Clinical characteristics and disease activity at baseline and after TAC treatment
The flowchart of the study was shown in Fig. 1. Baseline clinical and demographic characteristics are presented in Table. 1. Among the 77 patients with baseline Gal-9 data, there were 14 males and 63 females, with an average age of 52.0 ± 13.8 years. The average course of RA disease was 7.12 ± 6.84 years, and the average DAS28 score was 4.26 ± 1.3. There were 61 (79.2%) patients with moderate and severe disease activity. The clinical characteristics of 52 post-treatment patients and 33 RA patients with blood samples available before and after treatment are compared.
There were no significant differences in baseline demographic characteristics(gender, age, disease course)among the severe, moderate and mild groups of RA patients. During the 12-week treatment, there were no statistical differences in disease activity and drug response among patients. The patients who withdrew the study showed no differences from those finished the trial in baseline demographic characteristics. (results not shown).
Gal-9 expression in 77 RA patients at baseline
The gating strategy for different cell subsets is shown in Fig. 2. CD3+ cells were considered T lymphocytes, including the following subsets: CD4+ (CD3+CD4+CD8−), CD8+(CD3+CD4−CD8+), double-negative(CD3+CD4−CD8−), or Treg (CD4+CD25+CD127low).
The expression of Gal-9 in the PBMCs of the severe group was higher than in the moderate and mild groups. The DAS28 score of the severe group was higher than that of the moderate (Fig. 3A). Gal-9 expression in CD4+ T and Treg cell subsets of severe patients was higher than that of mild patients (Fig. 3B). The percentages of CD4+ T and Treg cells in PBMCs did not differ significantly among the three groups.
The expression of Gal-9 in the plasma of 77 RA patients was higher than in the control group. High and moderate disease activity groups of patients had higher plasma levels of Gal-9 than controls (P = 0.015 in severe group, P = 0.003 in moderate group; Fig. 3C). In addition, plasma concentrations of VEGF(56 ± 123 vs 594 ± 810 pg/ml), TNF-α (13.71 ± 29.15 vs 5.46 ± 2.41 pg/ml) and IL-6 (14.49 ± 23.42 vs 0.9 ± 0.74 pg/ml) were significantly higher in RA patients than in healthy controls (data not shown).
RA patients with high CDAI scores also expressed higher Gal-9 levels. The percentage of CD3+ T cells expressing Gal-9 in PBMCs was higher in the high CDAI group than in the low CDAI group (P = 0.003; Fig. 3E). Plasma Gal-9 levels were higher in the high CDAI group than in controls (P = 0.000), while no significant difference was shown between the two CDAI groups (Figs. 3F).
Gal-9 expression in 52 patients at week 12 post-treatment
After TAC-based treatment for 12 weeks, 30 of 52 RA patients showed good response, while 22 showed poor response. Good responders received TAC + M (n = 4, 13.3%), TAC + P (n = 14, 46.7%), or TAC + M + P (n = 12, 40.0%). Among the poor responders, one patient received only TAC (4.6%), two received TAC + M (9.1%), seven received TAC + P (31.8%), and 12 received TAC + M + P (54.6%).
The good response group (1.97 ± 0.83) showed lower DAS28 score than the poor response group at 12 weeks (3.81 ± 1.06, P < 0.01; Fig. 4A), as well as lower Gal-9 expression in CD3+ and CD4+ T cell subsets (P = 0.020, P = 0.030, respectively; Figs. 4B-C). Good responders also showed significantly lower plasma Gal-9 levels at 12 weeks (2672 ± 930 vs 4377 ± 3989 pg/ml;, P = 0.05; Fig. 4D). Together, the results indicate that low plasma Gal-9 expression levels and DAS28 score were performed in good response group.
Decrease in Gal-9 expression in PBMCs and plasma after TAC treatment in 33 patients
The disease activity parameters ESR, CRP, T28, SW28, PGA, DAS28-CRP, SDAI and CDAI decreased significantly between baseline and week 12 after TAC-based treatment in 33 RA patients for whom pre- and post-treatment samples were available (P < 0.01; Fig. 5A). This included one patient (3.03%) who received TAC, five (15.1%) who received TAC + M, 10 (30.3%) who received TAC + P, and 17 (51.5%) who received TAC + M + P.
After 12 weeks of treatment, the proportion of Gal-9 expressing cells in the following T cell subsets was significantly lower than at baseline: CD3+T, 8.81 ± 11.04% at week 12 vs. 25.15 ± 31.9% at baseline; CD4+T, 12.77 ± 12.05% vs. 24.16 ± 26.29%; CD8+T, 10.56 ± 10.18% vs. 22.99 ± 24.41%; and double-negative cells, 11.52 ± 12.09% vs. 22.26 ± 24.37% (Fig. 5B). Mean fluorescence intensity (MFI) of anti-Gal-9 staining was significantly lower at week 12 than at baseline in CD3+ T (3102 ± 4004 vs. 6243 ± 6373), CD4+ T (2575 ± 4604 vs. 6492 ± 9701) and CD8+ T cells (3666 ± 3843 vs. 6854 ± 7382) (all P < 0.05), but not double-negative cells (Fig. 5C). There were no significant changes in the percentages of CD3+, CD4+, CD8+ or double-negative T cell subsets after TAC treatment.
The percentage of Treg cells among CD4+ T cells was significantly lower at week 12 (3.57 ± 1.72) than at baseline (4.54 ± 2.04, P = 0.041), but the percentage and MFI of Gal-9 expression in Tregs were not significantly different between the two time points.
Next, we evaluated changes in Gal-9 and cytokine levels in plasma. Baseline plasma Gal-9 levels were significantly higher in RA patients than in healthy controls (Fig. 5D). In RA patients, the concentration of plasma Gal-9 was lower at 12 weeks (2938 ± 1255 pg/ml) than at baseline (3613 ± 1569 pg/ml, P = 0.026; Fig. 5D). The decrease in plasma Gal-9 levels and DAS28 score between baseline and week 12 was significant in moderate RA patients but not in mild or severe patients (Fig. 5E -F). The decrease in plasma Gal-9 levels and DAS28 score between baseline and week 12 was significant in good responders but not in poor responders (Fig. 5G -H).
Both baseline and post-treatment levels of VEGF and TNF-α in plasma were higher in RA patients than in healthy controls. No significant differences were observed in cytokine levels between baseline and week 12 in the 33 RA patients with samples from both time points (data not shown).
Comparison between treatment response groups
RA patients in the severe group had the highest disease activity at both baseline and week 12 (Fig. 5E). At week 12, plasma Gal-9 expression levels were higher in severe RA patients than in mild group (Fig. 5F).
Disease activity was lower among good responders than poor responders at week 12 (Fig. 5G). Plasma Gal-9 levels were lower in good responders (2245 ± 670 pg/mL) than in poor responders (3558 ± 1431 pg/ml, P = 0.006; Fig. 5H).
Correlation of Gal-9 and other factors with disease activity
At baseline, Gal-9 levels in both PBMCs and plasma showed a moderate positive correlation with disease activity in 77 RA patients. Plasma Gal-9 levels showed a moderate positive correlation with CRP, SDAI and DAS28 in RA patients at baseline(Fig. 6A-C). Levels of CRP correlated with Gal-9 expression in CD4+, CD8+, Tregs and double-negative T cell subsets, as well as with MFI of Gal-9 expression in CD4+ and Treg cell subsets. MFI of Gal-9 expression on CD4+ T cells also showed a moderate positive correlation with SDAI (Table 2).
Table 1
Patients demographic characteristics both at baseline and after 12 weeks of treatment.
| All patients (N = 96) | Patients analyzed pre-treatment (n = 77) | Patients analyzed post-treatment (n = 52) | Patients analyzed pre- and post-treatment (n = 33) |
Female,n(%) | 79 (82.3) | 63(81.8) | 45(86.5) | 29(87.9) |
Age(years) | 51.1 ± 14.1 | 51.97 ± 13.78 | 49.88 ± 14.41 | 51.42 ± 13.91 |
Disease Duration(years) | 7.06 ± 7.14 | 7.12 ± 6.84 | 6.25 ± 6.92 | 5.92 ± 6.02 |
Moderate and severe patients,n(%) | 76 (79.2) | 61(79.2) | 40(76.9) | 25(75.8) |
Previous medication (P + D + B),n(%) | | | | |
0 | 18 (18.7) | 16(20.8) | 5(9.6) | 3(9.1) |
1、2 | 32 (33.3) | 27(35.1) | 18(34.6) | 12(36.4) |
》3 | 46 (47.9) | 34(44.2) | 29(55.8) | 18(54.6) |
ESR(mm/h) | 51.58 ± 29.47 | 51.27 ± 29.04 | 53.87 ± 29.06 | 56.52 ± 27.72 |
CRP(mg/L) | 37.36 ± 37.20 | 40.21 ± 39.93 | 34.3 ± 34.76 | 39.18 ± 40.43 |
T28 | 7.40 ± 6.59 | 7.13 ± 6.28 | 7.13 ± 6.72 | 5.85 ± 4.7 |
SW28 | 5.36 ± 5.36 | 5.43 ± 5.31 | 4.96 ± 5.32 | 4.42 ± 4.26 |
PGA | 5.59 ± 1.68 | 5.55 ± 1.63 | 5.63 ± 1.67 | 5.61 ± 1.46 |
PHGA | 5.29 ± 1.84 | 5.32 ± 1.8 | 5.23 ± 1.83 | 5.3 ± 1.63 |
HAQ | 0.87 ± 0.57 | 0.88 ± 0.57 | 0.83 ± 0.55 | 0.76 ± 0.53 |
SDAI | 60.01 ± 42.99 | 63.63 ± 46.4 | 57.26 ± 38.47 | 21.18 ± 9.71 |
CDAI | 23.65 ± 13.66 | 23.43 ± 13.3 | 22.96 ± 13.47 | 60.37 ± 44.99 |
DAS28-CRP | 4.20 ± 1.29 | 4.26 ± 1.3 | 4.08 ± 1.23 | 4.1 ± 1.2 |
Tacrolimus treatment,n(%) | | | |
TAC | 1 (1.04) | 1(1.3) | 1(1.9) | 1(3.0) |
TAC + M | 15 (15.6) | 14(18.2) | 6(11.5) | 5(15.2) |
TAC + P | 37(38.5) | 26(33.8) | 21(40.4) | 10(30.3) |
TAC + M + P | 38(39.6) | 31(40.3) | 24(46.1) | 17(51.5) |
UK | 5 (5.2) | 5(6.5) | - | - |
P + D + B: numbers of previous drugs, including prednisone (P), Disease Modifying Antirheumatic Drug (D) and biologics (B). Abbreviations: ESR: erythrocyte sedimentation rate, CRP: C-reactive protein, T28: tender joints of 28 counted, SW28: swollen joints of 28 counted, PGA: patient global assessment, PHGA: physician global assessment, HAQ: health assessment questionnaire, SDAI: simplified disease activity index, CDAI: clinical disease activity index, DAS28: disease activity score in 28 joints, TAC: tacrolimus, M: methotrexate, P: prednisone, UK: uncertain drugs combined with tacrolimus. Values are shown as mean ± standard deviation or n (%). |
Table 2
Correlations of galectin 9 expression with disease activity in 77 pre-treatment and 33 post-treatment patients.
| Baseline (n = 77) | | After 12 weeks of treatment (n = 33) |
| ESR | CRP | DAS28-CRP | SDAI | | ESR | CRP | DAS28-CRP | SDAI |
Gal-9 | 0.057 | 0.428** | 0.294* | 0.430** | | 0.035 | 0.527** | 0.496** | 0.511** |
VEGF | 0.386** | 0.223 | 0.163 | 0.218 | | 0.346 | 0.216 | 0.185 | 0.198 |
IL-6 | -0.244 | 0.239 | 0.311 | 0.255 | | 0.306 | 0.609** | 0.559** | 0.571** |
TNF-α | 0.298* | -0.086 | -0.034 | -0.064 | | -0.181 | 0.038 | -0.105 | -0.010 |
CD3+ Gal+ | 0.190 | 0.228 | 0.123 | 0.201 | | -0.062 | -0.101 | 0 | -0.073 |
CD4+ Gal+ | 0.144 | 0.295* | 0.130 | 0.192 | | -0.327 | -0.184 | -0.012 | -0.117 |
CD8+ Gal+ | 0.112 | 0.244* | 0.044 | 0.154 | | -0.150 | -0.331 | -0.222 | -0.297 |
Treg+ Gal+ | 0.153 | 0.254* | 0.180 | 0.214 | | -0.259 | -0.169 | -0.036 | -0.073 |
DNT+ Gal+ | 0.160 | 0.299* | 0.106 | 0.228 | | -0.322 | -0.236 | 0.030 | -0.096 |
CD3+ G-M | 0.042 | 0.207 | 0.154 | 0.206 | | -0.239 | -0.069 | -0.156 | -0.060 |
CD4+ G-M | 0.209 | 0.299* | 0.144 | 0.247* | | -0.066 | -0.209 | -0.098 | -0.125 |
CD8+ G-M | 0.081 | 0.179 | 0.156 | 0.187 | | -0.122 | 0.125 | 0.078 | 0.124 |
Treg+ G-M | 0.188 | 0.288* | 0.066 | 0.172 | | -0.188 | -0.174 | -0.096 | -0.166 |
DNT+ G-M | 0.165 | 0.235 | 0.060 | 0.159 | | -0.181 | -0.190 | -0.048 | -0.112 |
If both factor are in Normal distribution, the correlation between two factors were analyzed by Pearson correlation test, otherwise by Spearman correlation test, A value of P < 0.05 was considered to be statistically significant.**P < 0.01,*P < 0.05. |
At week 12, plasma Gal-9 levels in 33 patients post-treatment showed a moderate positive correlation with CRP, DAS28, and SDAI(Fig. 6D-F). However, neither the percentage nor MFI of Gal-9 expression in T cell subsets correlated with disease activity in the 33 patients. Taken together, these results indicate that plasma Gal-9 levels showed a strongest positive correlation with disease activity than percentage and MFI of Gal-9 expression in T cell subsets.