• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of clinexpimmunolLink to Publisher's site
Clin Exp Immunol. Oct 2010; 162(1): 131–137.
PMCID: PMC2990938

The T helper type 17/regulatory T cell imbalance in patients with acute Kawasaki disease

Abstract

The study is designed to investigate the changes and roles of T helper type 17/regulatory T cells (Th17/Treg) in the immunological pathogenesis of Kawasaki disease (KD). In addition, we explore the alteration and significance of Th17 cells in patients with intravenous immune globulin-resistant KD. Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA levels of interleukin (IL)-17A/F, retinoic acid-related orphan receptor (ROR)-γt and forkhead box P3 (FoxP3) in CD4-positive cells. The proportions of Th17 cells and CD4+CD25+FoxP3high Tregs were analysed by flow cytometry. Plasma cytokine [IL-17A, IL-6, IL-23 and transforming growth factor (TGF)-β] concentrations were measured by sandwich enzyme-linked immunosorbent assay. Our data demonstrate that Th17 proportions and expression levels of cytokines (IL-17, IL-6 and IL-23) and transcription factors (IL-17A/F, ROR-γt) were up-regulated significantly, while Treg proportions and expression levels of Tregtranscription factor (FoxP3) were down-regulated significantly in children with acute KD (P < 0·01). Compared with the sensitive group, the Th17 proportions were up-regulated significantly during the acute phase in immune globulin-resistant KD (P < 0·01). The plasma IL-17A, IL-6 and IL-23 concentrations in patients with KD were significantly higher compared with the concentrations in normal controls (NC) and infectious disease (ID). Plasma TGF-β concentrations were markedly lower in the KD group than the NC and ID groups (P < 0·05). These results suggest that Th17/Treg cells imbalance exists in the patients with KD. Th17/T cells imbalance may be important factors causing disturbed immunological function and resulting in immunoglobulin-resistant KD.

Keywords: immunoglobulin, interleukin-6, -17, -23, Kawasaki disease, Th17 cells, Treg cells

Introduction

Kawasaki disease (KD) is an acute vasculitis that affects infants and children, and is the leading cause of acquired heart disease in the paediatric age group. The immunopathogenesis of KD needs to be investigated. A great many studies have found that the levels of many proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-6 are elevated in acute KD, but the mechanisms resulting in aberrant immune function or over-expression of proinflammatory cytokines are not clear [15].

Recently, CD4+CD25+ regulatory T cells (Treg) and T helper type 17 (Th17) cells have been described as two distinct subsets from Th1 and Th2 cells. Treg cells expressing the forkhead/winged helix transcription factor P3 (FoxP3) have an anti-inflammatory role and maintain tolerance to self-components by contact-dependent suppression or releasing anti-inflammatory cytokines [IL-10 and transforming growth factor (TGF)-β1][6], while Th17 cells expressing retinoic acid-related orphan receptor γt (ROR-γt) play critical roles in the development of autoimmunity and allergic reactions by producing IL-17 [7]. IL-17A and IL-17F produced by Th17 cells have proinflammatory properties and act on a broad range of cell types to induce the expression of cytokines [IL-6, TNF-α, IL-8, granulocyte–macrophage colony-stimulating factor (GM-CSF)], chemokines (cxcl1, cxcl10) and metalloproteinases, thus perpetuating inflammation of the tissues [8,9]. As there are few data available on Th17 Treg cells in KD, we investigate the frequency of Th17 and Treg cells in peripheral blood and the cytokines affecting Th17 and Treg differentiation in plasma to evaluate whether the Th17/Treg balance was disrupted in patients with KD.

Materials and methods

Forty-five children with acute febrile stage of KD (28 males and 17 females; mean age: 25·2 ± 14·4 months; age range: 8 months–4·7 years), age-matched control subjects including 18 patients with active infections disease (ID) (six with measles, seven with influenza, one with rotavirus, two with adenovirus and two with Epstein–Barr virus) (10 males and eight females; mean age: 15·8 ± 13·1 months; age range: 8 months–3 years) and 20 age-matched normal controls (NC) (11 males and nine females; mean age: 24·0 ± 14·4 months; age range: 1–4·5 years) were enrolled into this study (Table 1). The patients described above comprise the sensitive group who received intravenous immunoglobulin (IVIG) therapy. In addition, we selected 10 children (six males and four females; mean age: 15·0 ± 12·8 months; age range: 5 months–2·5 years) who were IVIG-resistant KD (patients were defined as resistant if their fever continued > 24 h after IVIG, or rose again within 48 h) to evaluate the effect of Th17 cells on IVIG treatment response. Informed consent was obtained from their parents and the study was approved by the medical ethics hospital committee. The diagnosis was made according to the clinical criteria of the Kawasaki Disease Research Committee of Japan. Blood samples from patients with KD before and 10 days after IVIG therapy were collected. Blood samples were analysed immediately without stimulation of mitogens or culture in vitro unless particularly indicated. All patients with KD received two-dimensional echocardiographic examination. Coronary artery lesion (CAL) was defined by internal diameter of artery > 3·0 mm (< 5 years); > 4·0 mm (≥ 5 years) or coronary artery aneurysms. Patients with KD were divided into the KD-CAL+ group (15) and the KD-CAL- group (30) according to the echocardiographic examination results (Tables 2 and and33).

Table 1
Characteristics of patients with Kawasaki disease (KD).
Table 2
Clinical data of patients with Kawasaki disease–coronary artery lesion (KD-CAL+) and KD-CAL-.
Table 3
Clinical data of patients with intravenous immunoglobulin (IVIG)-sensitive and IVIG-resistant Kawasaki disease.

Blood samples

Venous blood (5 ml) was taken from patients with KD, normal controls and patients with ID using ethylenediamine tetraacetic acid (EDTA) Na2 as anti-coagulant. Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient for flow cytometric analysis. Plasma was obtained after centrifugation and stored at −80°C for measurement of the enzyme-linked immunosorbent assay (ELISA). CD4+ T cells were isolated immediately from peripheral blood of the children participating in the study, according to the manufacturer's instructions for microbeads (111·49D; Dynal, Oslo, Norway). Purified cells were identified as > 97% with flow cytometry (FCM) (Fig. 1), while results of cell activity were > 95% by 0·05% trypan blue staining.

Fig. 1
Cell purity detected with flow cytometry after isolation with magnetic affinity cell sorting.

Total RNA extraction and cDNA synthesis

Total RNA from CD4+ T cells was prepared using the Versagene RNA Kit (0050C; Gentra, Minneapolis, MN, USA), according to the manufacturer's instructions. DNase I (0050D; Gentra) was used to eliminate the trace DNA during extraction. Isolated total RNA integrity was verified by an average optical density (OD) OD260/OD286 absorption to cDNA with oligodeoxythymidylic acid (oligo-dT) primer, using RevertAid™ H Minus Moloney murine leukaemia virus (MMLV) reverse transcriptase (K1632#; Fermentas, Vilnius, Lithuania). Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription reaction in the absence of reverse transcriptase.

LightCycler real-time polymerase chain reaction (PCR)

The cDNA levels of IL-17A/F, ROR-γt and FoxP3 and other genes were quantitated by real-time PCR using Quantitect™ SYBR green PCR Kit (204143; Qiagen, Hilden, Germany) and LightCycler II (Roche, Mannhein, Germany) according to the manufacturer's maximum method was performed for CP (cross point) determination using LightCycler Software version 3·5 (Roche Molecular Biochemicals, Mannhein, Germany). After normalization with Relative Quantification Software 1·0 (Roche Molecular Biochemicals), cDNA levels were identified as: target genes/β-actin (shown in Table 4).

Table 4
Primer for real-time polymerase chain reaction.

Flow cytometric analysis of Th17 and Treg

For analysis of Th17 and Treg, PBMCs were suspended at a density of 2 × 106 cells/ml into tubes and washed once in phosphate-buffered saline (PBS). For Th17 analysis, the cells were incubated with phycoerythrin (PE) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treganalysis, the cells were incubated with peridinin chlorophyll (PerCP) anti-human CD4 and fluorescein isothiocyanate (FITC) anti-human CD25. After surface staining, the cells were stained with FITC anti-human IL-17A for Th17 detection or PE anti-human FoxP3 for Treg detection after fixation and permeabilization according to the manufacturer's instructions. Isotype controls were given to enable correct compensation and confirm antibody specificity. All the antibodies were from eBioscience. Stained cells were analysed by flow cytometric analysis using an Epics-XL4 cytometer equipped with EXPO32 ADC software (Beckman Coulter, San Diego, CA, USA).

ELISA detection of plasma IL-17, IL-6, IL-23 and TGF-β

The plasma levels of IL-17, IL-6, IL-23 and TGF-β were measured by ELISA using an ELX-800 microplate reader (Biotek Corporation, Winooski, VT, USA), following the manufacturer's instructions (eBioscience). All samples were measured in duplicate.

Statistical analysis

The software used for statistical analysis was spss for Windows version 13·0 (SPSS, Chicago, IL, USA). Data are presented as mean ± standard deviation (s.d.). Differences between the values were determined using Student's t-test. A value of P < 0·05 was regarded as a significant difference.

Results

Circulating Th17 and Treg frequencies

As shown in Fig. 2, compared with healthy controls the Th17 cells proportions were up-regulated significantly in the ID group (1·53 ± 0·40% versus 0·28 ± 0·15%, P < 0·01). The frequencies of Th17 (CD4+IL-17+/CD4+ T cells) were markedly higher in patients with KD than those in the ID group (2·72 ± 0·71% versus 1·53 ± 0·40%, P < 0·01) and the proportions of Th17 cells had a tendency to decrease after treatment with IVIG (2·72 ± 0·71% versus 1·38 ± 0·40%, P < 0·01). It was found that Th17 proportions in the KD-CAL+ group were markedly higher than those in the KD-CAL- group (2·81 ± 0·74% versus 2·06 ± 0·53%, P < 0·01).

Fig. 2
The proportion of regulatory T cells (Treg) and T helper type 17 (Th17) cells in patients with Kawasaki disease.

The frequencies of Treg (CD4+CD25+FoxP3high/CD4+ T cells) were significantly lower in the KD group compared with the NC group (4·12 ± 1·24% versus 8·15 ± 2·60%, P < 0·01). The frequencies of Treg cells in the ID group were almost equal to those in the NC group (8·06 ± 2·53% versus 8·15 ± 2·60%, P > 0·01). The proportions of Treg cells increased gradually after treatment with IVIG (6·27 ± 2·04%). There was no obvious difference between the KD-CAL+ and KD-CAL- groups in the proportions of Treg cells (P > 0·05).

Expression of IL-17A/F, ROR-γt and FoxP3 in CD4+ T from patients with KD

IL-17A/F is a prominent cytokine produced by Th17 cells. ROR-γt is an important transcription factor for the differentiation of Th17, while FoxP3 is the master transcription factor in Treg. We thus investigated the expressions of ROR-γt and FoxP3 in CD4+ T from patients with KD. As shown in Fig. 3, compared with the NC group, the levels of IL-17A/F and ROR-γt expression were up-regulated significantly in the ID group [IL-17A(8·42 ± 2·13) × 10−6versus (3·82 ± 1·45) × 10−6, P < 0·01, IL-17F (3·25 ± 0·87) × 10−4versus (1·71 ± 0·50) × 10−4, P < 0·01, ROR-γt (5·31 ± 1·35) × 10−5versus (3·48 ± 1·03) × 10−5, P < 0·01]. The levels of IL-17A/F and ROR-γt expression were much higher in the acute KD patient group than those in the ID group [IL-17A (17·56 ± 7·43) × 10−6, IL-17F (7·96 ± 3·04) × 10−4, ROR-γt (8·57 ± 2·65) × 10−5] and remarkably lower after treatment with IVIG [IL-17A (10·68 ± 3·15) × 10−6, IL-17F (3·07 ± 1·12) × 10−4, ROR-γt (5·81 ± 1·68) × 10−5, P < 0·01]. The levels of IL-17A/F and ROR-γt expression in the KD-CAL+ group were markedly higher than those of the KD-CAL- group [IL-17A (20·00 ± 5·47) × 10−6versus (15·88 ± 4·63) × 10−6, P < 0·01, IL-17F (9·36 ± 3·01) × 10−4versus (7·37 ± 2·15) × 10−4, P < 0·01, ROR-γt (9·15 ± 2·82) × 10−5versus (7·48 ± 2·11) × 10−5, P < 0·01]. In contrast, the expression levels of FoxP3 mRNA was markedly lower in the KD group compared with the NC group [(3·69 ± 1·04) × 10−5versus (17·84 ± 5·12) × 10−5, P < 0·01], while there was no obvious difference between the KD-CAL+ group and KD-CAL- group in the levels of FoxP3 expression (P > 0·05). The expression levels of FoxP3 mRNA in the ID group were almost equal to those in the NC group (P > 0·05).

Fig. 3
Expression of transcription factors and cytokines in patients with Kawasaki disease; NC (normal controls) versus infectious disease (ID): aP < 0·05; ID versus KD: bP < 0·05, eP > 0·05; KD–coronary ...

Plasma cytokine concentrations in patients with KD

Plasma concentrations of IL-17A, IL-6, IL-23 and TGF-β were detected in each group (Table 5). The IL-17A, IL-6 and IL-23 concentrations in patients with KD were significantly higher compared with the concentrations in the NC and ID groups. It was found that serum IL-17A, IL-6 and IL-23 had a tendency to decrease after treatment with IVIG, but there was no significant difference in the concentrations of the cytokines before or after treatment with IVIG. Plasma TGF-β concentrations were markedly lower in the KD group than in the NC group (P < 0·01), while there was no obvious difference between the ID group and the NC group in concentrations of TGF-β (P > 0·05).

Table 5
Expression of cytokines associated with T helper type 17 (Th17) and regulatory T cell (Treg) differentiation in patients with Kawasaki disease.

Plasma concentrations of IL-17A, IL-6 and IL-23 in the KD-CAL+ groups were significantly higher than those of the KD-CAL- group, but there was no obvious difference to be found in concentrations of TGF-β between the KD-CAL+ and KD-CAL- groups.

Alteration of Thl7 cells in IVIG-resistant KD

Compared with the IVIG-sensitive group, the proportions of Th17 cells were up-regulated significantly during the acute phase in IVIG-resistant KD (4·53 ± 1·42% versus 2·72 ± 0·71%, P < 0·01).

Plasma concentrations of IL-17A and IL-6 in IVIG-resistant KD were found to be higher in comparison with the sensitive group before IVIG treatment (IL-17A 62·5 ± 18·4 pg/ml versus 30·3 ± 10·2 pg/ml, P < 0·01; IL-6 180·6 ± 50·5 pg/ml versus 71·6 ± 21·9 pg/ml, P < 0·01). The IVIG-resistant group was found to maintain continuously high levels of IL-17A IL-6 after IVIG treatment. There was no significant difference in the severity of decrease between both groups, but it was found that plasma concentrations of IL-17A and IL-6 had a tendency to decrease after treatment with IVIG (IL-17A 48·3 ± 14·5 pg/ml versus 28·1 ± 8·5 pg/ml, P > 0·05; IL-6 150·1 ± 46·7 pg/ml versus 65·9 ± 18·2 pg/ml, P > 0·05).

Discussion

KD is currently recognized as an acute vasculitis led by immune dysfunction disorder with cytokine cascade and endothelial cell lesion. Most research regarding cytokine networks in KD has focused upon inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-8, interferon (IFN)-α and TNF-α[15]. These inflammatory cytokines are elevated during the acute phase of KD, and their changes parallel the clinical presentation of the systemic vasculitis, but the mechanism triggering the cascade response of proinflammatory cytokine production needs further clarification.

Recently, the Th17 cell has a lineage that is distinctly different from that of the Th1 and Th2 cells, which regulate tissue inflammation [7]. Its identification emerges from the discovery of a new type of cytokine: IL-17A/F [8]. IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. IL-17 produced by Th17 cells induces activation of neutrophils, stimulates monocytes and fibroblasts to produce proinflammatory cytokines such as IL-6, TNF-α, IL-8 and IL-1β, thus perpetuating inflammation of the tissues [8]. Therefore, Th17 cells and their effector cytokines may contribute to the pathogenesis of inflammation, autoimmune diseases and graft-versus-host disease (GVHD) [9]. Treg cells have been demonstrated efficiently in the control of autoimmune disease. Treg cells exert their function partly through the secretion of anti-inflammatory cytokines (IL-10 and TGF-β) [6]. The number of Treg cells decreases in several autoimmune diseases, and adoptive transfer of purified Treg cells improves autoimmune disorders [10,11]. Recent data in humans suggest that Th17/Treg cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and asthma [12,13].

Sohn et al. have reported that IL-17 and IL-17-induced cytokines (IL-6 and IL-8) increased significantly in KD patients [14]. Several studies have reported that the proportions of Treg cells are decreased and their functional properties compromised in patients with KD [15,16]. In this study, we observed Th17/Treg cells on different levels, including cell proportions, related cytokine secretion and key transcription factors. Th17 proportions and expression levels of cytokines (IL-17A/F) and transcription factors (ROR-γt) were up-regulated significantly, while Treg proportions and expression levels of Treg transcription factor (FoxP3) were down-regulated significantly in children with acute KD. Although there was no obvious difference to be found in Treg cells between the KD-CAL+ and KD-CAL- groups, we found that Th17 proportions in the KD-CAL+ group were markedly higher than those of the KD-CAL- group. Results mentioned above indicate that a Th17/Treg cell imbalance exists in patients with KD, implicating a potential role for Th17/Treg imbalance in an acute vasculitis syndrome of KD.

In this study, we investigate further the proportions of Th17 cells in peripheral blood from IVIG-sensitive and -resistant patients. Compared with the sensitive group, Th17 proportions were up-regulated significantly during the acute phase in immunoglobulin-resistant KD. There was a significant difference in plasma concentrations of IL-17A and IL-6 between the resistant and sensitive groups before IVIG treatment. The resistant group still maintained significantly high levels of IL-17A and IL-6 after IVIG, suggesting that aberrant activation of Th17 cells may be one of the factors causing IVIG-resistant KD based on the inflammatory properties of Th17 cells.

Previous studies have shown that there is a reciprocal relationship between FoxP3+ Treg and Th17 cells, in which IL-6 plays a pivotal role in dictating whether the immune response is dominated by pathogenic Th17 cells or protective Treg cells [13,16]. Naive T cells exposed to TGF-β up-regulate FoxP3 and become induced Treg cells, while Th17 cells are differentiated from naive T cells in response to IL-6 plus TGF-β, and need the presence of IL-23 for their expansion and/or maintenance [6,10,17,18]. In this study, we found that the concentrations of IL-6 and IL-23 in the patients with KD were significantly higher compared with the concentrations in NC and ID, while plasma TGF-β concentration was markedly lower in KD. Therefore, it can be speculated that over-expression of IL-6 might contribute to the Th17/ Treg imbalance in KD. However, the mechanisms causing disturbed expression of IL-6 and TGF-β need further investigation.

In summary, our data demonstrate that a Th17/Treg cell imbalance exists in patients with acute KD. Therefore, the dynamic interaction between Th17 and Treg cells may be important in the development of KD, suggesting a potential role for Th17/Treg imbalance in the pathogenesis of the KD acute vasculitis syndrome. Because of the reciprocal developmental pathway for the generation of Th17 and Treg cells, and their opposite effects, Th17/Treg subsets may have been evolved to induce or regulate tissue inflammation, analogous to the dichotomy of Th1/Th2 T cell subsets. We need to confirm our conclusion in a larger-scale population, and ongoing efforts should be made to identify the precise effect and mechanism of the Th17/Tregimbalance in KD. A better understanding of the nature, regulation and function of Th17 and Treg cells in KD immunity may provide a new target for the treatment of children with KD.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (no. 30600234) and Science Project of Shenzhen (no. 30600234).

Disclosure

None.

References

1. Fu JR, Li CR, Zhou RF, Zhou YD. Effect of NF-kB on endothelial damage mediated by inflammatory cytokines in Kawasaki disease. Zhonghua Er Ke Za Zhi. 2002;40:72–4.
2. Wang GB, Li CR, Zhu Y, Yuan XW. The role of activation of toll-like receptors in immunological pathogenesis of Kawasaki disease. Zhonghua Er Ke Za Zhi. 2006;44:333–6. [PubMed]
3. Rowley AH, Shulman ST. Pathogenesis and management of Kawasaki disease. Expert Rev Anti Infect Ther. 2010;8:197–203. [PMC free article] [PubMed]
4. Wang CL, Wu YT, Liu CA, et al. Expression of CD40 ligand on CD4+ T-cells and platelets correlated to the coronary artery lesion and disease progress in Kawasaki disease. Pediatrics. 2003;111:E140–7. [PubMed]
5. Suganami Y, Kawashima H, Hasegawa D, Sato S, Hoshika A. Clinical application of rapid assay of serum interleukin-6 in Kawasaki disease. Pediatr Int. 2008;50:264–6. [PubMed]
6. Workman CJ, Szymczak-Workman AL, Collison LW, Pillai MR, Vignali DA. The development and function of regulatory T cells. Cell Mol Life Sci. 2009;66:2603–22. [PMC free article] [PubMed]
7. Crome SQ, Wang AY, Levings MK. Translational mini-review series on Th17 cells: function and regulation of human T helper 17 cells in health and disease. Clin Exp Immunol. 2010;159:109–19. [PMC free article] [PubMed]
8. Afzali B, Mitchell P, Lechler RI, John S, Lombardi G. Translational mini-review series on Th17 cells: induction of interleukin-17 production by regulatory T cells. Clin Exp Immunol. 2010;159:120–30. [PMC free article] [PubMed]
9. Sarkar S, Cooney LA, Fox DA. The role of T helper type 17 cells in inflammatory arthritis. Clin Exp Immunol. 2010;159:225–37. [PMC free article] [PubMed]
10. Tran DQ, Shevach EM. Therapeutic potential of FOXP3+ regulatory T cells and their interactions with dendritic cells. Hum Immunol. 2009;70:294–9. [PubMed]
11. Shevach EM. Mechanisms of FoxP3+ T regulatory cell-mediated suppression. Immunity. 2009;30:636–45. [PubMed]
12. Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17(Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol. 2007;148:32–46. [PMC free article] [PubMed]
13. Aerts NE, Dombrecht EJ, Ebo DG, Bridts CH, Stevens WJ, De Clerck LS. Activated T cells complicate the identification of regulatory T cells in rheumatoid arthritis. Cell Immunol. 2008;251:109–15. [PubMed]
14. Sohn MH, Noh SY, Chang W, Shin KM, Kim DS. Circulating interleukin 17 is increased in the acute stage of Kawasaki disease. Scand J Rheumatol. 2003;32:364–6. [PubMed]
15. Fukazawa R, Ogawa S. Long-term prognosis of patients with Kawasaki disease: at risk for future atherosclerosis? J Nippon Med Sch. 2009;76:124–33. [PubMed]
16. Furuno K, Yuge T, Kusuhara K, et al. CD25+CD4+ regulatory T cells in patients with Kawasaki disease. J Pediatr. 2004;145:385–90. [PubMed]
17. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-β-induced FoxP3+CD4+CD25+ regulatory T cells are not mirror images of each other. Trends Immunol. 2008;29:429–35. [PubMed]
18. Voo KS, Wang Y-H, Santori FR, et al. Identification of IL-17-producing FOXP3+ regulatory T cells in humans. Proc Natl Acad Sci USA. 2009;106:4793–8. [PMC free article] [PubMed]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...