Patient baseline clinical characteristics
A total of 280 patients (male 163, 67.2%; female 117, 32.8%; median age 31 y), including 30 chronic HBV infection in immune tolerance (IT group, n=30) and hepatitis B envelope antigen (HBeAg) positive CHB (CHB group, n=250), were enrolled. The basic demographic and clinical characteristics of the subjects were shown in Table 1 and Figure 1. There were significant differences in the median age (27 vs. 31y, P= 0.004; Z= 3.893), ALT level (29.85 vs. 234.70 U/L, P=0.000; Z = 8.488), AST level (23.40 vs. 114.90 U/L, P=0.000; Z=8.493), HBsAg level (4.79 vs. 3.88 log10 IU/ml, P=0.000; Z = -7.820), HBeAg (1606.36 vs. 862.47 S/CO, P= 0.000; Z=-8.108) and HBV DNA load (8.17 vs 6.71 log10 IU/ml, P=0.000; Z=6.762) between the IT and CHB group. In the CHB group, 143 patients had a worsening ALT level (5 times higher than the upper limit of normal), 57 patients had flare (3-5 times the upper limit of normal), the remained 50 patients had obvious chronic liver inflammation (1-3 times the upper limit of normal value).
Cytokine levels in IT and CHB
The levels of Flt3-L, IFN-a2, IFN-γ, IL-10, IL-17A, IL-6, TGF-b1, TGF-b2 and TGF-b3 were shown in Table2 and Figure2. The median levels of Flt3-L, IFN-γ, IL-17A, and TGF-β1 in IT group were significantly higher than those in CHB group (Flt3-L: 41.62 vs. 27.47 pg/ml, P=0.008/Z=-2.536; IFN-γ: 42.48 vs. 33.18 pg/ml, P=0.008/Z=-2.636; IL-17A: 15.66 vs. 8.90 pg/ml, P=0.002/Z=-3.037; TGF-β1: 4921.50 vs. 2234 pg/ml, P=0.000, Z=-4.404), while the median levels of IFN-a2, TGF-β3, and IL-10 in the IT group were significantly lower than those in the CHB group (IFN-a2: 15.24 vs 35.78 Pg/ml, P = 0.000/Z = 3.727; TGF-β3: 131.69 vs 1.62.61 pg/ml, P=0.025, Z=2.245; IL-10: 5.02 vs. 7.9 pg/ml, P=0.012/Z=2.498).
we divided CHB patients into three groups as follows :Group A, 50 patients had obvious chronic liver inflammation (1-3 times the upper limit of normal value);Group B, 57 patients had flare (3-5 times the upper limit of normal);Group C, 143 patients had a worsening ALT level (5 times higher than the upper limit of normal). The cytokine levels among the three groups were shown in Table s1. The median levels of IL-10 in group A were significantly lower than those in group B and C (respectively 5.070 vs 10.920 Pg/ml, P =0.021/Z =-2.304; 5.070 vs 9.170 Pg/ml, P = 0.003/Z =-2.954). The median levels of IFN-γ in group A were significantly lower than those in group B (12.940 vs 42.000 Pg/ml, P =0.028/Z =-2.203).
3. Logistic regression analysis of risk factors of chronic HBV infection
We included all cytokines in the risk factor analysis and compared with immune tolerant patients. All cytokines were not statistically significant in univariate logistic regression analysis, but in adjusted multivariate logistic regression analysis, TGF-β 1 and TGF-β 2 were found to be significant (OR= 0.999, 95% CI 0.999-1.000, P< 0.001; OR=1.008, 95% CI 1.004-1.012, P< 0.001, Table 3).
Table 1. Clinical characteristics between the two groups
|
All(n=280)
|
IT(n=30)
|
CHB(n=250)
|
P (IT vs CHB)
|
Male(%)
|
163(67.2%)
|
14/16(87.5%)
|
149/101(59.6%)
|
P=0.175; χ2=1.842
|
Age(y)
|
31(28, 37.75)
|
27(25.75, 31.25)
|
31(28, 37.25)
|
P=0.004;Z=3.893
|
ALT(U/L)
|
234.70(129.30, 355.28)
|
29.85(21.83, 39.85)
|
234.70(129.30, 354.75)
|
P=0.000;Z=8.488
|
AST(U/L)
|
113.80(63.40, 17440)
|
23.40(19.45, 26.83)
|
114.90(63.40, 174.40)
|
P=0.000;Z=8.493
|
TBIL(µmol/L)
|
14.30(11.63, 20.10)
|
NA
|
14.30(11.63, 20.10)
|
NA
|
ALB(g/L)
|
45.20(42.60, 47.40)
|
NA
|
45.20(42.60, 47.40)
|
NA
|
HBsAg(log10 IU/ml)
|
3.88(3.61, 4.11)
|
4.79(4.59, 4.93)
|
3.88(3.62, 4.12)
|
P=0.000;Z=-7.820
|
HBeAg(S/CO)
|
861.26(468.04, 1195.97)
|
1606.36(1556.53, 1679.44)
|
862.47(469.18, 1196.43)
|
P=0.000;Z=-8.108
|
HBV DNA(log10 IU/ml)
|
6.71(6.34, 7.39)
|
8.17(7.75, 8.42)
|
6.71(6.35, 7.39)
|
P=0.000;Z=6.762
|
Table 2. The comparison of Cytokine levels between IT and CHB groups
|
All(n=280)
|
IT(n=30)
|
CHB(n=250)
|
P (IT vs CHB)
|
FlT3-L (pg/ml)
|
29.26(13.42, 83.99)
|
41.62(21.47, 124.87)
|
27.47(12.56, 82.42)
|
P=0.008; Z=-2.536
|
IFN-a2 (pg/ml)
|
32.35(9.12, 67.54)
|
15.24(4.07, 30.73)
|
35.78(10.59, 68.80)
|
P=0.000; Z=3.727
|
IFN-γ (pg/ml)
|
33.99(12.94, 93.03)
|
42.48(26.24, 111.56)
|
33.18(5.02, 93.03)
|
P=0.008; Z=-2.636
|
IL-10 (pg/ml)
|
7.26(3.08, 17.00)
|
5.02(2.98, 10.13)
|
7.92 (3.25, 18.03)
|
P=0.012; Z=2.498
|
IL-17A (pg/ml)
|
12.30(3.62, 44.74)
|
15.66 (9.84, 44.46)
|
8.90(3.34, 45.06)
|
P=0.002; Z=-3.037
|
IL-6 (pg/ml)
|
2.28(1.10, 9.32)
|
3.17 (1.53, 18.33)
|
2.26(0.98, 9.29)
|
P=0.061; Z=-1.872
|
TGFβ1 (pg/ml)
|
2274.50(3.63, 4751.75)
|
4921.50 (3840.75, 7689.50)
|
2234 (3.56, 3387)
|
P=0.000; Z=-4.404
|
TGFβ2 (pg/ml)
|
425.81(348.70, 647.41)
|
419.01 (311.80, 580.51)
|
435.48 (348.70, 681.08)
|
P=0.398; Z=0.846
|
TGFβ3 (pg/ml)
|
156.35(131.69, 198.01)
|
131.69 (112.78, 171.94)
|
162.61 (135.16, 207.50)
|
P=0.025; Z=2.245
|
Table 3. Logistic regression analysis of risk factors of chronic HBV infection
Single factor logistic regression analysis
|
cytokines
|
OR(95%CI)
|
P value
|
Flt3-L
|
1.000(0.9981-001)
|
0.715
|
IFN-α2
|
1.008(0.999-1.017)
|
0.083
|
IFN-γ
|
0.999(0.998-1.000)
|
0.188
|
IL-10
|
1.045(1.000-1.092)
|
0.052
|
IL-17A
|
0.999(0.996-1.003)
|
0.774
|
IL-6
|
1.002(0.995-1.008)
|
0.664
|
TGF-β1
|
1.000
|
0.088
|
TGF-β2
|
1.000(0.999-1.001)
|
0.889
|
TGF-β3
|
1.008(0.999-1.018)
|
0.097
|
Multiple factor Logistic regression analysis
|
TGF-β1
|
0.999(0.999-1.000)
|
P <0.001
|
TGF-β2
|
1.008(1.004-1.012)
|
P<0.001
|
OR: Odds ratio; 95% CI: confidence interval