neutrophil chemoattractant; LIX, lipopolysaccharide-induced CXC chemokine; MIP-2, macrophage inflammatory protein-2.
Neutrophil infiltration is a hallmark of inflammatory injury after myocardial IR injury (17). Therefore, we investigated the function of IL-17A on neutrophil recruitment. As determined by myeloperoxidase (MPO) activity and fluorescence-activated cell sorting analysis of CD11b+Gr-1+ neutrophils, myocardial IR injury induced a surge in neutrophil recruitment to myocardium and anti–IL-17A mAb or anti-γδTCR mAb treatment reduced neutrophil recruitment (Fig. 3D,E). The CXC glutamic acid-leucine-arginine chemokines KC, MIP-2, and LIX are not only potent neutrophil chemoattractants but also IL-17A target genes (18). Myocardial IR injury caused a significant induction of mRNA level of all the three chemokines. Moreover, neutralization of IL-17A or γδTCR had an opposite effect on expression of these chemokines after myocardial IR injury (Fig. 3F).
IL-23 Is Required for the Generation of IL-17A
To investigate the role of IL-23 in the production of IL-17A, IL-23p19, one subunit of IL-23, was measured. IL-23p19 mRNA expression in cardiac isografts was significantly increased after IR injury (Fig. 4A). To further determine whether IL-23 is required for the production of IL-17A, we neutralized its function using an anti–IL-23p19 antibody. IL-17A mRNA expression was significantly decreased after neutralizing IL-23 (Fig. 4B). In our in vitro experiment, supernatant IL-17A from spleen lymphocytes was increased after stimulation with IL-23, which was further enhanced by IL-23+IL-1β stimulation (Fig. 4C). We further investigated the effect of IL-23 neutralization on IR injury; the results showed that the expression of TnT and MPO and the number of TUNEL-positive cardiomyocytes were significantly decreased with anti–IL-23p19 antibody treatment (Fig. 4D–F).
FIGURE 4: IL-23 is critical for the generation of IL-17A in vivo and in vitro. A, IL-23p19 mRNA levels were analyzed by real-time PCR in cardiac isografts from sham and cardium IR injury group for different times (n=6). B, IL-17A levels in cardiac isograft were measured from control IgG and anti–IL-23p19 mAb-treated recipient day 1 after transplantation (n=6). C, increase in the secretion of IL-17A from spleen lymphocytes stimulated with IL-23 in vitro. After 48 hr of stimulation, the supernatants were collected, and IL-17A concentrations were measured by ELISA kits (n=3). D, serum TnT was measured in control IgG and anti–IL-23p19 mAb-treated recipients day 1 after transplantation (n=6). E, percentages of TUNEL-positive nuclei over total number of nuclei in cardiac isograft from control IgG and anti–IL-23p19 mAb-treated recipient at day 1 after transplantation (n=6). F, cardiac MPO activity in cardiac isograft from control IgG and anti–IL-23p19 mAb-treated recipient at day 1 after transplantation (n=6). *P<0.05 versus sham or control group.
Hmgb1-TLR4 Mediates the Production of IL-23 by Macrophages
To understand whether macrophages mediate the production of IL-23, we inhibited macrophages with GdCl3. The expression of IL-23 and IL-17A was reduced after depletion of macrophages (Fig. 5A) and could not be induced in TLR4-/- mice (Fig. 5B) after IR injury. TLR4-/- also showed reduced TnT expression after IR injury (Fig. 5C). Concurrently, Hmgb1, a damage-associated molecule released from injured cardiomyocytes, increased after IR injury (Fig. 5D, E). Use of Hmgb1 inhibitor glycyrrhizin markedly reduced the production of IL-23 and IL-17A, and recombined mouse IL-23 administration abrogated the reduced IL-17A expression induced by Hmgb1 inhibitor (Fig. 5F). Furthermore, glycyrrhizin reduced TnT, MPO expression, and cardiomyocyte apoptosis (Fig. 5G–I).
FIGURE 5: Hmgb1-TLR4 pathway mediates the production of IL-23 by macrophages. A, IL-23p19 and IL-17A mRNA levels were analyzed by real-time PCR in cardiac isografts day 1 after transplantation. Mice were treated with GdCl3 or equal volumes of PBS as a control (n=6). B, IL-23p19 and IL-17A mRNA levels were analyzed by real-time PCR in cardiac isografts from TLR4-/- mice and C57BL/6 mice day 1 after transplantation. (n=6). C, serum cTnT was measured in TLR4-/- mice and C57BL/6 mice day 1 after transplantation (n=6). D, Hmgb1 mRNA level was analyzed by real-time PCR in cardiac isografts from sham and cardium IR injury group for different times (n=6). E, representative photographs of Hmgb1-positive staining in cardiac isografts from sham and cardium IR injury group. Arrows indicate positive Hmgb1 staining. F, IL-23p19 and IL-17A mRNA levels were analyzed by real-time PCR in cardiac isografts day 1 after transplantation. Mice were treated with GL, GL+rmIL-23, or PBS (n=6). G, serum cTnT was measured in PBS- and GL-treated recipients day 1 after transplantation (n=6). H, percentages of TUNEL-positive nuclei over total number of nuclei in cardiac isograft from PBS- and GL-treated recipient at day 1 after transplantation (n=6). I, cardiac MPO activity in cardiac isograft from PBS- and GL-treated recipient at day 1 after transplantation (n=6). *P<0.05 versus sham, PBS, or B6 group. GL, glycyrrhizin; PBS, phosphate-buffered saline; rmIL-23, recombined mouse IL-23.
DISCUSSION
This study revealed a crucial role for the Hmgb1-TLR4-IL-23-IL-17A axis in cardiomyocyte IR injury. Hmgb1 stimulates the production of IL-23 by macrophages in a TLR4-dependent manner; IL-23 promotes the expression of IL-17A, which was mainly generated by γδT cells. IL-17A then aggravates cardiomyocyte apoptosis and neutrophil infiltration.
IR triggers a vigorous inflammatory response, augmented by the generation and release of various cytokines, which ultimately exacerbates tissue injury, although the precise mechanism of the IR injury has not been fully revealed (19). Increasing evidence indicates that the elements of both adaptive immunity and innate immunity participate in IR injury (20). Notably, IL-17A acts as a bridge between adaptive and innate immunity through the potent induction of a gene expression program typical of the inflammatory response, presenting a unique position in the immune response process (2). IL-17A has been implicated in IR injury (6–9). Recently, it has been shown that IL-17A is produced by diverse T-cell subsets, including CD4+ T cells, CD8+ T cells, γδT cells, NK cells, NKT cells, and neutrophils (21, 22). In our experiment, we found that elevated circulating levels of IL-17A in cardiac isografts, and whereas small numbers of CD4+ T cells, CD8+ T cells, NK cells, and neutrophils produced IL-17A, there were much more IL-17A–producing γδT cells after IR injury. Therefore, our finding provided clear evidence that γδT cells represented a dominant IL-17A–producing lymphocyte subset after IR injury. Furthermore, both neutralization of IL-17A and γδTCR markedly ameliorated IR injury, as demonstrated by reduced TnT levels and improved cardiac function.
Apoptosis has been proposed to be an important mechanism for a significant amount of cell death in reperfused ischemic myocardium (16). It could be regulated by oxygen free radicals, cytokines, and neutrophil accumulation (23). Our experiment showed that neutralization of IL-17A or γδTCR could regulate cardiomyocyte apoptosis, as confirmed by the change of TUNEL-positive cardiomyocytes and caspase-3 activity. Neutrophil recruitment plays a major role in myocardial damage after IR injury (24). Neutrophil chemotaxis and activation might be strongly regulated by CXC chemokines (25). IL-17A has been shown to induce KC, MIP-2, and LIX, which are rodent homologues of CXCL1, CXCL2, and CXCL5 (26). Our result demonstrated that inhibition of IL-17A or γδTCR markedly decreased cardiac KC, MIP-2, and LIX expression and neutrophil infiltration in mouse IR myocardium.
The heterodimeric cytokine IL-23, which was secreted mainly by activated dendritic cells and macrophages in response to TLR activation, stimulate T-cell differentiation and function in linking innate and adaptive immunity (27). IL-23 contributes to autoimmunity and host defense through IL-23/IL-17–dependent pathways (28). Recent article showed that an IL-23/IL-17 pathway was activated in kidney IR injury (8). Our result indicated that IL-23 expression was significantly increased on day 1 after transplantation and blockade of IL-23 with anti–IL-23p19 significantly reduced the production of IL-17A and ameliorated IR injury after cardiac transplantation. Furthermore, both IL-17A and IL-23 production was ameliorated by depletion of macrophages. This was consistent with previous study, indicating that macrophages can quickly respond to endogenous stimulating factors after tissue injury and play a pathogenic role through their secretion of proinflammatory factors (29, 30).
Hmgb1, a highly conserved nuclear protein, served as an early mediator of inflammation and cell injury and plays a key role in many pathogenic states, including myocardial IR. Hmgb1 performs these functions via interactions with TLRs and the receptor for advanced glycation end products (31, 32). TLR4, crucial innate immune pattern recognition receptors, plays an important role in regulating the immune response and inflammatory reaction (13). The effect of TLR4 in myocardial IR injury is mainly through the TLR4 signal transduction pathway (33), which includes c-Jun N-terminal kinases, p38 kinases, and the nuclear factor-κB pathway and can activate the secretion of inflammatory cytokines, chemokines, and adhesion molecules secretion in myocardial IR injury (34, 35). In our experiment, either TLR4 deficiency or Hmgb1 blockade significantly reduced production of IL-17A and IL-23, and exogenous IL-23 abrogated the reduced IL-17 expression induced by Hmgb1 blockade. This indicated that Hmgb1-TLR4 pathway contributed to secretion of IL-17A dependent on IL-23 and then promote myocardial IR injury. All these data suggested that the Hmgb1-TLR4-IL-23-IL-17A axis plays a pivotal role during myocardial IR injury in cardiac transplantation, and blocking any portion of this axis will attenuate myocardial IR injury.
In summary, our study provides evidence that macrophage-γδT-neutrophil cascading response is involved in myocardiac IR injury after cardiac transplantation via an Hmgb1-TLR4-IL-23-IL-17A axis, which plays an important role in the link between innate and adaptive immune response. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in the immunoregulation, Hmgb1-TLR4-IL-23-IL-17A axis, which stands at the crossroads between innate and adaptive immunity, contributes to cardiomyocyte apoptosis, neutrophil accumulation, and IR injury in cardiac transplantation.
MATERIALS AND METHODS
Animals
Inbred male C57BL/6 mice, used as donors and recipients, were from the Center of Experimental Animals, Tongji Medical College of Huazhong Science and Technology University (Wuhan, China). TLR4-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were male at 15 to 20 g in weight, which were housed in specific pathogen-free facility with regular food and water ad libitum. Experiments were approved by the Institutional Animal Care and Use Committee at Tongji Medical College (Wuhan, China).
Heterotopic Cardiac Transplantation and Posttransplantation Therapies
Syngeneic heart transplantation was performed in C57BL/6 and TLR4-/- mice by a modified nonsuture cuff technique described previously by Heron et al. Hearts were stored in cold Bretschneider solution for 8 hr before transplantation with consecutive in vivo reperfusion for 1, 3, or 7 days (1). For neutralization of endogenous IL-17A or IL-23, 0.2 mg neutralizing rabbit anti-mouse IL-17A (Biolegend, San Diego, CA) or neutralizing rabbit anti-mouse IL-23p19 (eBioscience, San Diego, CA) was administered intravenously 5 min before reperfusion. Control rabbit IgG was used as isotype control (6). For deletion of γδT cells, mice were injected intravenously with 0.5 mg of an anti-γδTCR mAb (American Type Culture Collection, Manassas, VA) 5 min before reperfusion. For inhibition of macrophages, mice were injected intravenously with GdCl3 at 20 mg/kg (Sigma-Aldrich, St. Louis, MO) 5 min before reperfusion. For inhibition of Hmgb1, mice were treated with glycyrrhizin (TCI, Shanghai, China) at 5 mg/mouse 5 min before reperfusion (31). To investigate the role of IL-23 in Hmgb1-induced IL-17A production, recombined mouse IL-23 was injected intravenously to mice at 2 μg/mouse 5 min before reperfusion (36).
Function Assessment
To assess graft function, transplanted hearts were evaluated using an isolated working heart apparatus as described previously (1). After 8 hr of ischemia and 24 hr of in vivo reperfusion, hearts were excised and mounted on the isolated working heart apparatus. Hearts were perfused with a preload pressure of 8 mm Hg and exposed to an after-load pressure of 80 mm Hg while being paced at 250 beats/min. Pressure–volume ratio was performed as follows: after-load pressure was increased stepwise by 10 mm Hg from 10 up to 180 mm Hg of after-load pressure every 5 s.
Serum Analysis of Cardiac TnT
Analysis of cardiac serum TnT levels was performed as follows: 1 mL heparinized blood was centrifuged to obtain plasma and stored at −30°C until assayed. TnT was measured using the cardiac reader system according to the manufacturer’s instruction.
Myocardial Apoptosis
TUNEL staining was performed as described previously (6). Hearts were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5-μm-thick sections, and treated as instructed in the In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany). Total nuclei were stained with 4′,6-diamidino-2-phenylindole. Cardiac caspase-3 activity was measured as described previously (6) using a caspase colorimetric assay kit following the manufacturer’s instructions (Chemicon, Temecula, CA). The absorbance of the p-nitroaniline cleaved by caspase was measured at 405 nm using a microplate reader (ELx800; Bio-Tek Instruments, Winooski, VT). Results were standardized to the sham group for comparison of the fold change in caspase-3 activity.
Immunohistochemistry
Cardiac isograft tissues were prepared and stained for Hmgb1 as described previously (37).
MPO Assay
On day 1 after transplantation, tissue samples from cardiac isografts were assessed for MPO activity (6). Samples were homogenized in hexadecyltrimethyl ammonium bromide (Sigma-Aldrich) and dissolved in potassium phosphate. After centrifugation, supernatants were collected and mixed with o-dianisidine dihydrochloride (Sigma-Aldrich) and H2O2 in phosphate buffer. The activity of MPO was measured spectrophotometrically at 470 nm using microplate reader (ELx800; Bio-Tek Instruments) and expressed as units per 100 mg tissue. MPO standards (Sigma-Aldrich) were measured concurrently with the tissue samples.
Fluorescence-Activated Cell Sorting Analysis
Accordingly, in a previous study reported by Gorbacheva et al. (38), infiltrated cells in the isograft were isolated. For measurement of cardiac IL-17A–producing leukocytes, CD45+ cells were isolated using anti-CD45 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and then stained with intracellular cytokine combined with various surface markers as described previously (39). For detection of the number of cardiac infiltrating neutrophils, cells were stained with PerCP-Cy5.5 anti-mouse CD45, PE-Cy7 anti-mouse CD11b, and PE anti-mouse Ly-6G/Gr-1 and measured by FACSCalibur flow cytometry.
Western Blotting
The protein level of IL-17A was determined by Western blotting using primary anti-mouse IL-17A antibody. Protein extracted from cells or tissue was separated on 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Pierce, Rockford, IL). After being blocked with 5% nonfat milk in Tris-buffered saline for 3 hr, the membranes were incubated with indicated primary antibodies (0.2 μg/mL) at 4°C overnight followed by incubation with horseradish peroxidase–conjugated secondary antibody (1:5000) for 3 hr. β-Actin was used as a loading control for comparison between samples.
Real-time PCR
Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology) with ABI PRISM 7900 Sequence Detector System (Applied Biosystems, Foster City, CA). Each reaction was performed in duplicate, and changes in relative gene expression normalized to β-actin levels were determined using the relative threshold cycle method. Primer sequences are shown in Table S1 (see SDC, https://links.lww.com/TP/A815).
Measurement of IL-17A in Supernatant
Spleen mononuclear cells were stimulated in vitro with IL-1β (50 ng/mL), IL-23 (50 ng/mL), or the combination for 48 hr. The supernatants were collected for measurement of IL-17A with ELISA kit from Dakewe Biotech (Shenzhen, China).
Statistics
Data are presented as means±SEM. Differences were evaluated using unpaired Student’s t test between two groups and one-way ANOVA for multiple comparisons followed by a post hoc Student’s–Newman–Keuls’ test when necessary. All analyses were done using SPSS 13.0 (SPSS, Chicago, IL), and statistical significance was set at P<0.05.
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