Halofuginone Treatment reduces interleukin-17A and ameliorates features of chronic lung allograft dysfunction in a mouse orthotopic lung transplant model
Hisashi Oishi, MD, PhD,a Tereza Martinu, MD,a Masaaki Sato, MD, PhD,a,b Yasushi Matsuda, MD, PhD,c Shin Hirayama, MD, PhD,d Stephen C. Juvet, MD, PhD,a Zehong Guan,a Tomohito Saito, MD, PhD,e Marcelo Cypel, MD, MSc,a David M. Hwang, MD, PhD,a Tracy L. Keller, PhD,f Malcolm R. Whitman, PhD,f Mingyao Liu, MD, MSc,a and Shaf Keshavjee, MD, MSca
Abstract
BACKGROUND: Increasing evidence suggests that interleukin (IL)-17A plays an important role in chronic lung allograft dysfunction (CLAD), characterized by airway and lung parenchymal fibrosis, after lung transplantation. Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation. The purpose of this study was to examine the effect of halofuginone on CLAD development using a minor alloantigen‒mismatched mouse orthotopic lung transplant model.
METHODS: C57BL/6 recipient mice received an orthotopic left lung transplant from C57BL/10 donors, mismatched for minor antigens. Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant.
RESULTS: Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 4.7% vs 52.5
13.8%, p o 0.01), as well as significantly reduced parenchymal fibrosis (5.5 2.3% vs 35.9 10.9%, p o 0.05). Immunofluorescent staining for IL-17A demonstrated a decreased number and frequency of IL-17A‒ positive cells in halofuginone-treated lung grafts on Day 28, as compared with controls. Halofuginone treatment also decreased IL-17A and IL-22 transcripts at Day 14, transforming growth factor-β1 and matrix metalloproteinase-2 transcripts at Days 14 and 28.
CONCLUSION: The beneficial effect of halofuginone on development of airway and lung parenchymal fibrosis in the mouse lung transplant model highlights the important role of IL-17A in CLAD and merits further preclinical and clinical studies.
KEYWORDS:
chronic lung allograft dysfunction; halofuginone; IL-17; obliterative bronchiolitis; restrictive allograft syndrome
Introduction
Lung transplantation is an effective therapy for patients chronic lung allograft dysfunction (CLAD).1 Clinically, CLAD may have either obstructive (bronchiolitis obliterans, OB) or restrictive (restrictive allograft syndrome, RAS) features, or a mixture of these phenotypes.2
Animal models are key to the advancement of our mechanistic understanding of the pathogenesis of CLAD.3 Recently, minor alloantigen‒mismatched orthotopic mouse single lung transplantation has been developed as a novel model of OB.4 Transplantation of C57BL/10 donor lungs to C57BL/6 recipients, without the use of immunosuppression, was reported to yield airway obliteration in approximately 50% of allografts 3 to 4 weeks after transplantation, in association with severe acute rejection.4 Moreover, neutralization of interleukin (IL)-17A and IL-17F reduced the incidence of OB lesions in this model.4 In a previous study we used this minor-mismatched mouse orthotopic lung transplant model to show that lentivirus-mediated IL-10 gene therapy attenuates acute rejection and reduces expression of T-helper 17 (Th17)-associated, genes such as IL-17A, IL-23 and retinoic acid–related orphan receptor γt.5
Recently, Th17 cells have been described as a new subset of Th cells, which derive from a CD4þ T-cell lineage and are characterized by the production of IL-17A as well as IL-17F, IL-21 and IL-22. Increasing evidence suggests that Th17 cells and IL-17A play important roles in chronic inflammation and destructive tissue damage after organ transplantation.6,7 As such, understanding the role of IL-17A and Th17 cells, as well as how Th17 cell differentiation is regulated during CLAD pathogenesis, may lead to novel strategies to prevent or treat CLAD after lung transplantation.
Halofuginone (HF) is a small molecule that has been shown to have anti-fibrotic and anti-Th17 activities. It is a racemic halogenated derivative of febrifugine, the active ingredient found in the roots of Dichroa febrifuga, fundamental herb of traditional Chinese medicine commonly used to treat malarial fevers. HF has been drawing attention because of its anti-fibrotic and anti-cancer effects in vivo.8–14 A Phase I clinical study of oral administration of HF was conducted in patients with advanced solid tumors.15 A Phase II clinical study of topically administered HF in patients with acquired immunodeficiency syndrome (AIDS)related Kaposi sarcoma showed a significant decrease in Type I collagen in HF-treated lesions.16 Furthermore, HF was reported to selectively inhibit the differentiation of Th17 cells both in vitro and in vivo,17 and to inhibit fibroblast activation.17,18
We hypothesized that inflammation and fibrosis are mediated by Th17 and IL-17A in the lung allograft and play an important role in OB development after lung transplantation. Herein we report that HF treatment in the minormismatched mouse orthotopic lung transplantation model decreases OB and lung parenchymal fibrosis and reduces the expression of Th17- and fibrosis-associated genes.
Methods
Animal procedures
Eight- to 12-week-old male C57BL/6J (B6) and C57B/10J (B10) mice (Jackson Laboratory, Bar Harbor, ME) were maintained on a standard diet and kept in a pathogen-free environment. B10 donor left lungs were transplanted orthotopically into B6 recipients (B10-B6) following the technique previously described by Okazaki et al.19 All animals received care in compliance with the Guide to the Care and Use of Experimental Animals, formulated by the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care Committee of the Toronto General Research Institute. Animals were killed on Days 7, 14 and 28. One quarter of each left lung graft was frozen in liquid nitrogen and preserved at –801C for RNA analysis. The rest of the lung allograft was inflated with 10% formalin, fixed, embedded in paraffin, and sectioned for pathologic assessment.
HF treatment
After lung transplantation, mice were randomly allocated to either the HF or control group. HF was reconstituted in dimethylsulfoxide (DMSO). Recipient mice in the HF group were injected intraperitoneally with 2.5 μg of HF dissolved in 300 μl of lactated Ringer’s solution (DMSO final concentration 0.1%) daily, beginning on the day of transplant. Recipient mice in the control group were injected with an equivalent concentration of DMSO in 300 μl of lactated Ringer’s solution.
Microcomputed tomography
Recipient mice were lightly anesthetized by isoflurane inhalation. Microcomputed tomography (micro-CT) was performed on Days 1, 14 and 28 on a GE micro-CT device (GE Locus Ultra Micro-CT; GE Healthcare, London, ON, Canada) to evaluate the whole lung. Resulting images were analyzed using MICROVIEW version 2.2 (GE Healthcare) software. Volumetry of the lung graft on CT images was performed by manual tracing of the lung graft boundary and summation of the lung area on each section using Image J version 1.47 (National Institutes of Health, Bethesda, MD). The percentage of total lung volume accounted for by the graft at each time-point was calculated.
Grading of rejection, airway obliteration and lung parenchymal fibrosis
After hematoxylin–eosin and Masson’s trichrome staining of paraffin-embedded lung tissue, acute rejection was graded by a pathologist (D.H.) based on the International Society for Heart and Lung Transplantation (ISHLT) criteria.20 Furthermore, for each lung sample, the number of obliterated and non-obliterated airways in the lung graft on histopathology was determined (by T.M.) in a blinded fashion with respect to treatment assignment. The percentage of obliterated airways was calculated. The percentage of the area of the lung section affected by parenchymal fibrosis was also estimated in a blinded manner.
Immunofluorescent staining for IL-17A and α-smooth muscle actin
Immunofluorescence labeling of IL-17A and α-smooth muscle actin (SMA) was conducted as described elsewhere (see Table S1 in the Supplementary Material, available online at www.jhltonline. org).21 Images were acquired using a spinning disk confocal system (WaveFX; Yokogawa Electric Corp., Japan). After staining for IL-17A, 10 high-power field images were randomly acquired from 3 sections per animal and the number of because of the severity of the fibrosis. IL-17A‒positive cells was counted in a blinded manner. The number of total cells in each image was counted automatically using Image J software.
Gene transcript analysis
Total RNA was extracted from lung tissue using the RNeasy Mini Kit (Qiagen, Inc., Mississauga, ON, Canada) and converted into cDNA (Transcriptor First Strand cDNA Synthesis Kit; Roche Applied Science, Indianapolis, IN). cDNA was amplified using SYBR Green I Master (Roche Applied Science) in a LightCycler 480 (Roche). All primers were designed across introns (see Table S2 in Supplementary Material online). Threshold cycle (Ct) values were determined using a real-time polymerase chain reaction system (LightCycler 480 RealTime PCR with LightCycler 480 software version 1.5). Change in expression was calculated using the 2–ΔΔCt method normalized to peptidylprolyl isomerase A expression and expressed as fold-change compared with the control group.
Statistical analysis
Data are expressed as mean SEM. A t-test was used to compare the means of 2 groups. A non-parametric Mann–Whitney U-test was performed for comparison of median acute rejection grades. For micro-CT lung volume assessment, repeated-measures 2-way analysis of variance (ANOVA) was performed. Linear regression was used to describe associations between the ratio of the number of IL-17A–positive cells to total cells and the percentage of obliterated airways or the percentage of parenchymal fibrosis. All statistical analyses were performed using PRISM 5 (GraphPad Software, Inc., La Jolla, CA). p o 0.05 was considered significant.
Results
HF prevents lung volume loss observed on micro-CT imaging
We tested the effects of HF therapy using the minormismatched B10-B6 mouse orthotopic lung transplant model. Micro-CT scans were performed on recipient mice on Days 1, 14 and 28 (Figure 1A–E). Lung grafts in the control group demonstrated volume reduction on Days 14 and 28 when compared with Day 1 (Figure 1A). Conversely, lung grafts in the HF-treated group maintained their volumes close to the Day 1 baseline (Figure 1B). Expressed as a percentage of total lung volume, in the control group, the lung graft volume was 27.4 1.5% on Day 1, 14.3 0.9% on Day 14 and 11.6 2.3% on Day 28. Lung grafts in the HF-treated group had significantly less lung volume loss over time compared with the control group, with lung graft volumes of 23.7 2.1% on Day 1, 25.9 1.1% on Day 14 and 23.3 2.2% on Day 28 (Figure 1G).
HF decreases airway obliteration and parenchymal fibrosis, without affecting acute rejection histology, on Day 28 post-transplant
We then evaluated the effects of HF therapy on development of airway and lung fibrosis in the minor-mismatched B10B6 mouse orthotopic lung transplant model. In the control animals, on Day 28, prominent airway obliteration with fibrous tissue was seen (Figure 2A and C). In addition, intense fibrosis in the alveolar parenchyma and thickening of the visceral pleura were observed (Figure 3A and C). In contrast, in the HF-treated mice, on Day 28, airway obliteration (Figure 2B and D) and parenchymal fibrosis (Figure 3B and D) were significantly reduced compared with the control group. Blinded semi-quantitative pathologic assessment confirmed that the percentage of obliterated airways was significantly reduced (Figure 2E) and that the extent of parenchymal fibrosis was significantly diminished in the HFtreated group compared with the control group (Figure 3E). In addition, independent blinded scoring according to the ISHLT criteria showed no difference in rejection A grades on Days 7, 14 and 28 post-operatively between the 2 groups (Figure 4).
HF decreases accumulation of α-SMA–positive myofibroblasts and expression of pro-fibrotic mediators in lung grafts
Myofibroblasts, characterized by α-SMA expression, are an activated differentiated form of fibroblasts and are thought to be key in the pathogenesis of fibrotic diseases such as CLAD.22 Immunofluorescence staining for α-SMA was performed on the lung grafts to test for the presence of myofibroblasts. In the control group, we observed numerous α-SMA‒positive cells in obliterative airway lesions and in the parenchymal areas (Figure 5A). In the HF group, there was a paucity of α-SMA‒positive myofibroblasts in the lungs (Figure 5B). Importantly, we were able to determine that these were not vascular smooth muscle cells because they were not found in regions delineated by expression of the endothelial marker CD31 (Figure 5C and D). The mRNA expression levels of fibrosis-related genes transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-2 (MMP-2) were significantly reduced in the HF group on Days 14 and 28 (Figure 5E and F).
HF reduces Th17-related cytokines in the transplanted lung
HF has previously been shown to function as a Th17 inhibitor. We therefore aimed to evaluate the effect of HF on Th17-derived cytokines IL-17A, IL-17F, IL-21 and IL-22. HF-treated lungs had significantly reduced gene expression of IL-17A and IL-22 on Day 14 (Figure 6A and D). The mRNA expression levels of IL-17F and IL-21 were not significantly different between the control and HF groups (Figure 6B and C).
HF reduces number of IL-17A–positive cells in lung grafts, which correlates with severity of airway obliteration and parenchymal fibrosis
As described earlier, HF decreases airway obliteration and parenchymal fibrosis. However, the control and HF groups showed similar lymphocyte infiltration around vessels of transplanted lungs on Day 28, as demonstrated by ISHLT acute rejection grading. The severity of fibrosis was thus not proportional to the level of overall inflammation. We therefore hypothesized that the composition of the cellular infiltrate may be important in development of fibrosis. In an attempt to estimate the contribution of Th17 cells within the acute rejection infiltrates, we performed immunofluorescence staining for IL-17A in lung grafts extracted on Day 28 (Figure 7A and B). Semi-quantification of IL-17A–positive cells demonstrated that the number of IL-17A–positive cells per 10 high-power fields in the HF groups was significantly lower compared with the control group (Figure 7C). This difference was not simply due to a higher cellularity as the total number of cells per 10 high-power fields was similar between the 2 groups (Figure 7D). In fact, the ratio of the number of IL-17A‒positive cells to total cells was significantly reduced in the HF group (Figure 7E). In addition, the ratio of the number of IL-17A–positive cells to total cells correlated positively with the percentage of obliterated airways and the extent of parenchymal fibrosis (Figure 7F and G).
Discussion
We have shown that HF treatment decreases the development of OB as well as parenchymal lung fibrosis in a minormismatched orthotopic mouse lung transplant model, along with an associated reduction in Th17-related cytokines.
The minor-mismatched orthotopic mouse lung transplant model from B10 to B6 mice was first reported as a model of OB with obliterative airway lesions found in about 50% of allografts at 3 to 4 weeks after transplantation.4 Consistent with this earlier report, our current study showed severe airway obliteration (480% airways obliterated) in 4 of 9 mice (44%) in the control group; additional mice had smaller numbers of OB lesions. This is similar to the most common pattern of human CLAD pathology, where OB lesions with relatively intact parenchyma are characteristic of bronchiolitis obliterans syndrome (BOS). Interestingly, we found that the mouse lung grafts also showed fibrosis in the parenchymal lung tissue, reminiscent of that described in RAS, a sub-type of CLAD characterized by restrictive lung physiology and diffuse lung fibrosis.2 The mouse lung graft fibrosis is further consistent with the reduced lung volume seen on CT imaging in the control group. Evaluation of CT lung volumetry allows longitudinal monitoring of dynamic changes in the same animal over time. This type of evaluation could facilitate drug discovery and evaluation in future studies without the need to kill the animals.
Immunofluorescent staining for IL-17A in the lung grafts extracted on Day 28 demonstrated numerous IL-17A– positive cells in the control group. HF dramatically reduced IL-17A–positive cells in this model and also decreased expression of IL-17A and IL-22 on Day 14. HF did not have a significant effect on IL-17F and IL-21, which are also produced by Th17 cells. The reason for this differential effect of HF on Th17 cytokine expression is unclear and we speculate that it may be explained by other cellular sources contributing to IL-17F and IL-21 cytokine production. The specific effect of HF on different subsets of IL-17‒ producing cells requires further investigation. Overall, our findings suggest that the Th17 pathway is inhibited in this model, consistent with earlier studies showing the potential of HF to block Th17.17,18 It has been shown that HF blocks Th17 by activating the amino acid starvation reaction17 through direct inhibition of the prolyl-tRNA synthetase activity of glutamyl-prolyl-tRNA synthetase (EPRS).18 It is likely that HF is acting on the same pathway in Th17 cells in our transplant model. The importance of Th17 in CLAD is further substantiated by clinical observations of elevated IL17A and Th17 differentiating/stimulating cytokines in the bronchoalveolar lavage from lung transplant recipients with BOS.23 In our study, HF attenuated both airway obliteration and parenchymal fibrosis, implying that IL-17A may contribute to several concurrent post-transplant fibroproliferative processes.
HF has also been shown to activate the amino acid starvation reaction in fibroblasts,18,24 which can lead to decreased differentiation from fibroblasts to myofibroblasts.25,26 α-SMA– positive myofibroblasts are key cells in the production of extracellular matrix in fibrosing diseases such as pulmonary fibrosis.27 In our previous study we demonstrated that modulating MMP led to regression of established fibrosis in a rat intrapulmonary tracheal transplant model.28 In the present study, immunofluorescent staining for α-SMA of the lung grafts extracted on Day 28 in the control group showed α-SMA– positive myofibroblasts in the airway obliterative lesions as well as in the parenchyma, resembling the pattern previously shown in human CLAD pathology.2 HF reduces numbers of α-SMA– positive myofibroblasts and diminishes expression of the important fibroblast-related genes MMP-2 and TGF-β1, which have been previously identified as potential mediators of CLAD.28 Thus, HF may prevent CLAD development through both direct and indirect effects on fibroblast activation and matrix deposition. Although this study has shown that HF may represent a novel potential treatment option for CLAD, this work only assessed the efficacy of HF when treatment was initiated at the time of transplantation. It remains to be clarified, however, whether HF can have beneficial effects on already established OB and parenchymal fibrosis.
HF activation of the amino acid starvation response yields therapeutic benefit in multiple cell types. HF’s cellular target, EPRS, is expressed ubiquitously, likely accounting for the therapeutic effects of HF seen in an array of tissues, including endothelium, epithelium and vascular smooth muscle,10,18,29–31 in addition to fibroblasts and effector T cells. The distinctive therapeutic benefit of HF in OB likely derives from its ability to limit tissue damage in multiple cell types that participate in the fibrotic process. Investigation of the relative contribution of HF action in the different cell types involved in airway remodeling during OB is an important area for future investigation.
In conclusion, we have demonstrated that the beneficial effect of halofuginone on development of airway and parenchymal lung fibrosis in the mouse lung transplant model occurs in association with down-regulation of IL-17A and reduced fibroblast activation. HF represents a novel promising strategy for the prevention and treatment of CLAD and warrants further consideration and detailed study.
References
1. Yusen RD, Christie JD, Edwards LB, et al. The registry of the International Society for Heart and Lung Transplantation: thirtieth adult lung and heart‒lung transplant report—2013; focus theme: age. J Heart Lung Transplant 2013;32:965-78.
2. Sato M, Waddell TK, Wagnetz U, et al. Restrictive allograft syndrome (RAS): a novel form of chronic lung allograft dysfunction. J Heart Lung Transplant 2011;30:735-42.
3. Sato M, Keshavjee S, Liu M. Translational research: animal models of obliterative bronchiolitis after lung transplantation. Am J Transplant 2009;9:1981-7.
4. Fan L, Benson HL, Vittal R, et al. Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation. Am J Transplant 2011;11:911-22.
5. Hirayama S, Sato M, Loisel-Meyer S, et al. Lentivirus IL-10 gene therapy down-regulates IL-17 and attenuates mouse orthotopic lung allograft rejection. Am J Transplant 2013;13:1586-93.
6. Abadja F, Sarraj B, Ansari MJ. Significance of T helper 17 immunity in transplantation. Curr Opin Organ Transplant 2012;17:8-14.
7. Shilling RA, Wilkes DS. Role of Th17 cells and IL-17 in lung transplant rejection. Semin Immunopathol 2011;33:129-34.
8. Nagler A, Katz A, Aingorn H, et al. Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone. Kidney Int 1997;52:1561-9.
9. Pines M, Nagler A. Halofuginone: a novel antifibrotic therapy. Gen Pharmacol 1998;30:445-50.
10. Elkin M, Ariel I, Miao HQ, et al. Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone. Cancer Res 1999;59:4111-8.
11. Bruck R, Genina O, Aeed H, et al. Halofuginone to prevent and treat thioacetamide-induced liver fibrosis in rats. Hepatology 2001;33: 379-86.
12. McGaha T, Kodera T, Phelps R, et al. Effect of halofuginone on the development of tight skin (TSK) syndrome. Autoimmunity 2002;35: 277-82.
13. Pines M, Snyder D, Yarkoni S, et al. Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma. Biol Blood Marrow Transplant 2003;9:417-25.
14. Xavier S, Piek E, Fujii M, et al. Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone. J Biol Chem 2004;279:15167-76.
15. de Jonge MJ, Dumez H, Verweij J, et al. Phase I and pharmacokinetic study of halofuginone, an oral quinazolinone derivative in patients with advanced solid tumours. Eur J Cancer 2006;42:1768-74.
16. Koon HB, Fingleton B, Lee JY, et al. Phase II AIDS Malignancy Consortium trial of topical halofuginone in AIDS-related Kaposi sarcoma. J Acquir Immune Defic Syndr 2011;56:64-8.
17. Sundrud MS, Koralov SB, Feuerer M, et al. Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response. Science 2009;324:1334-8.
18. Keller TL, Zocco D, Sundrud MS, et al. Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol 2012;8:311-7.
19. Okazaki M, Krupnick AS, Kornfeld CG, et al. A mouse model of orthotopic vascularized aerated lung transplantation. Am J Transplant
20. Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26:1229-42. 27.
21. Sato M, Liu M, Anraku M, et al. Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling. Am J Transplant
22. Ramirez AM, Shen Z, Ritzenthaler JD, et al. Myofibroblast transdifferentiation in obliterative bronchiolitis: TGF-β signaling through Smad3-dependent and -independent pathways. Am J Transplant 2006;
23. Vanaudenaerde BM, De Vleeschauwer SI, Vos R, et al. The role of the IL23/IL17 axis in bronchiolitis obliterans syndrome after lung trans-
24. Kamberov YG, Kim J, Mazitschek R, et al. Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells. BMC Res Notes 2011;4:381. 31.
25. Sheffer Y, Leon O, Pinthus JH, et al. Inhibition of fibroblast to myofibroblast transition by halofuginone contributes to the chemotherapy-mediated antitumoral effect. Mol Cancer Ther 2007;6: 570-7.