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Original Basic Science—General

IL-17A Is Critical for CD8+ T Effector Response in Airway Epithelial Injury After Transplantation

Zhang, Ruochan1; Fang, Huihui1; Chen, Rongjuan1; Ochando, Jordi C. PhD2,3; Ding, Yaozhong PhD1; Xu, Jiangnan MD1

Author Information
doi: 10.1097/TP.0000000000002452
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The development of obliterative bronchiolitis (OB) is the major factor leading to long-term morbidity and mortality after lung transplantation, significantly restricting and hindering the clinical application of lung transplantation.1 Although the pathogenesis of the disease remains incompletely understood, the evidences of airway epithelial cells as a dominating immune target in obliterative airway diseases have been documented in humans and animal models,2,3 and the degree of epithelial injury is correlated with the development of OB after transplantation.4 Antecedent literatures have sufficiently showed that T cell-mediated adaptive immune responses of the recipients to the allografts play a key role in the immunopathogenesis of OB.5-8 Subsequent studies demonstrated that CD8+ effector T cells are the dominant mediators in the progression of OB.9-11 More correlative studies showed that graft-infiltrating CD8+ T cells predominate over CD4+ T cells in mouse lung transplant and heterotopic trachea transplant allograft models.9,12-13 Airway epithelium expresses the major histocompatibility complex (MHC) class I mainly on the apical surface, and their presentation of alloantigen leads to direct CD8+ T cell activation in the airway, and their proliferative response can be observed as early as 48 hours after transplantation.14

Upon activation, CD8+ T cells develop into 2 main functional effector subsets. Type 1 CD8+ T (Tc1) cells produce IFNγ with high cytotoxic activity; type 17 CD8+ T (Tc17) cells produce IL-17 with enhanced renewal ability but less cytotoxic capacity.15 Tc17 development is governed by the similar molecular mechanisms for type 17 CD4+ helper T (Th17) cells, that cytokines IL-6, IL-21, IL-23, TGFβ, and transcription factors STAT3 and RORγt are critical for their differentiation.16,17 Later studies demonstrated that there are also distinct regulatory mechanisms for Tc17 development. Unlike Th17 cells, development of Tc17 cells in vivo does not require TGFβ signals.18

IL-17 and CD8+ T cells are associated with the early postlung transplantation time period,19 and high numbers of Tc17 cells are presented in the airway of lung transplant patients with lymphocytic bronchiolitis.20 Direct evidences of Tc17 cells in mediating allograft rejection in animal models have been observed when the master type 1 CD4+ helper T (Th1) cell transcription factor, T-bet, is absent.21,22 All these studies point to the important roles of IL-17 and CD8+ T cells in lung transplant rejection and OB pathogenesis; however, their precise role and the underneath cellular and molecular mechanism remain to be further elucidated.

Various animal and in vitro models of lung transplant complications have been developed.23 The histological changes of orthotopic mouse trachea allografts resemble the early stage of OB development, including airway epithelial disruption and submucosal lymphocytic inflammation, but no significant obstruction in the airway lumen24,25; Orthotopic mouse lung allografts display similar pathological features of injury of acute rejection in lung transplant patients,23 and it has recently been shown that the MHC-mismatched orthotopic lung transplant model under mild immunosuppression could be the most suitable model for investigating posttransplant chronic airway fibrosis.26

In this study, by using mouse orthotopic trachea and lung transplant models, we examined the roles of IL-17A and Th17 development signals in airway epithelial injury and OB pathogenesis after transplantation. We showed that elevated levels of IL-17A in the trachea allografts were associated with airway epithelial injury; neutralization of IL-17A by anti-IL-17A or devoid of IL-17A production by RORγt deficiency in the allograft recipients significantly reduced epithelial damage. When further dissecting the mechanistic insights into IL-17A-mediated airway destruction of the trachea and lung allografts, we revealed the critical role of IL-17A in early accumulation of CD8+ T cells and their cytotoxic activities that cause the airway epithelial injury and allograft rejection after transplantation.

MATERIALS AND METHODS

Mice

C57BL/6 and BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). RORγt−/− mice (C57BL/6 background) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred under specific-pathogen-free conditions. All the mouse experiments in this study were performed in conformity to Ethical Principles of Animal Welfare and approved by the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, China).

Orthotopic Trachea and Lung Transplantations

Murine orthotopic allogeneic trachea transplants were implemented in wild type (WT) or RORγt−/− C57BL/6 (H-2b) mice using BALB/c (H-2d) donors. Syngeneic transplant controls were performed in WT C57BL/6 mice using littermate donors. We used age- and sex-matched littermates at 8 to 12 weeks of age, and animals were assigned randomly to experimental groups. All surgical operations were performed using sterile technique and under the stereoscopic microscope (Olympus SZ61, Japan). Both donors and recipients were anesthetized with pentobarbital sodium (80 mg/kg) by intraperitoneal (i.p.) injection. In brief, a tracheal segment of 8 cartilage rings was removed from the donor and stored in cold normal saline. After anesthesia, an appropriate incision was operated in the middle cervical region of the recipient, and the strap muscles were separated for exposing the entire trachea. The recipient's trachea was then transected and the donor's tracheal segment was implanted end-to-end and stitched with 10-0 nylon suture. The cervical incision was closed with 5-0 prolene suture. After operation, the recipient mouse was placed in a warm incubator until awake and monitored daily.

Orthotopic lung transplantations were performed using a cuffed technique as we described previously.27 Donor and recipient mice were sedated with pentobarbital sodium (80 mg/kg) by i.p. injection before surgery. All micro operations were performed under the stereomicroscope (Nikon SMZ1270, Japan).

In Vivo Treatments of Transplant Recipients

Anti-IL-17A (clone 17F3), anti-IFNγ (clone XMG1.2), and anti-CD8 (clone 2.43) monoclonal antibodies (mAbs) were all purchased from Bio X Cell (West Lebanon, NH). STAT3 inhibitor (C188-9) was purchased from Merck Millipore (Darmstadt, Germany).

For specific cytokine neutralization, recipients received 200 μg of anti-IL-17A or anti-IFNγ by i.p. injected on days -1, 1, 3, 5 and 7 after trachea transplantation (day 0 being the day of transplantation). To deplete CD8+ T cells, recipients were administrated with 500 μg of anti-CD8 i.p. on days -1, 2, and 7. In separate experiments, trachea or lung transplant recipients received 1 mg of C188-9 daily from day -1 to 7 by i.p. injection.

Quantitative Real-time RT-PCR

Trachea grafts were harvested on indicated days after transplantation, and ground in lysis solution. Lung grafts were harvested 10 days after transplantation, and graft-infiltrating cells were isolated. Total RNA was isolated using the AxyPrep Multisource Total RNA Miniprep kit (Corning, New York, NY), and cDNA was reverse transcribed using 5× All-In-One RT MasterMix (Applied Biological Materials, Richmond, British Columbia, Canada). The primers for quantitative real-time PCR were as follows: IL-17A, CTC CAG AAG GCC CTC AGA CTA and AGC TTT CCC TCC GCA TTG ACA; IFNγ, TGG CTC TGC AGG ATT TTC ATG and TCA AGT GGC ATA GAT GTG GAA GAA; CXCL9, GGA GTT CGA GGA ACC CTA GTG and GGG ATT TGT AGT GGA TCG TGC; CXCL10, CAT CCT GCT GGG TCT GAG TG and AGG CTC TCT GCT GTC CAT CC; CXCL11, GGC TTC CTT ATG TTC AAA CAG GG and GCC GTT ACT CGG GTA AAT TAC A; CCL20, GCC TCT CGT ACA TAC AGA CGC and CCA GTT CTG CTT TGG ATC AGC; Granzyme B, CCA CTC TCG ACC CTA CAT GG and GGC CCC CAA AGT GAC ATT TAT T; Perforin, AGC ACA AGT TCG TGC CAG G and GCG TCT CTC ATT AGG GAG TTT TT; Cyclophilin A, AGG GTG GTG ACT TTA CAC GC and ATC CAG CCA TTC AGT CTT GG. Reactions were performed using the SYBR Green PCR kit (Qiagen, Valencia, CA) on the Rotor-Gene Q system (Qiagen). All experiments were repeated at least 3 times. Target gene expression was normalized to the expression of the housekeeping gene (cyclophilin A) using the comparative Ct (2−ΔΔCt) method and presented as the fold change relative to the control group.

Histopathology and Quantitative Analysis of Posttransplant Graft Injuries

Trachea and lung grafts were harvested on day 14 and day 10, respectively, after transplantation, fixed in 4% formaldehyde, embedded in paraffin wax, and cut into 5-μm sections followed by hematoxylin and eosin (H&E) staining and Masson's Trichrome staining.

For trachea grafts, the histopathology was blindly reviewed under a modified histology scoring system to evaluate the degree of OB development.28 All qualitative histological changes were noted, and 3 easily identifiable pathological processes were scored on a scale of 0 to 4 (0, normal; 1, mild; 2, moderate; 3, severe; and 4, very severe): (a) airway epithelial flattening and loss, (b) deposition of collagen fibrils, and (c) leukocyte infiltration. An overall score of OB (scale, 0-12) was obtained based on the summation of all the scores, and then an average score (mean ± SD) was generated from the cohort of trachea grafts (5 grafts/group, 3 sections/graft).

For lung grafts, a modified quantitative scoring system based on perivascular and interstitial mononuclear infiltrates was served to grade the severity of acute rejection pathology.22,29 The following 4 characteristics were scored independently on a scale of 0 to 4 (0, none; 1, minimal; 2, mild; 3, moderate; and 4, severe): (a) peribronchial/perivascular inflammatory cell infiltrate, (b) interstitial inflammatory cell infiltrate, (c) alveolar inflammatory cell infiltrate, and (d) intraluminal airway inflammation. The final score (range 0-4) is the mean of the points for the 4 characteristics. Data are presented as the average score (mean ± SD) generated from the cohort of lung grafts (5 grafts/group, 3 sections/graft).

Immunohistochemistry

For immunohistochemistry (IHC) assays, trachea grafts were harvested on day 3 after transplantation, fixed in 4% formaldehyde, embedded in paraffin wax, and cut into 5-μm sections. The tissue sections were probed with rabbit anti-mouse CD8 or anti-mouse IL-17A polyclonal primary Abs (Abcam, Cambridge, MA) and goat anti-rabbit IgG-HRP conjugate secondary Abs (Abcam), and the brown precipitating HRP substrate DAB was used for detection. CD8+ and IL-17A+ cell infiltration in the trachea grafts was evaluated by the detection of CD8 and IL-17A, respectively. The average numbers (mean ± SD) of CD8+ cells per microscopic field (×400) were calculated from the cohort of trachea grafts (4 grafts/group, 3 sections/graft, 5 fields/section).

Statistical Analysis

Statistical significances between groups were determined by the Student t test and 1-way analysis of variance using GraphPad Prism. P less than 0.05 was considered to indicate a significant difference.

RESULTS

Elevated Levels of IFNγ and IL-17A Expression in the Trachea Allografts Are Associated With Airway Epithelial Injury

Pathological features of orthotopic trachea allografts represent the early stage of OB development, including airway epithelial disruption and submucosal lymphocytic inflammation.25 Murine orthotopic allogeneic trachea transplants were implemented in WT C57BL/6 mice using MHC fully mismatched BALB/c mice as donors; syngeneic transplants were performed in WT C57BL/6 mice using C57BL/6 donors. Grafts were harvested and histopathology was performed 14 days after transplantation. As shown in Figure 1A, the airway epithelia of isografts maintained normal without any inflammation or injury, whereas significant airway destruction was observed in the allografts, characterized by transformation of the pseudostratified ciliated columnar epithelia into flat epithelia, along with inflammatory cell infiltration and collagen fibril proliferation in submucosal tissue, confirming that orthotopic trachea allograft rejection resembles early histopathological features of OB, and the average pathological score was greatly increased in allografts compared with isograft controls (Figure 1B). Assessment of the kinetics of early IFNγ and IL-17A expression after transplantation showed rapid and significant increases for both cytokines from the allografts, as compared with the syngeneic controls. Their patterns of expression were different. IFNγ peaked at day 2, whereas IL-17A peaked at days 1 and 3 (Figure 1C), which confirmed the rapid alloresponse.14 Furthermore, the elevated levels of IFNγ and IL-17A expression in the trachea allografts correspond to the epithelial damage, suggesting their potential roles in airway epithelial injury after transplantation.

FIGURE 1
FIGURE 1:
Alloresponse leads to airway epithelial injury and elevated IL-17A and IFNγ expression after trachea transplantation. Tracheas from C57BL/6 or BALB/c donors were transplanted into WT C57BL/6 recipients as isograft or allograft, respectively. A, Trachea grafts were harvested on day 14 after transplantation for Masson's Trichrome staining (original magnification, ×200) and H&E staining (original magnification, ×400). Arrows indicate the normal airway pseudostratified ciliated columnar epithelia of the isograft, and the transformed flattened epithelia of the allograft. Representative sections are shown from 5 mice in each group. B, Pathological score of OB was used to evaluate quantitative histological changes of the trachea isografts and allografts according to the scoring system described in Materials and Methods. Data are presented as the average pathological score (mean ± SD) of 5 grafts in each group (3 different sections for each graft) (**P < 0.01). C, Quantitative RT-PCR was performed for IL-17A and IFNγ expression in trachea grafts on days 0, 1, 2, and 3 after transplantation (day 0 being the day of transplantation). Data were normalized to the expression of cyclophilin A as the housekeeping gene. Target gene expression in isografts (n = 3 for each time point) and allografts (n = 3 for each time point) is presented as the fold change (mean ± SD) relative to the control group (day 0, untransplanted normal trachea, n = 3) (*P < 0.05, **P < 0.01).

Neutralization of IL-17A or Devoid of IL-17A Expression by RORγt Deficiency Significantly Reduces Epithelial Damage of the Trachea Allografts

To further dissect the roles of IFNγ and IL-17A in airway epithelial injury, we administrated the allograft recipients with anti-IFNγ or anti-IL-17A neutralizing mAbs. As shown in Figure 2A, allografts harvested from recipients with anti-IL-17A treatment showed that the structure of airway epithelia approached the normal morphology, and the average pathological score of OB was significantly decreased and closer to isografts (Figure 2B); In contrast, the allografts of anti-IFNγ–treated recipients still displayed flattened epithelia (Figure 2A), and there was no significant change of the average pathological score compared with the allografts of untreated recipients (Figure 2B). Quantitative RT-PCR data showed that anti-IL-17A, but not anti-IFNγ, significantly reduced IL-17A expression in the trachea allografts (Figure 2C); IHC for IL-17A revealed that the early posttransplant IL-17A was mostly produced by mononuclear cells, and neutralization of IL-17A greatly reduced IL-17A production in the allografts (Figure 2D). These results indicate the dominant role of IL-17A in airway epithelial injury after transplantation.

FIGURE 2
FIGURE 2:
Neutralization of IL-17A or devoid of IL-17A expression by RORγt deficiency in the allograft recipients significantly reduces epithelial damage of the trachea allografts. Syngeneic trachea transplants were performed in WT C57BL/6 mice using littermate donors. Allogeneic BALB/c tracheas were transplanted into WT or RORγt−/− (C57BL/6 background) recipients. For specific cytokine neutralization, the WT recipients were administrated with 200 μg of anti-IL-17A or anti-IFNγ mAbs on days −1, 1, 3, 5, and 7. A, Masson's Trichrome staining of trachea grafts harvested 14 days after transplantation (original magnification, ×200). Arrows indicate the difference of airway epithelial morphology between isografts and allografts from WT and RORγt−/− recipients (top), and the different changes of epithelial structure after each indicated treatment (bottom). Representative sections from 5 mice in each group are shown. B, Pathological score for quantitative histological changes of trachea allografts from WT recipients, with or without anti-IFNγ or anti-IL-17A treatment, and RORγt−/− recipients, as compared with isograft controls. Data are presented as the average pathological score (mean ± SD) of 5 grafts in each group (3 different sections for each graft) (*P < 0.05, **P < 0.01). C, Quantitative RT-PCR was performed for IL-17A expression in trachea grafts 3 days after transplantation. Data were normalized to the expression of cyclophilin A as the housekeeping gene. IL-17A expression in allografts (n = 5 in each experimental group) is presented as the fold change (mean ± SD) relative to the isograft control group (n = 5) (*P < 0.05, **P < 0.01). D, IHC staining for IL-17A in trachea grafts on day 3 after transplantation (original magnification, ×200 and ×400, inset). Representative sections from 4 mice in each group are shown.

RORγt is the master transcription factor for Th17 differentiation and IL-17 production.30 To further elucidate the roles of Th17 development signals in airway epithelial injury, we performed orthotopic allogeneic trachea transplants using RORγt−/− (C57BL/6 background) recipients. Histopathological data showed that, compared with WT recipients, RORγt deficiency significantly improved the integrity of airway epithelia in the allografts (Figure 2A), along with a much lower average pathological score of OB (Figure 2B). Quantitative RT-PCR data showed that RORγt deficiency abolished IL-17A expression in the allografts (Figure 2C). Together, these results demonstrate the essential role of IL-17A in airway epithelial injury after transplantation.

In Vivo Depletion of CD8+ T Cells Prevents Airway Epithelial Injury of the Trachea Allografts

CD8+ T cells are the major T-cell subset promoting acute lung allograft rejection and the allogeneic response is initiated by airway epithelial cells.14 To further dissect the functional roles of CD8+ T cells in airway epithelial injury and the early pathogenesis of OB, allograft recipients received anti-CD8 depleting mAbs. Histopathologic analysis of trachea allografts showed that depletion of CD8+ T cells completely prevented airway epithelial injury (Figure 3A), and there was no significant difference in the average pathological score between the allografts from anti–CD8-treated recipients and the isograft controls (Figure 3B). Quantitative RT-PCR analysis showed that depletion of CD8+ T cells by anti-CD8 resulted in decreased IL-17A expression (Figure 3C). These data demonstrate that CD8+ T cells not only lead the assault on the airway epithelium during the early stage of OB development but also contribute to the early IL-17A production after allotransplantation.

FIGURE 3
FIGURE 3:
In vivo depletion of CD8+ T cells prevents airway epithelial injury. Syngeneic trachea transplants were performed in WT C57BL/6 mice using littermate donors. Allogeneic BALB/c tracheas were transplanted into WT C57BL/6 recipients with or without 500 μg of anti-CD8 depleting mAbs on days −1, 2, and 7. A, Histopathological analysis of trachea grafts harvested 14 days after transplantation following by Masson's Trichrome staining (original magnification, ×200). The arrow marks the significant improvement of airway epithelial integrity in trachea allografts by anti-CD8 treatment (right), as compared with allografts from untreated recipients (middle). Representative sections from 5 mice in each group are shown. B, Pathological score for quantitative histological changes of trachea allografts from recipients with or without anti-CD8 treatment, as compared with isograft controls. Data are presented as the average pathological score (mean ± SD) of 5 grafts in each group (3 different sections for each graft) (**P < 0.01). C, Quantitative RT-PCR was performed for IL-17A expression in trachea grafts 3 days after transplantation. Data were normalized to the expression of cyclophilin A as the housekeeping gene. IL-17A expression in allografts (n = 5 in each experimental group) is presented as the fold change (mean ± SD) relative to the isograft control group (n = 5) (**P < 0.01).

Neutralization of IL-17A Inhibits the Accumulation of CD8+ T Cells in the Trachea Allografts

To further determine whether and how IL-17A regulates CD8+ T cell-driven airway epithelial injury, IHC for CD8 was performed 3 days after transplantation on the trachea isografts and allografts from recipients with or without anti–IL-17A administration, and the numbers of CD8+ cells per microscopic field (×400) were calculated. As shown in Figures 4A and B, neutralization of IL-17A significantly attenuated CD8+ cell infiltration in the allografts, when compared with the untreated group, demonstrating the critical role of IL-17A in mediating CD8+ T cell accumulation and consequent airway epithelial injury after transplantation.

FIGURE 4
FIGURE 4:
Neutralization of IL-17A inhibits the accumulation of CD8+ T cells in the trachea allografts. Syngeneic trachea transplants were performed in WT C57BL/6 mice using littermate donors. Allogeneic transplants were implemented in WT C57BL/6 recipients using BALB/c donors, with or without 200 μg of anti-IL-17A mAbs on days −1 and 1. A, IHC staining for CD8 in trachea grafts on day 3 after transplantation (original magnification, ×200 and ×400, inset). Representative sections from 4 mice in each group are shown. B, Quantitative analysis of CD8+ T-cell infiltration in the trachea grafts was evaluated by the average number (mean ± SD) of CD8+ cells per microscopic field (×400) calculated from the cohort of trachea grafts (4 grafts in each group, 3 sections for each graft, 5 fields per section) (*P < 0.05). C, Quantitative RT-PCR was performed for the expression of CXCR3 chemokine ligands (CXCL9, CXCL10, CXCL11) and CCR6 ligand (CCL20) in trachea grafts 3 days after transplantation. Data were normalized to the expression of cyclophilin A as the housekeeping gene. Target gene expression in allografts (n = 5 in each experimental group) is presented as the fold change (mean ± SD) relative to the isograft control group (n = 5) (*P < 0.05).

Tc1 cells mainly express CXCR3, the shared receptor for chemokines CXCL9, CXCL10 and CXCL11; while Tc17 cells express CCR6, the receptor for CCL20. CXCR3 and its ligands play critical role in the pathogenesis of OB.28 To investigate how IL-17A may affect CD8+ T effector accumulation in the airway, we examined the expression of CXCL9, CXCL10, CXCL11 and CCL20 in the trachea grafts 3 days after transplantation with or without anti-IL-17A treatment. As shown in Figure 4C, anti-IL-17A significantly reduced the expression of all 4 chemoattractants for both CD8+ T effectors, Tc1 and Tc17. These results suggest that IL-17A may mediate CD8+ T effector accumulation in the airway through chemotaxis after allotransplantation.

Inhibition of IL-17A Production Through Blocking STAT3 Activation Is Not Sufficient to Improve Epithelial Integrity of the Trachea Allografts

STAT3 is a key transcription factor for RORγt regulation, Th17/Tc17 development and IL-17 production.31 To further verify the effects of Th17 development signal cascade on airway epithelial injury, we treated the allograft recipients with a small molecule compound, C188-9, an inhibitor of STAT3 activation.32 As shown in Figure 5A, STAT3 inhibitor attenuated inflammatory cell infiltration and extracellular matrix deposition in the allografts, paralleling the decreased average pathological score of OB (Figure 5B); however, it was less effective for epithelial protection (Figure 5A). Quantitative RT-PCR data showed that blocking STAT3 activation by C188-9 resulted in markedly reduced IL-17A, but significantly increased IFNγ, granzyme B and perforin expression in the trachea allografts, as compared with the untreated group (Figure 5C), which suggests that inhibition of Th17/Tc17 development signal leads to increased Th1/Tc1 response and cytotoxic activities, causing the airway epithelial injury after allotransplantation.

FIGURE 5
FIGURE 5:
Inhibition of IL-17A production by STAT3 inhibitor is not sufficient to improve epithelial integrity in the trachea allografts. Syngeneic trachea transplants were performed in WT C57BL/6 mice using littermate donors. Allogeneic transplants were implemented in WT C57BL/6 recipients using BALB/c donors, with or without 1 mg of STAT3 inhibitor (C188-9) daily on days −1 to 7. A, Histopathological analysis of trachea grafts harvested 14 days after transplantation for Masson's Trichrome staining (original magnification, ×200). Arrows indicate the similar flattened airway epithelia of allografts from C188-9–treated (right) and untreated (middle) recipients, along with the normal airway pseudostratified ciliated columnar epithelia of the isograft controls (left). Representative sections from 5 mice in each group are shown. B, Pathological score for quantitative histological changes of trachea allografts from recipients with or without C188-9 treatment, as compared with isograft controls. Data are presented as the average pathological score (mean ± SD) of 5 grafts in each group (3 different sections for each graft) (*P < 0.05, **P < 0.01). C, IL-17A and IFNγ expression in trachea grafts was examined on day 3, and cytotoxic-mediator (granzyme B and perforin) gene expression was evaluated on day 7 after transplantation. Data were normalized to the expression of cyclophilin A as the housekeeping gene. Target gene expression in allografts (n = 5 in each experimental group) is presented as the fold change (mean ± SD) relative to the isograft control group (n = 5) (*P < 0.05, **P < 0.01).

STAT3 Inhibition Increases Cytotoxic Activities and Results in More Vigorous Lung Allograft Rejection

Similar to the trachea allografts, our previous data showed rapidly elevated IL-17A expression after transplantation in the lung allografts, and neutralization of IL-17A impaired the characteristics of acute rejection.33 To further dissect the role of Th17/Tc17 development signal in lung transplantation, mouse orthotopic syngeneic and allogeneic lung transplants were performed, with or without C188-9 administration to the recipients. Grafts were harvested 10 days after transplantation; histopathologic characteristics and rejection status were assessed; graft-infiltrating cells were isolated and real-time RT-PCR was performed for the expression of IL-17A, IFNγ, granzyme B and perforin.

As shown in Figure 6A, no obvious inflammation, fibrosis or airway lesion was observed in the lung isografts 10 days after transplantation. Allografts from untreated recipients largely maintained intact airway epithelia, despite developing acute rejection; while the allograft rejection of C188-9–treated recipients appeared more vigorously, along with an increased pathological score of acute rejection (Figure 6B). Quantitative RT-PCR data also showed that blockade of STAT3 activation resulted in a reduction of IL-17A while an elevation of IFNγ and granzyme B expression in the lung allografts, when compared with the untreated group (Figure 6C), further confirming that inhibition of Th17/Tc17 development signal leads to increased Th1/Tc1 response and cytotoxic activities for lung allograft rejection.

FIGURE 6
FIGURE 6:
STAT3 inhibition increases cytotoxic activities and results in more vigorous lung allograft rejection. Syngeneic lung transplants were performed in WT C57BL/6 mice using littermate donors. Allogeneic transplants were implemented in WT C57BL/6 recipients using BALB/c donors, with or without 1 mg of STAT3 inhibitor (C188-9) daily on days -1 to 7. A, Lung grafts were harvested on day 10 after transplantation for Masson's Trichrome staining (original magnification, ×200) and H&E staining (original magnification, ×400). There is no obvious inflammation, fibrosis or airway damage in the lung isografts (left); allografts from untreated recipients develop acute rejection (middle); the allorejection of C188-9–treated recipients display more vigorously (right). Representative sections from 5 mice in each group are shown. B, Pathological score for acute rejection of lung allografts from recipients with or without C188-9 treatment, as compared with isograft controls. Data are presented as the average pathological score (mean ± SD) of 5 grafts in each group (3 different sections for each graft) (*P < 0.05). C, Lung graft-infiltrating cells were isolated and quantitative RT-PCR was performed for IL-17A, IFNγ and cytotoxic-mediator (granzyme B and perforin) gene expression 10 days after transplantation. Data were normalized to the expression of cyclophilin A as the housekeeping gene. Target gene expression in allografts (n = 5 in each experimental group) is presented as the fold change (mean ± SD) relative to the isograft control group (n = 5) (*P < 0.05).

DISCUSSION

In this study, we demonstrated that allogeneic CD8+ T cells are the key effectors for airway epithelial injury after transplantation, and revealed the dominant role of IL-17A in controlling the expression of CD8+ T effector chemoattractants and, consequently, the accumulation of CD8+ T cells in the airway, and regulating allogeneic cytotoxicity after trachea and lung transplantations.

IL-17 and Th17 signals have been demonstrated to be involved in all the phases of allograft rejection after organ transplantation.34 Previous studies showed that IL-17 is critical in mediating early posttransplant lesions after trachea allotransplantation,35 and IL-17–deficient recipients treated with cyclosporine A are protected against developing obliterative airway disease after heterotopic allogeneic trachea transplantation.36 Neutralization of IL-17 has been shown to prevent the development of OB37; suppression of IL-17 production by regulatory T (Treg) cells results in attenuation of mouse orthotopic lung allograft rejection38; and inhibition of IL-17 by halofuginone, a plant derivative, ameliorates the features of chronic lung allograft dysfunction in a minor alloantigen-mismatched murine orthotopic lung transplant model.39 However, there were also reports showing that IL-17–mediated immunity is only involved in the inflammatory component,40 and CD4+ T cells but not Th17 cells are required for OB development after lung transplantation.41 The precise roles of IL-17 in lung transplantation and the cellular and molecular mechanisms of OB pathogenesis, as well as their association, remain to be elucidated. In this study, we found that increased levels of IL-17A expression in the allografts are associated with airway epithelial injury after orthotopic trachea transplantation. Neutralization of IL-17A or devoid of IL-17A production by RORγt deficiency in the recipients significantly improved epithelial integrity of the trachea allografts (Figure 2A), which supports the notion that IL-17A plays a critical role in airway inflammatory responses after transplantation and provides evidence that IL-17A is directly linked to the airway epithelial injury of the trachea allografts.

It has been demonstrated that CD4+ and CD8+ effector T cells use distinct allorecognition pathways during obliterative airway diseases. Alloantigen-specific CD4+ T cells are activated predominantly through indirect allorecognition, whereas CD8+ T cells are capable of initiating robust response via direct allorecognition, with substantial enhancement in the presence of accessory cells, and their proliferative response can be observed as early as 2 days after transplantation.10,14 There is an emerging understanding that tissue-resident memory T cells that persist in epithelial barrier tissues at interfaces with the environment, including skin and lung, play an important role in tissue-specific immune and inflammatory responses.42 Endogenous memory T cells generated during responses to infectious or environmental antigens could cross-react with donor allogeneic MHC molecules, quickly and robustly respond to allografts, and directly mediate graft tissue injury and failure.43,44 It has also been reported that upon skin antigen challenge, CD8+ T cells rapidly produce IL-17 and IFNγ to initiate the innate immune response which subsequently regulates the infiltration of effector T cells into the skin.45 Our results that allogeneic IL-17A and IFNγ response occurs rapidly (Figure 1C) and depletion of CD8+ T cells reduces IL-17A expression in the trachea allografts (Figure 3C) are in agreement with these notions and indicate that CD8+ T cells contribute to the early posttransplant IL-17A expression.

In addition to Th17/Tc17 cells, γδ T cells and type 3 innate lymphoid cells (ILC3) are also identified to be important sources of IL-17.46-48 IL-17 can specifically and selectively recruits neutrophils into the airways via the release of C-X-C chemokines from the bronchial epithelial cells.49 Elevated levels of CXCL9, CXCL10 and CXCL11, chemokine ligands of CXCR3, in human bronchoalveolar lavage fluid from transplantation recipients are associated with acute and chronic lung allograft rejection,50,51 and in vivo neutralization of CXCR3 or its ligands inhibits the intragraft recruitment of mononuclear cells and attenuates the pathogenesis of bronchiolitis obliterans syndrome in a murine model of heterotopic trachea transplantation.28 Our results showed that neutralization of IL-17A reduced the expression of all 3 CXCR3 chemokine ligands and consequently abolished CD8+ T cell accumulation in the allografts after orthotopic trachea transplantation (Figure 4). These data reveal a novel role of IL-17A in controlling the expression of CXCR3 ligands, CXCL9, CXCL10 and CXCL11, in the airway after allotransplantation, consistent with the findings that the promoters of CXCL9, CXCL10, and CXCL11 are regulatory targets of IL-17.52,53 In addition to early adaptive response, surgical and ischemia-reperfusion injury during transplantation could also lead to IL-17 production. Together IL-17 produced in these processes stimulates the airway epithelial cells to release C-X-C chemokines for CD8+ T cell accumulation. Furthermore, after stimulation, neutrophils produce numerous cytokines and chemokines, and consequently mediate the recruitment of T cells into sites of immune reactions.54 It has been shown that neutrophils are critical for CD8+ T effector infiltration into the cutaneous antigen challenge site,45,55 and we and others have recently shown that high level of IL-17 expression is accompanied by an intense infiltration of neutrophils into the allografts after orthotopic lung transplantation.22,27 Thus, it is also possible that IL-17 may mediate CD8+ T-cell accumulation in the allografts and airway epithelial injury through neutrophil activation. These possibilities are currently under investigation.

Recent studies also demonstrate that IL-17 plays positive regulatory roles in CD8+ T-cell immunity. IL-17A is required for optimal adaptive CD8+ T-cell response against bacterial infection through acting on dendritic cells to upregulate their expression of MHC class I molecules as well as cytokines IL-12, IL-6, and IL-1β, thus facilitating cross-priming and proliferation of CD8+ T cells.56 IL-17A–deficient (Il17a−/−) mice are more susceptible to West Nile virus infection, developing a higher viral burden than wild-type mice, and CD8+ T cells isolated from infected Il17a−/− mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo.57 Our results that neutralization of IL-17A abolishes CD8+ T-cell accumulation in the airway (Figures 4A, B) and reduces epithelial damage of the trachea allografts (Figure 2A) demonstrate that IL-17A also plays important role in regulating allogeneic CD8+ T-cell responses after trachea and lung transplantations.

IL-17 and Th17 signals may also play roles in restraining the cytotoxicity of CD8+ T cells. It has been reported that IL-17 and IL-6 synergistically promote viral persistence by protecting virus-infected cells from apoptosis and CD8+ T cell-mediated target destruction.58 Tc17 cells are highly plastic. Some Tc17 cells convert into Tc1 cells after transferring in vivo for tumor therapy,59-62 and in vitro generated Tc17 cells could serve as a reservoir of Tc1 cells in vivo.63 It has recently been demonstrated that Th17 development signals restrain cytotoxic gene expression in CD8+ T cells responding to viral infection in vivo through STAT3 activation, and STAT3-induced RORγt represses cytotoxic genes by inhibiting the functions of transcription factors T-bet and Eomesodermin.64 Our data (Figures 5, 6) that inhibition of STAT3 activation by C188-9 suppresses IL-17A while enhances IFNγ and cytotoxic gene expression support the notion that IL-17A/Th17/Tc17 signals negatively regulate allogeneic CD8+ T cell response and cytotoxicity, and also implicate the possibility that allogeneic Tc17 cells may convert to and serve as reservoirs for the generation of Tc1 cells.

The role of STAT3 in T cell immunity is complex. STAT3 is a critical determinant of whether the naive T-cell differentiates into Treg cell or inflammatory Th17 cell lineage. Loss of STAT3 in donor CD4+ T cells prevents the development of sclerodermatous chronic graft-versus-host disease by enhancing CD4+CD25+Foxp3+ T-cell reconstitution65; However, STAT3 is also required for Treg cells to control Th17 responses.66 Our data that STAT3 inhibitor markedly reduces IL-17A expression and attenuates inflammatory cell infiltration and extracellular matrix deposition in the trachea allografts (Figure 5A) support the proinflammatory activities of STAT3; whereas inhibition of STAT3 also significantly increases IFNγ, perforin and granzyme B expression (Figures 5C, 6C), and fails to prevent epithelial injury of the trachea allografts or acute rejection of the lung allografts (Figures 5A, 6A), further demonstrating that STAT3 plays a complex role in inflammatory responses and its regulatory activities depend on specific cell type and immune microenvironment.

The differential effects of airway protection between RORγt deficiency and STAT3 inhibition (Figures 2, 5, 6) were observed in the trachea or lung allografts. One possible explanation is that, in addition to Th17 development and IL-17A production, RORγt is also involved in early T-cell development and CD8+ T effector function.67-69 Thus, absence of IL-17A expression couples with impaired CD8+ T effector function in RORγt-deficient allograft recipients result in the protection of airway epithelium after transplantation. Together, our results demonstrate that IL-17A plays critical roles in regulating allogeneic CD8+ T-cell response through affecting their migration, activation, and cytotoxicity, and indicate that IL-17A could serve as the key therapeutic target for airway epithelial protection and allograft survival after trachea and lung transplantations.

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