As presented in Figure 1, a total of 270 patients received IFX treatment for moderate-to-severe active CD in our hospital. Among them, 197 patients were excluded: 101 because of data incompleteness, 38 owing to previous exposure to other drugs/agents as aforementioned exclusive criterion, 52 due to concomitant use of corticosteroids during the administration of IFX, and 6 were found to have other autoimmune diseases. Finally, 73 patients were enrolled in our study.
The demographics characteristics of CD patients are summarized in Table 1. The median (IQR) age was 29 (21-37) years, and 64.4% of subjects were male. Smoking habits presented in only 5 patients (6.8%). Most of the enrolled patients had L1 diseases and B1/B2 behaviors. Of them, 23 patients (31.5%) had perianal lesions and 4 patients (5.5%) had upper GI involvement. Except for 2 patients with severe activity, the remaining 71 patients had moderate disease activity at the time of enrollment, with a median (IQR) HBI of 10 (9–12). Seven patients (9.6%) underwent surgical intervention due to intestinal complications, and 19 patients (26.0%) started the combination therapy with AZA (1-2 mg/kg·d) at the fourth times of IFX infusion as they had the predictors related to poor prognosis, such as age <40 years old and upper GI involvement .
Thirty-seven patients received vitD3 supplementation (D3-patients), while 36 did not (non-D3-patients). There were no significant differences in baseline characters between D3-patients and non-D3-patients (P>0.05) (Table 1, Figure 2a). Serum levels of inflammatory parameters (Hb, CRP, ESR, Alb, PLT) at baseline were also similar between the two groups (all P>0.05) (Figure 2b-f).
Influence of vitD3 supplementation on the change of serum 25(OH)D
As described in Figure 3a, the baseline level of 25(OH)D was 15.07±6.70 ng/mL in D3-patients, while 18.01±13.48 ng/mL in non-D3-patients (P=0.086, student’s t-test). There was a clear different change pattern of 25(OH)D between the two groups. The mean serum concentration of 25(OH)D basically stayed stable in non-D3-patients throughout the duration of the study (baseline vs. week 54: 18.01±13.48 vs. 17.79±7.10, P=0.841, paired t-test). In D3-patients, the average concentration of 25(OH)D significantly raised from baseline 15.07 ng/mL to 20.33 ng/mL at week 54 (P<0.001, paired t-test). After 54-week of vitD3 supplementation, D3-patients manifested a greater increase of 25(OH)D than non-D3-patients did (5.24±7.01 vs. -0.22±6.44, P=0.001, student’s t-test) (Figure 3a).
Based on the above findings, the linear regression model was further established to identify the contributors for the increase of 25(OH)D in D3-patients. The independent variables including age, gender, smoking status, disease extent, disease behavior, perianal lesions, upper GI involvement, prior surgical history, concomitant use of AZA, BMI, Hb, PLT, CRP, ESR, Alb, baseline 25(OH)D and HBI. We found that both upper GI involvement and baseline 25(OH)D concentration were negatively correlated to the increase of 25(OH)D (B=-12.797, P=0.020; B=-1.144, P<0.001, respectively) (Table 2). In other words, the patients with upper GI involvement poorly responded to vitD3 supplementation, while the patients with lower baseline 25(OH)D had a greater increase of 25(OH)D after oral vitamin D3 supplementation.
In addition, there was 73.0% (27/37) patients was deficient in vitamin D in D3-patients, and 63.9% (23/36) patients in non-D3-patients at baseline (P=0.404). After 54-week of oral administration of vitD3, the frequency of vitamin D deficiency was reduced from 73.0% to 51.4% in D3-patients (P=0.055), while from 63.9% to 61.1% in non-D3-patients (P=0.808). Either in D3-patients or non-D3-patients, however, no statistical difference was found in terms of the distribution of vitamin D deficiency between baseline and 54-week (all P>0.05) (Figure 3b).
Influence of vitamin D supplementation on disease activity
On the follow-up visit for 54 weeks, 72.6% (53/73) patients receiving IFX treatment achieved clinical remission, and 27.4% (20/73) were degraded into mild activity. The overall median HBI was 10 at baseline, and decreased to 4 at week 54 (P<0.001, paired Wilcoxon’s signed rank test). However, the increase of remission rate in D3-patients was more significant than in non-D3-patients at week 54 (83.8% vs. 61.6%, P=0.030, chi-square test). The similar trend was obtained for the decrease of HBI between baseline and week 54 in D3-patients when compared with non-D3-patients (7.41±3.0 vs. 6.28±2.75, P=0.023, student’s t-test) (Figure 4a). In addition, the average/median levels of BMI, Hb and Alb significantly increased, while CRP and ESR significantly decreased in either patients with or without oral vitD3 supplementation after 54-week IFX treatment (week 54 vs baseline, all P<0.05, paired student’s t-test or paired Wilcoxon’s signed rank test). However, the differences in changes of these parameters did not reach statistical significance between D3-patients and non-D3-patients at week 54 (all P>0.05, student’s t-test or Wilcoxon’s signed rank test) (Figure 2a-f).
After adjustment of age, gender, smoking status, disease extent, disease behavior, perianal lesions, upper GI involvement, prior surgical history, concomitant use of AZA, the multivariable logistic regression analysis was further conducted to determine the association of vitD3 supplementation with the achievement of clinical remission in the total cohort. The results suggested that vitD3 supplementation was an independent factor for improving the remission rate of IFX-treated CD patients (β=-1.667, P=0.015) (Table 3).
In D3-patients, the multivariable linear regression model was established to explore the influence factors for the beneficial role of vitD3 supplementation. The following variables were included: age, gender, smoking status, disease extent, disease behavior, perianal lesions, upper GI involvement, prior surgical history, concomitant use of AZA, BMI, Hb, PLT, CRP, ESR, Alb, baseline 25(OH)D and HBI. As a result, the concomitant use of AZA and baseline HBI were shown to be independently and positively correlated with the decrease of HBI in those CD patients (B=0.645, P=0.028; B=1.090, P<0.001, respectively) (Table 4).
Influence of vitD3 supplementation disease activity stratified by baseline vitamin D status
To clarify whether vitamin D status at baseline influenced the beneficial effect of vitD3 supplementation in CD patients, the change of disease activity and relevant biochemical parameters were separately analyzed in the subgroups of vitamin D deficient (<20 ng/mL, n=50) and non-deficient (≥20 ng/mL, n=23). In each subgroup, CD patients were further divided into supplementary (D3-patients) and non-supplementary group (non-D3-patients). Overall, D3-patients and non-D3-patients were well balanced with respect to demographic and disease characteristics in either vitamin D deficient or non-deficient group, respectively (all P>0.05, student’s t-test, Wilcoxon’s signed rank test or chi-square test) (Table 1, Figure 2g-r). The exception is the gender distribution in non-deficient group, which presented a higher ratio of male in sub-non-D3-patients than in sub-D3-patients (P=0.007, chi-square test) (Table 1).
When comes to vitamin D deficient subgroup, after 54-week of vitD3 supplementation, D3-patients manifested a greater increase of 25(OH)D than non-D3-patients did (1.65±5.64 vs. 7.19±6.25, P=0.002, student’s t-test) (Figure 3c). Moreover, the remission rate, together with the decrease of HBI, in D3-patients was higher than that in non-D3-patients (P=0.028, chi-square test) (Figure 4b). The multivariable logistic regression model showed that vitD3 supplementation was an independent factor for improving the remission rate of patients under IFX therapy (B=-1.919, P=0.019), after adjustment of age, gender, smoking status, disease extent, disease behavior, perianal lesions, upper GI involvement, prior surgery, concomitant use of AZA (Table 5). However, this significant difference of changes in 25(OH)D concentration and remission rate between D3-patients and non-D3-patients did not appear in non-deficient group (all P>0.05, paired student’s t-test or chi-square test) (Figure 3d, Figure 4c)
Effects of vitD3 supplementation on metabolism of calcium and phosphorus
Serum calcium and phosphorus levels at baseline and week 54 were also examined. No statistical differences in the two parameters at baseline were observed between D3-patients and non-D3-patients (P=0.617, 0.172, respectively). At week 54, changes of serum calcium or phosphorus also did not reach statistical significance between D3-patients and non-D3-patients (P=0.074, P=0.567, respectively) (Figure 5a, b). Despite the mean concentration of calcium in D3-patients significantly raised at week 54 (P=0.018, paired t test), all data did not exceed the upper limit of normal calcium level (Figure 5a).
Influence of vitD3 supplementation on the expression profiles of Th-cell-related cytokines in CD patients
Among 73 enrolled patients, 9 patients (4 patients without vitD3 supplementation, 5 patients supplemented by vitD3) had blood sample at baseline and week 54. Serum TNF-α and IL-6 level in non-D3-patients significantly reduced at 54-week compared to their baseline values (P=0.032, 0.022, respectively). There were no significant differences of IL-2, IL-4, IL-10 and IFN-γ between baseline and week 54 (all P>0.05). However, the cytokine profiles in D3-patients were quite distinct from non-D3-patients. Serum level of IL-10 at week 54 had a remarkable increase in D3-patients (P=0.037). No statistical significant changes was found in other cytokines (Figure 6).