© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Clin Transplant 2015: 29: 547–554 DOI: 10.1111/ctr.12551
Clinical Transplantation
Role of Toll-like receptor 4 signaling in cutaneous chronic graft-versus-host disease Weng J, Lai P, Geng S, Luo C, Wu S, Ling W, Deng C, Huang X, Lu Z, Du X. Role of Toll-like receptor 4 signaling in cutaneous chronic graft-versus-host disease. Abstract: Cutaneous damage is one of the characterized manifestations in chronic graft-versus-host disease (cGVHD). When local effective immunity in the skin is altered to a dysimmune reaction, cutaneous injuries occur. Toll-like receptor 4 signaling is regarded as a central mediator of inflammation and organ injury. In this study, we found that TLR4 mRNA in peripheral blood from patients with cutaneous cGVHD was markedly increased compared with that from non-GVHD patients and healthy controls. In addition, NF-jB expression, TLR4 downstream signaling, and TLR4-mediated cytokines, including IL-6 and ICAM-1, were upregulated. Moreover, ICAM-1 was widely distributed in skin biopsies from patients with cutaneous cGVHD. We also found that LPS induced TLR4-mediated NF-jB activation and IL-6 and ICAM-1 secretion in human fibroblasts in vitro. Thus, TLR4, NF-jB, IL-6, and ICAM-1 contribute to the inflammatory response that occurs in cutaneous cGVHD, indicating the TLR4 pathway may be a novel target for cutaneous cGVHD therapy.
Jianyu Weng*, Peilong Lai*, Suxia Geng, Chenwei Luo, Suijing Wu, Wei Ling, Chengxin Deng, Xin Huang, Zesheng Lu and Xin Du Department of Hematology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Key words: chronic graft-versus-host disease – cutaneous graft-versus-host disease – IL-6 – intercellular adhesion molecule-1 – Toll-like receptor 4 Corresponding author: Xin Du, MD, PhD, 106. Zhongshan Er Road, Guangzhou 510080, China. Tel.: (86) 20 83827812 62122; fax: (86) 20 83889772; e-mail: [email protected] *These authors contributed equally to this work. Conflict of interest: The authors have no conflict of interests to disclose. Accepted for publication 30 March 2015
Chronic graft-versus-host disease (cGVHD) is a serious and increasingly common long-term complication of allogeneic hematopoietic stem cell transplantation (HSCT) (1). The most frequent and prominent manifestation of cGVHD, which is a multisystem disease, is debilitating fibrosing skin, which occurs in approximately 90–100% of patients (2). Clinical presentations of cutaneous cGVHD considerably vary and include pruritus, pain, dryness, burning, lichenoid, and erythematous, fibrotic, or sclerodermatous lesions, which arise as crucial complications affecting long-term survivors (3, 4). The pathophysiology of cutaneous cGVHD involves autoreactive lymphocytes and cytokine dysregulation (5). However, the exact mechanism involved in cutaneous chronic GVHD has not been clearly elucidated. Previous work has shown that the initiation and progression of chronic GVHD involves impaired cytokine cascades and autoreactive T cells homing to and destroying target organs (6, 7), including
skin, which is the most accessible and susceptible organ. Local cutaneous effective immunity transforms into a dysimmune reaction, permitting the fibrotic process. Similar to the fibrosis involved in systemic sclerosis in which a crucial inducible costimulator of T-cell proliferation and responses increases (8), patients with GVHD present with T cell-mediated cutaneous fibrotic damage. Neutralization of inflammatory cytokines, such as IL-6, TNF-a, IL-1, and IL-12 (9), has been reported to effectively treat an animal GVHD model. In addition, in vitro differentiated IL-17- and IL-22-producing Th17 cells induced severe cutaneous pathologic manifestations in acute GVHD mice. However, the inflammatory pathway mediated in cutaneous chronic GVHD remains unclear. Toll-like receptors (TLRs) have been established to play a fundamental role in the activation of innate immunity against microbial pathogens and subsequent induction of the adaptive immune response (10). It has become increasingly apparent
547
Weng et al.
that TLRs enhance the induction of GVHD reactions by amplifying a cytokine storm, increasing the amount of pro-inflammatory cytokines such as IL-1, TNF-a, and IL-6, which are also important for systemic sclerosis (11). Polymorphisms in TLR4 have been related to susceptibility to acute GVHD (12, 13), Behcet’s disease (14), and renal scarring (15). In addition, the TLR4-mediated loss in intestinal homeostasis accelerates GVHD severity (16). In this study, we aimed to determine whether TLR4 signaling contributes to inflammation in patients with cutaneous chronic GVHD. Considering the pathogenic role that TLR4 plays in autoimmune/chronic inflammatory diseases, we were interested in determining whether TLR4-mediated inflammation is important for the pathogenesis of cutaneous cGVHD. Here, using patient samples, we demonstrate that activation of TLR4-mediated NF-jB signaling leads to significant cutaneous manifestations in chronic GVHD. We also show that ICAM-1 expression increases in patients with cutaneous cGVHD. Thus, TLR4 pathway activation is possibly required for the development of cutaneous cGVHD.
Materials and methods Patient characteristics
As shown in Table 1, the study population included the following: 15 patients who met the criteria for establishing a diagnosis for cutaneous cGVHD (mean age: 28 yr old), 15 patients without GVHD after HSCT (mean age, 30 yr old), and 20 healthy controls (mean age, 27 yr old). Patients who received HSCT present with primary diseases such as acute lymphoblastic leukemia, chronic myelogenous leukemia, and acute myeloid leukemia in this study. The cGVHD diagnoses were based on National Institutes of Health (NIH) conTable 1. Baseline subject characteristics
Males:females Age Primary disease
HLA matched: 10 alleles matched 1–2 alleles mismatched
Cutaneous cGVHD
Non-GVHD
10:5 28.13 11.37 ALL: 2 CML: 4 AML: 9
9:6 29.73 6.81 ALL: 2 CML: 7 AML: 6
12 3
14 1
Healthy control 13:7 26.8 3.91
cGVHD, chronic GVHD; F, female; M, male; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; AML, acute myeloid leukemia.
548
sensus diagnostic criteria, which emphasize manifestations and not the time of onset (17). Patients with cGVHD manifesting with cutaneous damage were diagnosed with cutaneous cGVHD (Table 2), and other etiologies of skin eruptions were clinically excluded. Patients without GVHD after HSCT were regarded as non-GVHD controls, and healthy donors were considered as negative controls. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and our protocol was approved by the Ethics Committee of Guangdong General Hospital. Skin histology
Cutaneous biopsies (approximately 0.2 cm2) were performed from the clinically most representative area of skin from patients with cutaneous cGVHD who agreed to participate in the study and provided informed consent. For control non-cGVHD patients, we suggested that patients with clinically suspected cutaneous cGVHD have their skin biopsies tested, and only those who agreed and provided consent underwent skin biopsies. Skin samples were fixed in 4% formalin, embedded in paraffin, sectioned, slide-mounted, and stained with hematoxylin and eosin (H&E). Immunohistochemistry for ICAM-1 was performed according to standard protocols. Briefly, the skin tissues were fixed in 10% PFA, dehydrated in EtOH, and embedded in paraffin. Radial 4-lm sections were stored at 4°C, or histologic staining with hematoxylin and eosin was performed. For ICAM-1 staining, sections were dewaxed three times in xylene, rehydrated with a graded series of ethanol, and then heated with boiling water for 15 min for antigen retrieval. After cooling down, the sections were placed in 3% hydrogen peroxide for 10 min and then rinsed in distilled water. Donkey serum was added as a blocking agent, and the slides were stained with an antibody against ICAM-1 (Abcam) at a 1/100 dilution overnight. Biotinylated anti-rabbit IgG was then used and amplified with streptavidin-biotinylated horseradish peroxidase complexes. Signals were developed for visualization using 3,3’-diaminobenzidine (DAB) (Sigma Chemical Co.St. Louis, MO, USA), counterstained with hematoxylin, and then mounted with Depex (Serva, Heidelberg, Germany). TLR4, NF-jB, IL-6, and ICAM-1 analysis by real-time PCR
Quantitative real-time PCR analysis was performed to detect the TLR4, NF-jB, IL-6, and ICAM-1 mRNA levels in peripheral blood mononuclear cells (PBMCs). PBMCs were separated from ethylenedia-
TLR4 signaling in cutaneous cGVHD mine tetraacetic acid anticoagulated peripheral blood using the Ficoll-Hypaque gradient centrifugation method. Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) and converted into first-strand cDNA using random hexamer primers and the reverse transcriptase Superscript II Kit (Invitrogen) according to the manufacturer’s instructions. PCR was then performed in a total volume of 20 lL containing 2 lL cDNA, 10 lL 29 SYBR Premix Ex Taq, 0.8 lL 509 ROX Reference Dye (TaKaRa Biotechnology Co., Ltd, Dalian, China), and 10 lmol/L of the primer pairs. The primers are listed in Table S1. b-actin was used as a reference gene. The PCR amplification protocols consisted of 95°C for 30 s and up to 40 cycles of 95°C for 5 s and 60°C for 34 s according to the manufacturer’s instructions. The 2 DDCt method was used to normalize the expression of the genes of interest relative to an internal control gene. Cytokine analysis by ELISA
Venous blood samples (2–4 mL) and supernatants from human fibroblast cells in vitro were collected, centrifuged for 15 min, and stored at 80°C. IL-6, a classic proinflammatory cytokine, was measured by ELISA (eBioscience, San Diego, CA, USA) according to the standard protocol provided by the manufacturer. The minimum detection level was 0.92 pg/mL for IL-6. When the calculated cytokine concentration was below this sensitivity, it was considered undetectable.
Statistical analysis
Statistical analysis was performed using SPSS 13.0 software (Chicago, IL, USA). The independent samples t-test was used to test the probability of significant differences between cutaneous cGVHD and control samples. Bivariate correlation analysis was performed to evaluate changes in TLR4 relative to downstream components in the pathway, and the correlation coefficient r was measured to determine the strength and direction of the linear relationship between individual components in the TLR4 pathway. These data are from the same patients before and after the onset of cutaneous cGVHD. p-Values
Clin Transplant 2015: 29: 547–554 DOI: 10.1111/ctr.12551
Clinical Transplantation
Role of Toll-like receptor 4 signaling in cutaneous chronic graft-versus-host disease Weng J, Lai P, Geng S, Luo C, Wu S, Ling W, Deng C, Huang X, Lu Z, Du X. Role of Toll-like receptor 4 signaling in cutaneous chronic graft-versus-host disease. Abstract: Cutaneous damage is one of the characterized manifestations in chronic graft-versus-host disease (cGVHD). When local effective immunity in the skin is altered to a dysimmune reaction, cutaneous injuries occur. Toll-like receptor 4 signaling is regarded as a central mediator of inflammation and organ injury. In this study, we found that TLR4 mRNA in peripheral blood from patients with cutaneous cGVHD was markedly increased compared with that from non-GVHD patients and healthy controls. In addition, NF-jB expression, TLR4 downstream signaling, and TLR4-mediated cytokines, including IL-6 and ICAM-1, were upregulated. Moreover, ICAM-1 was widely distributed in skin biopsies from patients with cutaneous cGVHD. We also found that LPS induced TLR4-mediated NF-jB activation and IL-6 and ICAM-1 secretion in human fibroblasts in vitro. Thus, TLR4, NF-jB, IL-6, and ICAM-1 contribute to the inflammatory response that occurs in cutaneous cGVHD, indicating the TLR4 pathway may be a novel target for cutaneous cGVHD therapy.
Jianyu Weng*, Peilong Lai*, Suxia Geng, Chenwei Luo, Suijing Wu, Wei Ling, Chengxin Deng, Xin Huang, Zesheng Lu and Xin Du Department of Hematology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Key words: chronic graft-versus-host disease – cutaneous graft-versus-host disease – IL-6 – intercellular adhesion molecule-1 – Toll-like receptor 4 Corresponding author: Xin Du, MD, PhD, 106. Zhongshan Er Road, Guangzhou 510080, China. Tel.: (86) 20 83827812 62122; fax: (86) 20 83889772; e-mail: [email protected] *These authors contributed equally to this work. Conflict of interest: The authors have no conflict of interests to disclose. Accepted for publication 30 March 2015
Chronic graft-versus-host disease (cGVHD) is a serious and increasingly common long-term complication of allogeneic hematopoietic stem cell transplantation (HSCT) (1). The most frequent and prominent manifestation of cGVHD, which is a multisystem disease, is debilitating fibrosing skin, which occurs in approximately 90–100% of patients (2). Clinical presentations of cutaneous cGVHD considerably vary and include pruritus, pain, dryness, burning, lichenoid, and erythematous, fibrotic, or sclerodermatous lesions, which arise as crucial complications affecting long-term survivors (3, 4). The pathophysiology of cutaneous cGVHD involves autoreactive lymphocytes and cytokine dysregulation (5). However, the exact mechanism involved in cutaneous chronic GVHD has not been clearly elucidated. Previous work has shown that the initiation and progression of chronic GVHD involves impaired cytokine cascades and autoreactive T cells homing to and destroying target organs (6, 7), including
skin, which is the most accessible and susceptible organ. Local cutaneous effective immunity transforms into a dysimmune reaction, permitting the fibrotic process. Similar to the fibrosis involved in systemic sclerosis in which a crucial inducible costimulator of T-cell proliferation and responses increases (8), patients with GVHD present with T cell-mediated cutaneous fibrotic damage. Neutralization of inflammatory cytokines, such as IL-6, TNF-a, IL-1, and IL-12 (9), has been reported to effectively treat an animal GVHD model. In addition, in vitro differentiated IL-17- and IL-22-producing Th17 cells induced severe cutaneous pathologic manifestations in acute GVHD mice. However, the inflammatory pathway mediated in cutaneous chronic GVHD remains unclear. Toll-like receptors (TLRs) have been established to play a fundamental role in the activation of innate immunity against microbial pathogens and subsequent induction of the adaptive immune response (10). It has become increasingly apparent
547
Weng et al.
that TLRs enhance the induction of GVHD reactions by amplifying a cytokine storm, increasing the amount of pro-inflammatory cytokines such as IL-1, TNF-a, and IL-6, which are also important for systemic sclerosis (11). Polymorphisms in TLR4 have been related to susceptibility to acute GVHD (12, 13), Behcet’s disease (14), and renal scarring (15). In addition, the TLR4-mediated loss in intestinal homeostasis accelerates GVHD severity (16). In this study, we aimed to determine whether TLR4 signaling contributes to inflammation in patients with cutaneous chronic GVHD. Considering the pathogenic role that TLR4 plays in autoimmune/chronic inflammatory diseases, we were interested in determining whether TLR4-mediated inflammation is important for the pathogenesis of cutaneous cGVHD. Here, using patient samples, we demonstrate that activation of TLR4-mediated NF-jB signaling leads to significant cutaneous manifestations in chronic GVHD. We also show that ICAM-1 expression increases in patients with cutaneous cGVHD. Thus, TLR4 pathway activation is possibly required for the development of cutaneous cGVHD.
Materials and methods Patient characteristics
As shown in Table 1, the study population included the following: 15 patients who met the criteria for establishing a diagnosis for cutaneous cGVHD (mean age: 28 yr old), 15 patients without GVHD after HSCT (mean age, 30 yr old), and 20 healthy controls (mean age, 27 yr old). Patients who received HSCT present with primary diseases such as acute lymphoblastic leukemia, chronic myelogenous leukemia, and acute myeloid leukemia in this study. The cGVHD diagnoses were based on National Institutes of Health (NIH) conTable 1. Baseline subject characteristics
Males:females Age Primary disease
HLA matched: 10 alleles matched 1–2 alleles mismatched
Cutaneous cGVHD
Non-GVHD
10:5 28.13 11.37 ALL: 2 CML: 4 AML: 9
9:6 29.73 6.81 ALL: 2 CML: 7 AML: 6
12 3
14 1
Healthy control 13:7 26.8 3.91
cGVHD, chronic GVHD; F, female; M, male; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; AML, acute myeloid leukemia.
548
sensus diagnostic criteria, which emphasize manifestations and not the time of onset (17). Patients with cGVHD manifesting with cutaneous damage were diagnosed with cutaneous cGVHD (Table 2), and other etiologies of skin eruptions were clinically excluded. Patients without GVHD after HSCT were regarded as non-GVHD controls, and healthy donors were considered as negative controls. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and our protocol was approved by the Ethics Committee of Guangdong General Hospital. Skin histology
Cutaneous biopsies (approximately 0.2 cm2) were performed from the clinically most representative area of skin from patients with cutaneous cGVHD who agreed to participate in the study and provided informed consent. For control non-cGVHD patients, we suggested that patients with clinically suspected cutaneous cGVHD have their skin biopsies tested, and only those who agreed and provided consent underwent skin biopsies. Skin samples were fixed in 4% formalin, embedded in paraffin, sectioned, slide-mounted, and stained with hematoxylin and eosin (H&E). Immunohistochemistry for ICAM-1 was performed according to standard protocols. Briefly, the skin tissues were fixed in 10% PFA, dehydrated in EtOH, and embedded in paraffin. Radial 4-lm sections were stored at 4°C, or histologic staining with hematoxylin and eosin was performed. For ICAM-1 staining, sections were dewaxed three times in xylene, rehydrated with a graded series of ethanol, and then heated with boiling water for 15 min for antigen retrieval. After cooling down, the sections were placed in 3% hydrogen peroxide for 10 min and then rinsed in distilled water. Donkey serum was added as a blocking agent, and the slides were stained with an antibody against ICAM-1 (Abcam) at a 1/100 dilution overnight. Biotinylated anti-rabbit IgG was then used and amplified with streptavidin-biotinylated horseradish peroxidase complexes. Signals were developed for visualization using 3,3’-diaminobenzidine (DAB) (Sigma Chemical Co.St. Louis, MO, USA), counterstained with hematoxylin, and then mounted with Depex (Serva, Heidelberg, Germany). TLR4, NF-jB, IL-6, and ICAM-1 analysis by real-time PCR
Quantitative real-time PCR analysis was performed to detect the TLR4, NF-jB, IL-6, and ICAM-1 mRNA levels in peripheral blood mononuclear cells (PBMCs). PBMCs were separated from ethylenedia-
TLR4 signaling in cutaneous cGVHD mine tetraacetic acid anticoagulated peripheral blood using the Ficoll-Hypaque gradient centrifugation method. Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) and converted into first-strand cDNA using random hexamer primers and the reverse transcriptase Superscript II Kit (Invitrogen) according to the manufacturer’s instructions. PCR was then performed in a total volume of 20 lL containing 2 lL cDNA, 10 lL 29 SYBR Premix Ex Taq, 0.8 lL 509 ROX Reference Dye (TaKaRa Biotechnology Co., Ltd, Dalian, China), and 10 lmol/L of the primer pairs. The primers are listed in Table S1. b-actin was used as a reference gene. The PCR amplification protocols consisted of 95°C for 30 s and up to 40 cycles of 95°C for 5 s and 60°C for 34 s according to the manufacturer’s instructions. The 2 DDCt method was used to normalize the expression of the genes of interest relative to an internal control gene. Cytokine analysis by ELISA
Venous blood samples (2–4 mL) and supernatants from human fibroblast cells in vitro were collected, centrifuged for 15 min, and stored at 80°C. IL-6, a classic proinflammatory cytokine, was measured by ELISA (eBioscience, San Diego, CA, USA) according to the standard protocol provided by the manufacturer. The minimum detection level was 0.92 pg/mL for IL-6. When the calculated cytokine concentration was below this sensitivity, it was considered undetectable.
Statistical analysis
Statistical analysis was performed using SPSS 13.0 software (Chicago, IL, USA). The independent samples t-test was used to test the probability of significant differences between cutaneous cGVHD and control samples. Bivariate correlation analysis was performed to evaluate changes in TLR4 relative to downstream components in the pathway, and the correlation coefficient r was measured to determine the strength and direction of the linear relationship between individual components in the TLR4 pathway. These data are from the same patients before and after the onset of cutaneous cGVHD. p-Values
