Patient characteristics
A total of 54 patients with KD and 27 HCs were recruited based on our inclusion and exclusion criteria. Their characteristics are shown in Table 1. No significant differences in age and sex were observed between the groups. The WBC count, neutrophil count, prealbumin, and CRP levels were significantly higher in patients with KD pre-IVIG treatment than in HCs, whereas no significant difference was observed in the absolute lymphocyte count. After IVIG treatment, WBC and neutrophil count and the CRP level decreased rapidly to an almost normal level, whereas the prealbumin level remained lower. According to echocardiography parameters, 52 patients with KD were divided into two groups: KD without coronary artery lesion (CALs; KD-NCAL) group (n = 40) and KD with CAL (KD-CAL) group (n = 14) (Table 2). No significant differences were observed in terms of WBC, neutrophil, and lymphocyte counts; neutrophil to lymphocyte ratio (NLR); and CRP and prealbumin levels between the KD-CAL and KD-NCAL groups.
Table 1
Characteristics of the study population.
Parameters
|
Kawasaki disease
|
Healthy controls
|
Pre-IVIG
|
Post-IVIG
|
Number
|
54
|
54
|
27
|
Age, months
|
35.5 (18.75-56.25)
|
35.5 (18.75-56.25)
|
45 (36-56)
|
Sex, male/female
|
29/25
|
29/25
|
16/11
|
Fever duration before diagnosis
|
5 (4.0-6.25)
|
5 (4.0-6.25)
|
-
|
WBC, 109/L
|
13.67 (10.59-15.95)†,‡
|
7.54 (5.41-10.47)
|
7.38 (6.16-8.33)
|
Neutrophil, 109/L
|
8.21 (6.09-11.36)†,‡
|
2.59 (1.6-3.9)
|
2.58 (2.23-3.35)
|
Lymphocytes,109/L
|
3.21 (2.52-4.12)
|
3.38 (2.66-4.88)
|
3.68 (2.89-4.08)
|
CRP, mg/L
|
49.04 (34.42-85.57)†,‡
|
5.9 (3.12-11.86)†
|
0.16 (0.11-0.42)
|
NLR
|
2.52 (1.79-3.88)†,‡
|
0.78 (0.52-1.02)
|
0.77 (0.63-1.09)
|
Prealbumin, mg/L
|
75.5 (58.0-87.25)†,‡
|
166 (137.75-200)†
|
207 (190-225)
|
Baseline characteristics of the participants. Data shown are median (quartile spacing) or the number of cases. -, this data was not detected; CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; WBC, white blood cell counts.
†P < 0.05 vs. the healthy controls. ‡P < 0.05 vs. the post-IVIG treatment
|
Table 2
Characteristics of patients with KD classified according to coronary artery lesions or not.
Parameters
|
Kawasaki disease
|
P value
|
NCAL
|
CAL
|
Number
|
40
|
14
|
|
Age, months
|
33.5 (20.0-50.0)
|
45.4 (14.5-66.75)
|
0.4125
|
Sex, male/female
|
22/18
|
7/7
|
0.7468
|
Fever duration before diagnosis
|
6 (4-6.75)
|
4.5 (4-6.25)
|
0.2937
|
WBC, 109/L
|
13.67 (10.14-15.97)
|
13.96 (11.43-15.77)
|
0.5737
|
Neutrophil, 109/L
|
8.16 (6.09-11.95)
|
8.37(6.09-11.10)
|
0.8979
|
Lymphocytes,109/L
|
3.17 (2.53-4.11)
|
3.32 (2.19-4.30)
|
0.6427
|
CRP, mg/L
|
45.72 (29.14-79.61)
|
67.24 (38.95-105.28)
|
0.2690
|
NLR
|
2.52 (1.82-3.78)
|
2.36 (1.61-4.30)
|
0.7596
|
Prealbumin, mg/L
|
76 (59.5-88)
|
71.5 (52.25-80.25)
|
0.2645
|
Baseline characteristics of the participants. Data shown are median (quartile spacing) or the number of cases. CAL, coronary artery lesion; CRP, C-reactive protein; NCAL, without coronary artery lesion; NLR, neutrophil to lymphocyte ratio; WBC, white blood cell counts. |
Frequency and number of pDCs and CD1c+ mDCs
To identify DC subsets, the gating strategy of total DCs and their subsets was shown (Fig. 1a). Quantitative flow cytometric analysis revealed that the frequency and absolute number of pDCs and CD1c+ mDCs among pan-DCs significantly decreased in patients with KD pre-IVIG treatment compared with those in HCs (P < 0.0001, Fig. 1b; P < 0.0001, Fig. 1c; P < 0.0001, Fig. 1d; P < 0.0001, Fig. 1e; respectively). Importantly, we found significantly recovered frequency and absolute number of pDCs and CD1c+ mDCs in patients post-IVIG therapy (P = 0.0812, Fig. 1b; P = 0.0006, Fig. 1c; P < 0.0001, Fig. 1c; P < 0.0001, Fig. 1e; respectively). Although the percentage of both DC subsets in patients with KD did not recover to their levels observed in HCs (P < 0.0001, Fig. 1b; P < 0.0001, Fig. 1d; respectively), the absolute number of both DC subsets was same as that found in HCs. (P = 0.2255, Fig. 1c; P = 0.1730, Fig. 1e; respectively).
Phenotypic properties on pDCs and CD1c+ mDCs
We compared the expressions of the antigen presenting molecule HLA-DR and co-stimulatory molecules (CD40 and CD86) in the two DC subsets in the peripheral circulation of patients with KD and HCs. These molecules are critical for DCs to elicit adaptive immune responses.
Although no significant significance was observed between pre- and post-IVIG treatment in patients with KD and HCs in terms of pDCs, the MFI of the antigen presenting molecule HLA-DR exhibited a decreasing trend. (P = 0.1822, Fig. 2a; P = 0.3130, Fig. 2a; respectively). CD40 percentage on pDCs in patients pre-IVIG treatment and MFI considerably decreased. (P < 0.0001, Fig. 2g; P < 0.0001, Fig. 2h; respectively). IVIG treatment significantly elevated the percentage and MFI of CD40 expression on pDCs; however, they were not at normal levels as observed in HCs (P = 0.0010, Fig. 2g; P = 0.0170, Fig. 2h; respectively). Similarly, the percentage and MFI of CD86+ pDCs increased in patients post-IVIG treatment compared with that pre-IVIG treatment, with no difference between patients post-IVIG treatment and HCs. (P = 0.0016, Fig. 2c; P = 0.0091, Fig. 2d; P = 0.5288, Fig. 2c; P = 0.5158, Fig. 2d; respectively).
For CD1c+ mDCs, we observed significantly decreased MFI of HLA-DR in patients with KD pre-treatment compared with that in HCs, with no difference between post- treatment and HCs (P < 0.0001, Fig. 2b; P = 0.1368, Fig. 2b; respectively). Unlike pDCs, the co-stimulatory molecule on CD1c+ mDCs in the patients was CD86 rather than CD40. A drastic decrease in the MFI and of CD86+ percentage was observed on CD1c+mDCs in the patients pre- IVIG treatment. (P < 0.0001, Fig. 2e; P < 0.0001, Fig. 2f; respectively). IVIG treatment significantly elevated the percentage and MFI of CD86 expression on CD1c+ mDCs in patients with KD; however, these parameters were significantly lower than those in HCs (P < 0.0001, Fig. 2e; P < 0.0001, Fig. 2f; respectively). We did not observe obvious changes in terms of the percentage and MFI of CD40 on CD1c+ mDCs between patients with KD and HCs (P = 0.6815, Fig. 2i; P = 1609, Fig. 2j; respectively).
Collectively, data presented in Fig. 1 indicated that the quantity of DCs and functional molecules on DCs were impaired in patients pre-IVIG treatment. IVIG treatment restored the quantity of DCs and functional molecules on DCs to distinct levels, indicating a possible role of DCs in the recovery of patients after IVIG treatment.
Increased inhibitory receptor TIM-3 expression in patients with KD pre-IVIG treatment
Considering the impaired quantity and functional molecules on DCs in patients with acute KD patients, we further assessed CD4+ T cells because DCs are the critical regulators of CD4+ T cells. The percentage and absolute number of CD4+ T cells decreased in the peripheral blood of patients pre-IVIG treatment compared with those in HCs (P < 0.0001, Fig. 3a; P = 0.0034, Fig. 3b; respectively). Subsequently, the percentage and number of CD4+ T cells increased after IVIG treatment, returning to normal levels (P = 1027, Fig. 3a; P = 08239, Fig. 3b; respectively). Inhibitory receptors such as programmed cell death 1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), and T cell immunoglobulin and mucin domain 3 (TIM-3) are important molecules controlling T cell effector responses [17]. Inhibitory receptors have been involved in the pathophysiology of various human diseases, including autoimmune diseases [17, 18], sepsis [19], and cancer [20]. However, the expression of these inhibitory receptors on CD4+ T cells has not been reported previously in KD. To determine whether these inhibitory receptors are involved in the pathogenesis of KD,we used flow cytometry to assess the expressions of PD-1, TIGIT, and TIM-3 on peripheral CD4+ T cells. Representative flow cytometric analyses are shown in Fig. 3c, Patients with KD were found to exhibit a significantly higher expression of TIM-3 on CD4+ T cells pre-IVIG treatment than HCs, whereas no changes in PD-1 or TIGIT were observed between them. (P < 0.0001, Fig. 3f; P = 0.2990, Fig. 3d; P = 0.08123, Fig. 3e; respectively). IVIG treatment significantly reduced percentage TIM-3 expression on CD4+ T cells, but not to a normal level as seen in HCs. (P = 0.0034, Fig. 3f). However, the percentage of CD4+ T cells expressing the TIGIT was increased post-IVIG treatment (P = 0.0012, Fig. 3e).
Unaltered Th1/ Th2 polarisation of CD4+ T cells in patients with KD.
Although many studies have focused on T helper cytokines, most of them have determined the cytokines in plasma instead of CD4+ T cells. To understand the biology of CD4+ T cells, frequencies of different CD4+ T-cell subsets were analysed based on cytokine patterns after in vitro stimulation of the T-cell receptor (TCR) signal. Production of intracellular IFN-γ and IL-4, which are the representative factors of Th1 and Th2, respectively, in CD4+ T cells, was analysed in the peripheral blood of patients with KD and HCs. The gating strategy for determining Th1 and Th2 cells is shown in Fig. 4a. Neither the onset of KD nor IVIG treatment altered the percentages of Th1 and Th2 in CD4+ T cells. (P = 0.9851, Fig. 4b; P = 0.7776, Fig. 4b; P = 0.3980, Fig. 4c; P = 0.5509, Fig. 4c; respectively).
We further measured the level of a panel of Th subset-related cytokines in the plasma of patients with KD and HCs. Of these, the plasma level of IL-4 was very low or undetectable in either study population (date not shown). IL-17A, IFN-γ, TNF-α, IL-6, IL-10, and IL-2 levels were higher in patients with KD pre-IVIG than in HCs (P = 0.0464, Fig. 4d; P = 0.0020, Fig. 4e; P = 0.0084, Fig. 4f; P < 0.0001, Fig. 4g; P < 0.0001, Fig. 4h; P = 0.0138, Fig. 4i; respectively). These results are consistent with those previously reported [21, 22]. After IVIG treatment, plasma levels of IL-17A and IL-2 returned to normal (P = 0.2258, Fig. 4 d; P = 0.1145, Fig. 4i; respectively), IL-6 and IL-10 levels decreased, but levels were still significantly higher than in HCs (P < 0.0001, Fig. 4g; P = 0.0056, Fig. 4h; respectively). IFN-γ and TNF-α levels had a tendency to be reduced, but levels were still significantly higher than in HCs. (P = 0.0122, Fig. 4e; P = 0.0108, Fig. 4f; respectively).
Numbers and phenotypic properties on DC subsets and CD4+ T cells in patients with CAL and without CAL pre-IVIG treatment
To investigate the correlation among DC subsets, CD4+ T cells, and CAL, we performed a subgroup analysis comparing patients with and without CAL (KD-CAL and KD-NCAL groups). The number and proportion of circulating pDCs and CD1c+ mDCs were not significantly different in KD-CAL and KD-NCAL groups (Table 3). The expressions of HLA-DR, CD86, and CD40 on pDCs and CD1c+ mDCs did not differ significantly between KD-CAL and KD-NCAL groups (Table 4). Furthermore, no difference was observed in the number, proportion, and TIM-3 receptor expression on CD4+ T cells between the two groups (Table 5).
Table 3
Percentage and number of DC subsets between KD patients with and without CAL.
Parameters
|
Kawasaki disease
|
P value
|
NCAL (n = 40)
|
CAL (n = 14)
|
pDCs%
|
18.45 (11.82-32.28)
|
31.15 (14.24-41.48)
|
0.1671
|
pDCs (/ml)
|
4964 (2979-7212)
|
10425 (4024-24450)
|
0.0725
|
CD1c+ mDCs%
|
7.84 (4.72-14.93)
|
8.44 (5.69-15.28)
|
0.8668
|
CD1c+ mDCs (/ml)
|
2440 (900-4846)
|
3146 (1688-4937)
|
0.6498
|
Data shown are median (quartile spacing) or the number of cases. CAL, coronary artery lesion; NCAL, without coronary artery lesion. |
Table 4
HLA-DR, CD86 and CD40 expression on DC subsets between KD patients with and without CAL.
Parameters
|
Kawasaki disease
|
P value
|
NCAL (n = 29)
|
CAL (n = 13)
|
MFI of HLA-DR on pDCs
|
11826 (8605-15739)
|
16625 (13268-18988)
|
0.0586
|
MFI of HLA-DR on CD1c+ mDCs
|
16360 (13777-21717)
|
18561 (14376-22444)
|
0.5586
|
CD86+ expression on pDCs%
|
22.6 (15.8-27.2)
|
20.5 (15.25-28.0)
|
0.7543
|
CD86+ expression on CD1c+ mDCs%
|
47.8 (38.56-56.2)
|
47.7 (34.4-60.15)
|
0.6534
|
MFI of CD86 on pDCs
|
224 (142-294)
|
204 (149-242)
|
0.6147
|
MFI of CD86 on CD1c+ mDCs
|
915 (850-1145)
|
967 (738-1102)
|
0.3914
|
CD40+ expression on pDCs%
|
4.20 (2.05-7.61)
|
6.07 (3.41-11.65)
|
0.0866
|
CD40+ expression on CD1c+ mDCs%
|
8.29 (6.21-17.65)
|
6.45 (5.21-12.1)
|
0.1310
|
MFI of CD40 on pDCs
|
274 (239-404)
|
341 (274-430)
|
0.0997
|
MFI of CD40 on CD1c+ mDCs
|
430 (318-1007)
|
304 (265-577)
|
0.0643
|
Data shown are median (quartile spacing) or the number of cases. CAL, coronary artery lesion; NCAL, without coronary artery lesion. |
Table 5
CD4+ and TIM-3+ CD4+ T cells between KD patients with and without CAL.
Parameters
|
Kawasaki disease
|
P value
|
NCAL (n = 25)
|
CAL (n = 9)
|
CD4+ T cells %
|
6.47 (4.68-9.71)
|
3.83 (2.30-7.29)
|
0.0859
|
CD4+ T cells (/ml)
|
42034115
(24620739-62163389)
|
41448706
(24919683-74148861)
|
0.9842
|
TIM-3+ expression on CD4+ T cells %
|
5.58 (4.20-7.81)
|
6.93 (4.92-8.73)
|
0.2717
|
Data shown are median (quartile spacing) or the number of cases. CAL, coronary artery lesion; NCAL, without coronary artery lesion. |