# Abstract

 Vitamin D deficiency was common among patients with chronic hepatitis C (CHC) and had negative influence on treatment outcome. Correction of vitamin D deficiency improved treatment response. Interferon gamma-induced protein 10 (IP-10) and enzyme dipeptidyl peptidase-4 (DPP IV) involved in inflammatory responses in CHC. Their higher levels at pretreatment of CHC could predict poorer responses. Vitamin D suppressed expression of IP-10 from monocytes *in vitro*. In CHC patients, DPP IV involved in IP-10 regulation. We hypothesized that correction of vitamin D insufficiency or deficiency in CHC patients might restore immune dysregulation through a pathway linked to the TH1/Th2 cytokines, IP-10 or DPP IV. We conducted a double-blind, placebo-controlled trial. 80 CHC patients with vitamin D levels less than 30 ng/mL were assigned to receive vitamin D (40) or placebo (40) supplements for 6 weeks. The levels of 25-hydroxyvitamin D [25(OH)D], Th1/Th2 cytokines, IP-10 and DPP IV were measured at baseline and at the 6th week. At the end of study, the mean 25(OH)D level in vitamin D group was significantly increased and normalised. There were no changes in the level of Th1/Th2 cytokines. Our important finding revealed that upon correction of vitamin D insufficiency or deficiency, the serum IP-10 and DPP IV levels were decreased significantly as compare to the placebo group (delta changes; 83.27 vs -133.80; 95% CI [-326.910, -40.758], p = 0.0125, and 271.04 vs -518.69; 95% CI [-1179,15, -59.781], p = 0.0305, respectively. As previous evidences suggested that each factor individually influenced and predicted outcome of CHC treatment. Our results offer a new insight and help to piece the puzzle of vitamin D deficiency, IP-10 and DPP IV together in CHC. **Trial registration:** Thai Clinical Trials Registry TCTR20160429001 # Results 

## Study patients at baseline From January to December 2014, 80 patients were included into this study and randomly assigned to receive a placebo (n = 40) vitamin D supplement (n = 40). 42 patients were naïve cases without previous CHC treatment and 38 patients were previously failed therapy either relapsers or non-responders. Demographic and clinical characteristics were shown in [Table 1](#). The mean age was 52.39 years (range; 30--70). ::::table-wrap

10.1371/journal.pone.0174608.t001

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## Baseline characteristics of CHC patients in the placebo and vitamin D groups.

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Variables Placebo group (n = 40) Vitamin D group (n = 40) p values

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Mean age (year) 52.2 ± 11.1 52.6 ± 8.5 0.830
Male gender (%) 20 (50) 23 (57.5) 0.654
HCV treatment status 0.403
• Naïve (%) 21 (52.5) 21 (52.5)\
• Previously failed (%) 19 (47.5) 19 (47.5)\
HCV genotypes 0.647
• Genotype 1, n (%) 18 (45.0) 19 (47.5)\
• Genotype 3, n (%) 16 (40.0) 13 (32.5)\
• Others (%), n (%) 6 (15.0) 8 (20.0)\
HCV viral load, log C/mL 5.72 ± 0.88 5.76 ± 0.84 0.822
FIB4 score, n (%) 0.559
• < 1.45 9 (22.5) 6 (15.0)\
• 1.45--3.25 19 (47.5) 23 (57.5)\
• > 3.25 12 (30.0) 11 (27.5)\
Median AST (IU/L), (range) 76.3 (16--323) 65.1 (21--323) 0.421
Median ALT (IU/L), (range) 78.5 (15--217) 72.1 (11--183) 0.560
Mean BMI 24.56 ± 3.98 24.48 ± 3.37 0.520
Mean serum levels[\*](#) of\
• 25(OH)D[\*\*](#) 20.27 ± 4.83 20.88 ± 5.40 0.596
• IP-10 665.61 ± 665.61 770.27 ± 642.02 0.180
• DPP IV[\*\*](#) 6137.29 ± 1584.32 6551.06 ± 1757.14 0.387
• IL-2 13.07 ± 16.50 8.20 ± 24.30 0.245
• IL-4 3.93 ± 4.99 5.39 ± 5.39 0.246
• IL-5 4.69 ± 4.69 7.60 ± 11.95 0.157
• IL-10 7.56 ± 18.96 17.09 ± 44.51 0.216
• IL-12 29.25 ± 98.04 40.61 ± 111.33 0.629
• IL-13 4.68 ± 8.01 11.24 ±22.57 0.087
• IFN-γ 251.64 ± 334.47 328.31 ± 383.54 0.344
• TNF-α 602.45 ± 1374.21 651.52 ± 1267.72 0.869
• GM-CSF 21.96 ± 57.01 27.72 ± 43.74 0.614

- unit in pg/mL,

\*\* unit in ng/mL

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI,
body mass index; IP-10, inducible protein-10; IL, interleukin; IFN-γ,
interferon gamma; TNF-α, tumor necrosis factor alpha; GM-CSF,
granulocyte macrophage colony-stimulating factor. Data are expressed as
mean ± SD:::: In each group, 52.5% and 47.5% of patients were naïve HCV status and previously failed therapy respectively. The mean 25(OH)D level was 20.27 ± 4.83 ng/mL and 20.88 ± 5.40 ng/mL in placebo and vitamin D group respectively. Other parameters including HCV genotypes, HCV viral loads, transaminase levels and liver fibrosis scores were not significant between two groups ([Table 1](#)). The mean IP-10 level in placebo group was slightly lower than the levels in vitamin D group (665.61 pg/mL vs 770.27 pg/mL) without statistical differences. While the mean DPP IV levels in placebo was slightly higher than the level in vitamin D group (6551.1 ng/mL vs 6137.7 npg/mL) without statistical differences. There were no significant differences in the baseline serum Th1/2 cytokines and DPP IV between two groups ([Table 1](#)). 

## Changes of parameters at 6-week supplements At the end of 6-week supplements, the median and range of serum 25(OH)D levels in the placebo and vitamin D groups were 21.22 (10.4--30.2) ng/mL, 45.93 (14.5--81.1) ng/mL, respectively. Vitamin D deficiency were corrected in all 40 patients who received vitamin D supplement. While vitamin D levels remained low and were not changed from the baseline levels in the placebo group ([Table 2](#) and [Fig 2](#)). ::::table-wrap
10.1371/journal.pone.0174608.t002

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## Comparing mean serum levels of each parameter between pre-and post-supplements in placebo and vitamin D groups.

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Variables (Unit in pg/mL) Placebo (40) Vitamin D (40)

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Pre: Mean (SE) Post: Mean (SE) P-value Pre: Mean (SE) Post: Mean (SE) P-value\
25(OH)D[\*](#) 20.59 (0.76) 21.87 (0.84) 0.229 20.88 (0.85) 45.93 (2.43) \<0.001[^A^](#)
IP-10 665.61 (77.05) 748.88 (87.12) 0.130 770.27 (101.51) 636.47 (70.91) 0.036[^B^](#)
DPP IV[\*](#) 6137.29 (250.50) 6408.33 (288.11) 0.386 6,551.06 (277.83) 6,032.37 (214.90) 0.028[^C^](#)
IL-2 23.07 (12.09) 28.69 (16.29) 0.318 8.20 (3.85) 4.49 (2.11) 0.120
IL-4 3.99 (0.79) 3.99 (0.96) 1.000 5.39 (0.98) 5.57 (1.12) 0.750
IL-5 4.69 (0.76) 4.47 (0.73) 0.717 7.60 (1.89) 8.06 (2.50) 0.623
IL-10 7.56 (3.00) 7.05 (2.66) 0.635 17.09 (7.04) 20.01 (8.82) 0.342
IL-12 29.25 (15.50) 26.80 (14.56) 0.670 40.61 (17.60) 50.11 (21.98) 0.292
IL-13 4.68 (1.27) 5.48 (1.52) 0.575 11.24 (3.57) 11.76 (4.95) 0.841
IFN-γ 251.64 (52.88) 237.28 (48.87) 0.594 328.31 (60.64) 304.90 (60.82) 0.429
TNF-α 602.45 (217.28) 548.33 (191.46) 0.884 651.52 (200.44) 752.94 (298.86) 0.487
GM-CSF 21.96 (9.01) 23.18 (7.63) 0.787 27.77 (6.91) 29.33 (9.92) 0.781

- unit in ng/mL;

^*A*^ 95% CI [20.65, 29.45];

^*B*^ 95% CI [-9.32, -258.28];

^*C*^ 95% CI \[-60.42, -983.15\]:::: <figure> 10.1371/journal.pone.0174608.g002 <p><img src="" /></p> <figcaption>Mean changes of vitamin D levels.<br /> (A), IP-10 (B) and DPP IV (C) levels after 6-week supplements in placebo and vitamin D groups were demonstrated. The x-axis of each graph represents mean changes of the levels (delta). The blue and orange boxes represent placebo group and vitamin D group, respectively.</figcaption> </figure> There were no differences of the transaminase levels at baseline and no changes after 6 weeks of supplements with placebo or vitamin D (data not shown). There were no significant differences of Th1 and Th2 cytokines levels in between group and no changes were observed during 6-week period of the study ([Table 3](#)). ::::table-wrap
10.1371/journal.pone.0174608.t003

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## Comparing changes in serum levels (or delta) of each parameter in placebo and vitamin D groups during 6-week period of supplement.

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Changes of serum or delta (Δ) levels (unit in pg/mL) Placebo group Vitamin D group Estimate (SE)[^B^](#) *P* values [^A^](#) Effect size [^C^](#)

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25(OH)D[\*](#) 0.560 25.050 24.399 (2.233) [^D^](#) \<.0001 2.47
IP-10 83.270 -133.800 -183.834 (71.852) [^E^](#) 0.0125 0.59
DPP IV[\*](#) 271.04 -518.69 -619.464 (281.070) [^F^](#) 0.0305 0.55
IL-2 5.630 -3.710 -5.973 (5.399) 0.2720 0.35
IL-4 0.001 0.179 0.200 (0.780) 0.7983 0.08
IL-5 -0.211 0.467 0.171 (1.065) 0.8728 0.18
IL-10 -0.506 2.922 2.129 (3.092) 0.4931 0.22
IL-12 -2.451 9.495 11.598 (10.624) 0.2784 0.24
IL-13 0.797 0.520 -1.158 (2.967) 0.6974 0.00
IFN-γ -16.119 1.014 115.927 (182.167) 0.5264 0.05
TNF-α -14.351 -23.407 2.515 (38.146) 0.9476 0.08
GM-CSF 1.223 1.611 0.966 (7.282) 0.8949 0.02

- Unit in ng/mL;

^*A*^ Comparison between two groups was performed through ANCOVA. In
ANCOVA, the dependent variable is the post-test measure, and the
pre-test measure was a [covariate](#) and controlled for.

^*B*^ Estimate is the adjusted difference between treatment and placebo
group (pre-test measure was adjusted for).

^*C*^ Cohen's d effect size: 0.2 small, 0.5 medium, 0.8 large magnitude
of effects

95% CI of estimate

^*D*^ [19.953, 28.844];

^*E*^ [-326.910, -40.758];

^*F*^ \[-1179.15, -59.781\]:::: After 6 weeks, the delta value of IP-10 level was decreased significantly in vitamin D group, as compared to the increased level in placebo group, -133.8 vs 83.27 pg/mL; 95% CI [326.910, -40.758], p = 0.0125 ([Table 3](#) and [Fig 2](#)). The mean serum DPP IV levels were not significant difference between pre-and post-supplements in both groups. During 6-week period, the mean DPP IV level in placebo group had trend to increase, and on the opposite direction, the mean level was decreased in vitamin D group. Comparing the mean changes (or delta changes) between the two groups showed significant differences, placebo group 255.46 ± 216.35 vs vitamin D group -521.79 ± 228.09; 95% CI [-1179.15, -59.781], p = 0.03 ([Table 3](#) and [Fig 2](#)). An effect size analysis (Cohen's d) was used to a quantitative measure of the strength of the outcome. The result showed strong magnitude of vitamin d changes (2.47), and moderate effect of VD on the changes of IP-10 (0.59) and DPP IV (0.55) levels ([Table 3](#)). The results also suggested that this RCT was performed with reasonable number of cases. We try to assess whether in this total population (80 CHC cases), serum levels of VD would affect the IP-10 levels in general. As shown in the scatter plot ([Fig 3](#)), even the dots are quite scattered, the best fit line could be plotted and the regression equation of [IP-10 = 841.34--4.45 x VD] was demonstrated. In other words, we could postulate that serum IP-10 decreases with increase in serum VD levels, and each unit of VD increase will lead to 4.5 unit decrease in serum IP-10. <figure> 10.1371/journal.pone.0174608.g003 <p><img src="" /></p> <figcaption>Scatter plot of post treatment VD levels (x-axis) and post treatment IP-10 level (y-axis) including fit line and regression equation were demonstrated.</figcaption> </figure>


Extracted trial result:
Outcome: Tumor necrosis factor alpha (continuous)
Intervention: Vitamin D supplementation
Comparator: Control
Intervention group size: 40
Intervention mean: 752.94
Intervention standard deviation: 1890.16
Comparator group size: 40
Comparator mean: 548.33
Comparator standard deviation: 1210.9