Abstract

Background

   Vitamin D contributes to bone health and extra-skeletal effects. The
   mechanisms underlying vitamin D metabolism have not been extensively
   evaluated. The relationships between vitamin D and inflammatory
   cytokines are debated. Our objective was to investigate whether
   supplemental interferons are associated with longitudinal change of
   vitamin D status in humans.

Methods

   A total of 48 patients with 24 or 48 weeks of pegylated interferon-α
   plus ribavirin therapy were examined for serum 25-hydroxyvitamin D
   (25[OH]D) level before treatment, at the end of treatment, and 24 weeks
   after treatment. In addition, we analyzed publicly available RNA
   sequencing data from accession [40]GSE42697 and [41]GSE7123 in the Gene
   Expression Omnibus.

Findings

   The overall sustained virologic response (SVR) rate was 62.5%. There
   was no statistically significant association between baseline 25(OH)D
   concentrations and liver fibrosis. In patients with SVR, serum 25(OH)D
   increased markedly at end-of-treatment and decreased markedly by the
   end of the 24-week follow-up period. In the non-SVR group, this
   treatment-dependent change was lost. In gene expression analysis, the
   vitamin D biosynthesis process was activated in subjects with SVR, but
   not in patients without SVR. Furthermore, vitamin D receptor (VDR)
   signaling in peripheral blood mononuclear cells (PBMCs) was triggered
   in marked responders but not in poor responders.

Conclusion

   In the aggregate, these data suggest that interferons have a regulatory
   influence on vitamin D status that can contribute to VDR signaling in
   PBMCs.

   Keywords: cytokines, interferon-α, PBMCs, VDR signaling, vitamin D

Introduction

   Vitamin D is of crucial importance for bone health, and it appears to
   have extra-skeletal influences.[42]^1^,[43]^2 Vitamin D can be procured
   from solar ultraviolet B radiation (wavelength 290–315 nm), the diet,
   or dietary supplements.[44]^3 The two major varieties of vitamin D
   (calciferol) are the plant form referred to as vitamin D2
   (ergocalciferol) and the animal form known as vitamin D3
   (cholecalciferol). The 25-hydroxylation of both forms of vitamin D
   happens in the liver via the major enzyme cytochrome P-450 family 2,
   subfamily R, polypeptide 1 (CYP2R1) to 25-hydroxyvitamin D (25[OH]D)
   that can be stored.[45]^4 The serum level of 25(OH)D reflects the
   adequacy of nutritional vitamin D status. The 25(OH)D is then
   transported into the blood stream to the proximal tubule of the kidney
   where it is 1α-hydroxylated to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or
   calcitriol), which is its only active form.[46]^5

   A low level of 25(OH)D is associated with a number of
   skeletal[47]^6^,[48]^7 and extra-skeletal complications, including
   cardiovascular diseases,[49]^8^,[50]^9 cancers,[51]^10 decreased muscle
   strength,[52]^11^,[53]^12 and autoimmune disorders such as type 1
   diabetes,[54]^13 rheumatoid arthritis,[55]^14^,[56]^15 systemic lupus
   erythematosus[57]^16 and ulcerative colitis.[58]^9^,[59]^17^,[60]^18
   This is because vitamin D is an essential micronutrient that regulates
   inflammatory events, skeletal muscle size and function, and collagen
   metabolism.[61]^9 Serum 25(OH)D concentrations are influenced by a
   number of factors. Evidence is emerging about dietary, environmental,
   and body compositional factors. The mechanisms by which these factors
   may influence vitamin D metabolism are not well understood.[62]^2

   Vitamin D is thought to be a strong immuno-modulator and that
   reciprocally, inflammation modulates vitamin D metabolism because
   systemic inflammation contributes to the reduction in serum 25(OH)D
   concentrations.[63]^9^,[64]^19^,[65]^20 However, previous studies
   exploring the relationships between vitamin D and inflammatory
   cytokines only focused on interferon-γ, interleukin (IL)-4, IL-6, IL-8,
   IL-10, tumor necrosis factor (TNF)-α, and leptin and have yielded
   inconsistent results.[66]^21–27 The regulatory influence of supplemental
   cytokines on vitamin D is still not clear.

   Before the launch of interferon-free treatments in 2013, the mainstay
   of hepatitis C virus (HCV) therapy in Taiwan was different treatment
   durations of peginterferon/ribavirin based on “response‐guided
   therapy”.[67]^28 The potential impact of ribavirin on the observations
   of vitamin D status is not obvious because serum concentrations of
   total 25(OH)D were not affected by ribavirin in one animal
   study.[68]^29 In addition to treatment of hepatitis B and C,
   interferon-α is an approved therapy to treat more than 14 types of
   cancer, including hairy cell leukemia, melanoma and renal cell
   carcinoma.[69]^30 The relationship between vitamin D and inflammatory
   cytokines remains controversial, and the assessment of vitamin D status
   and treatment response may be a potential indicator that parallels the
   different cancers. To date, no intervention study has determined
   whether an association exists between supplemental interferons and
   longitudinal change of 25(OH)D concentrations in humans. The primary
   aim of this study was to investigate whether interferon supplementation
   is associated with longitudinal changes in vitamin D status in humans,
   and we therefore performed a secondary analysis of a subset of
   hepatitis C subjects enrolled in our observational study.[70]^31

Materials and Methods

Subjects

   This study was performed in Taipei City (latitude: 25.1°N), Taiwan,
   where sunshine was sufficient all year.[71]^31 Since studies from Asia
   provide evidence supporting the response-guided treatment strategy for
   Asian patients,[72]^28 treatment-naïve patients with chronic HCV
   infection without cirrhosis received response-guided pegylated
   interferon α-based therapy, which was reimbursed by the Bureau of
   National Health Insurance in Taiwan and involved a 24-week treatment
   for patients with a rapid viral response (RVR) and a 48-week treatment
   for patients without an RVR.[73]^32 Chronic HCV infection was diagnosed
   based on the presence of anti-HCV antibodies and detectable serum HCV
   RNA. Subjects with hepatitis B, cirrhosis (Child–Pugh score ≥6),
   hepatocellular carcinoma, co-infection with human immunodeficiency
   virus, and autoimmune hepatitis were excluded. All patients offered a
   written informed consent form in order to engage in the study, which
   was approved by the Institutional Review Board of Tri-Service General
   Hospital, National Defense Medical Center, Taipei, Taiwan (protocol no.
   098–05-068) and was performed in accordance with the revised Helsinki
   Declaration.

Data and Sample Collection

   Variables, including age, gender, and body mass index (BMI), were
   recorded. During the study course, blood samples were obtained at the
   time of pre-treatment evaluation, the day after treatment (end of
   treatment), and at a scheduled clinic visit 24 weeks after treatment
   (post-treatment week 24). Complete blood count, HCV viral load, HCV
   genotype, aspartate aminotransferase (AST), alanine aminotransferase
   (ALT), intact parathyroid hormone (iPTH), calcium, bone alkaline
   phosphatase (BAP), creatinine, and phosphorus were measured using
   previously reported methods.[74]^31 Serum total 25(OH)D is the best
   reflection of vitamin D status and is very stable. Thus, levels of
   25(OH)D were assessed using chemiluminescence immunoassays (Liaison^®
   XL Analyzer; Diasorin, USA). The time from the sampling of serum stored
   at 2–8 °C to measurement was less than 5 days.

Definition of Terms

   The sustained virologic response (SVR) was defined as undetectable
   serum HCV RNA determined by polymerase chain reaction at 24 weeks after
   treatment cessation. Non-SVRs were considered patients in whom HCV RNA
   titers dropped < 2 log[10] from baseline at week 12, those whose viral
   loads dropped >2 log[10] at week 12 compared baseline, and who still
   had positive HCV RNA at week 24, as well as those who experienced HCV
   RNA reappearance after negativization before or at the end of
   treatment.[75]^33 Fibrosis staging was assessed based on the
   noninvasive index FIB-4, calculated using the formula: age (years) ×
   AST [U/L]/(platelets [10^9/L] ×ALT [U/L])^1/2, computed from laboratory
   results in all patients. Low (<1.45, no or minimal fibrosis),
   intermediate (1.45–3.25), and high (>3.25, significant fibrosis) FIB-4
   indexes were defined as previously established.[76]^34

Gene Expression Profiling

   The microarray array data of [77]GSE42697 published by Tsubota et
   al[78]^35 and [79]GSE7123 published by Taylor et al[80]^36 were
   downloaded from the National Center for Biotechnology Information Gene
   Expression Omnibus (GEO) database
   ([81]http://www.ncbi.nlm.nih.gov/geo/). Both datasets analyzed the
   response of CHC patients to interferon treatment by using whole gene
   expression profiling.

   In Tsubota’s study, the patients infected with HCV genotype 1b who were
   treated with 48- or 72-week duration of pegylated interferon α-2b at
   Jikei University Kashiwa-affiliated hospitals, Japan, were
   enrolled.[82]^35 Liver specimens of those patients were obtained before
   treatment to perform global mRNA expression analysis. [83]GSE42697
   collected liver tissue samples from 12 CHC patients for analysis, which
   included 5 patients with SVR and 7 non-responders. In Taylor’s study,
   African-American and Caucasian American patients treated for up to 48
   weeks with pegylated interferon α-2a were recruited to collect
   peripheral mononuclear blood cells (PBMCs) during the first 28 days of
   therapy. Global gene expression in PBMCs was analysed.[84]^36
   [85]GSE7123 collected peripheral blood from 25 CHC patients with poor
   viral response (<1.5 log[10] IU/mL decrease at 28 days) and 44
   responders (>1.5 log[10] IU/mL) for analysis before and during
   antiviral therapy. The maximum expression values of multiple probe IDs
   that corresponded to one official gene symbol were computed, and these
   values were used to represent the expression level of the related gene
   symbol.

Pathway Enrichment Analysis

   Gene Set Variation Analysis (GSVA) is a widely used approach for
   computing enrichment scores of given gene sets in single
   samples.[86]^37 We compared the results produced by the GSVA using “The
   Process of Vitamin D Biosynthesis (GO: 0042368)” and “Vitamin D
   receptor (VDR) signaling pathway (GO: 0070561)” gene signatures
   proposed by the Gene Ontology resource.[87]^38 The scores were computed
   in the R Bioconductor package gsva (v 1.34.0) with default parameters.

Statistical Analysis

   Continuous variables were presented as means ± SEM. The results for
   categorical variables were expressed as percentages. Comparisons amid
   groups were made by use of the unpaired Student’s t-test or
   Kruskal–Wallis test. To analyze the p-value of VDR’s GSVA scores
   overtime with multiple testing, Bonferroni correction was used.

Results

Demographics and Baseline Features

   A total of 48 patients, 27 (56.3%) women and 21 men (43.7%), were
   consecutively enrolled ([88]Figure 1). The average age was 53.3 ± 1.9
   years old (range: 22–79). Fifteen patients (31.3%) had vitamin D
   insufficiency (11–20 ng/mL) and 2 patients (4.16%) had vitamin D
   deficiency (≦ 10 ng/mL). None of these got any vitamin D supplements.
   The mean pretreatment 25(OH)D was 21.51 ± 0.7 ng/mL. The baseline
   demographic features of the patient cohort are summarized in [89]Table
   1 according to patients’ SVR status. Eighteen patients (37.5%)
   completed 24 weeks of therapy, while 30 patients (62.5%) were treated
   for 48 weeks. Overall, 30 patients achieved an SVR (62.5%). The SVR
   rate among the subset of patients who completed 24 weeks of therapy was
   100% (18 of 18 patients). A lower SVR rate (40%, 12 of 30 patients) was
   observed among the subset of patients who completed 48 weeks of
   therapy. Non-SVR was detected in 18 patients (37.5%). There was no
   association between gender, BMI, hemoglobin, white blood cells,
   platelet count, AST, ALT, iPTH, calcium, BAP, phosphorus, creatinine,
   HCV genotype, HCV RNA viral load, or FIB-4 index and in relation to
   SVR.

Figure 1.

   [90]Figure 1
   [91]Open in a new tab

   Participant flow chart.

Table 1.

   Demographic and Baseline Characteristics of Patients
   Characteristics SVR Group Non-SVR Group p value
   24-Week Treatment Group (n = 18) 48-Week Treatment Group (n = 12) (n =
   18)
   Age (years)^a 49.7 ± 2.9 51.3 ± 4.6 58.2 ± 2.5 0.12
   Sex (F, %) 10, 55.6 5, 41.7 12, 66.7
   BMI (kg/m^2)^a 24 ± 0.9 25 ± 2.9 25.7 ± 4.4 0.39
   Hemoglobin (g/dL)^a 13.7 ± 0.3 14.6 ± 0.4 13.7 ± 0.3 0.29
   White blood cells (× 10^9/L)^a 5.8 ± 0.5 5.9 ± 0.4 6.0 ± 0.3 0.67
   Platelets (× 10^9/L)^a 182.2 ± 15.2 175.3 ± 12.3 160.8 ± 12.7 0.59
   AST (U/L)^a 73.6 ± 16.8 83.7 ± 17.9 70.6 ± 9.4 0.88
   ALT (U/L)^a 115.2 ± 34.9 166.6 ± 44.3 91.1 ± 17.9 0.24
   iPTH (pg/mL)^a 31.4 ± 4.2 42.2 ± 13.2 28 ± 5.8 0.5
   Calcium (mg/dL)^a 9.3 ± 0.1 9.6 ± 0.1 9.5 ± 0.1 0.35
   BAP (U/L)^a 34.2 ± 3.5 53.2 ± 6.8 45.3 ± 9.3 0.08
   Phosphorus (mg/dL)^a 3.6 ± 0.1 3.8 ± 0.1 3.6 ± 0.1 0.63
   Creatinine (mg/dL)^a 0.8 ± 0.05 0.8 ± 0.06 0.8 ± 0.04 0.85
   HCV subtype
   1a (n, %) 1, 5.6 1, 8.3 0
   1b (n, %) 6, 33.3 5, 41.7 14, 77.8
   2a (n, %) 7, 39.9 3, 25 4, 22.2
   2b (n, %) 2, 11.1 2, 16.7 0
   1b+2a (n, %) 2, 11.1 1, 8.3 0
   Baseline HCV RNA > 10,000,000 IU/mL, n (%)^a 2 (11.1) 2 (16.7) 5 (27.8)
   0.43
   FIB-4 index^a 2.6 ± 0.6 2.2 ± 0.4 3.9 ± 0.9 0.17
   [92]Open in a new tab

   Notes: ^aMean ± s.e. of the mean.

   Abbreviations: 25(OH)D, 25-hydroxyvitamin D; ALT, alanine
   aminotransferase; AST, aspartate amino transaminase; BAP, bone alkaline
   phosphatase; BMI, body mass index; FIB-4, fibrosis-4; HCV, hepatitis C
   virus; iPTH, intact parathyroid hormone; SVR, sustained virologic
   response.

Vitamin D Status and Hepatic Fibrosis

   The mean FIB-4 index at baseline showed 2.89 ± 0.4. FIB-4 index
   prevalence was: low in 33.3% (n = 16), intermediate in 35.4% (n = 17),
   and high in 31.2% of the patients (n = 15), as shown in [93]Figure 2A.
   Mean 25(OH)D level did not significantly differ amid fibrosis stage (no
   or minimal fibrosis: 21.35 ± 1.5 ng/mL, intermediate FIB-4 index: 21.61
   ± 1.4 ng/mL, significant fibrosis: 21.56 ± 0.7 ng/mL; P = 0.98), as
   illustrated in [94]Figure 2B. There was no significant relationship
   amid 25(OH)D concentrations and liver fibrosis (r = −0.016, P = 0.91)
   as illustrated in [95]Figure 2C.

Figure 2.

   [96]Figure 2
   [97]Open in a new tab

   FIB-4 index and 25(OH)D level. (A) The proportion among patients with
   different scores of FIB-4 index showed an equal distribution. (B) Mean
   25(OH)D concentrations did not vary amid high, intermediate, and low
   FIB-4 index. (C) Mean 25(OH)D levels did not vary amid stages of
   fibrosis according to FIB-4 index.

Vitamin D and Viral Response

   Antiviral therapy led to a significant increase in the baseline 25(OH)D
   by the end of treatment in patients completing 24 weeks of therapy with
   SVR (18.90 ± 0.99 ng/mL vs 23.74 ± 1.19 ng/mL; P = 0.0084, [98]Figure
   3A). Likewise, 25(OH)D raised significantly by the end of treatment in
   comparison to baseline levels in the patients completing 48 weeks of
   therapy with SVR (21.74 ± 1.40 ng/mL vs 27.42 ± 2.29 ng/mL; P = 0.0487,
   [99]Figure 3B).

Figure 3.

   [100]Figure 3
   [101]Open in a new tab

   25(OH)D serum concentrations of 48 HCV patients are shown at baseline,
   end of treatment, and 24 weeks after treatment completion. In patients
   completing 24 (A) or 48 (B) weeks of therapy with SVR, an increase in
   the mean 25(OH)D level (ng/mL) was observed with significant difference
   at the end of treatment compared with at baseline. A decrease in the
   mean 25(OH)D level (ng/mL) was also observed with significant
   difference at 24 weeks after end of therapy compared with at the end of
   treatment. (C) Patients without SVR had no significant changes of
   vitamin D concentrations at the end of treatment or 24 weeks after
   cessation of treatment. *p < 0.05 compared with baseline, **p < 0.01
   compared with baseline, ^#p < 0.05 compared with end-of-treatment.

   Abbreviations: ns, not significant; SVR, sustained virologic response.

   All 48 patients underwent additional 25(OH)D measurements by the end of
   the 24-week follow-up period. In comparison with end-of-treatment
   values, 25(OH)D tended to diminish in the majority of patients with SVR
   after completing 24 weeks of therapy (23.74 ± 1.19 ng/mL vs 18.53 ±
   1.28 ng/mL; P = 0.0102, [102]Figure 3A). Similarly, a significant
   portion of the patients with SVR after completing 48 weeks of therapy
   had lower 25(OH)D by the end of the 24-week follow-up period as
   compared with end-of-treatment values (27.42 ± 2.29 ng/mL vs 22.92 ±
   1.24 ng/mL; P = 0.0320, [103]Figure 3B).

   In contrast, patients with non-SVR had no on-treatment change of
   25(OH)D during antiviral therapy or thereafter by the end of the
   24-week follow-up period ([104]Figure 3C).

Vitamin D Biosynthetic Process and Viral Response

   To ascertain the influences of interferons on vitamin D synthesis and
   metabolism, we analyzed the metadata of the GEO. “The Process of
   Vitamin D Biosynthesis (GO: 0042368)” was used to assess the effect of
   interferon therapy on the ability of the liver to synthesize vitamin D
   in CHC patients with or without SVR. The vitamin D synthesis ability in
   the liver of each CHC patient in [105]GSE42697 was quantified using the
   GSVA method. Patients with SVR had significantly higher GSVA scores of
   the vitamin D biosynthesis process as compared to those with non-SVR
   ([106]Figure 4).

Figure 4.

   [107]Figure 4
   [108]Open in a new tab

   Gene expression profiles reveal an association between vitamin D
   biosynthesis process and the viral response to interferon-based
   treatment. The gene set variation analysis (GSVA) score of the vitamin
   D biosynthesis process was significantly greater in patients with SVR
   in comparison with those with no response. *p < 0.05.

   Abbreviation: SVR, sustained virologic response.

Vitamin D Biosynthetic Process and Vitamin D Receptor (VDR) Signaling in
Peripheral Blood Mononuclear Cells (PBMCs)

   Vitamin D status contributes to VDR protein levels in PBMCs, which
   respond to interferon-based therapy.[109]^39^,[110]^40 To investigate
   the association between the interferon-induced vitamin D biosynthesis
   and activation of VDR signaling in PBMCs, “VDR signaling pathway (GO:
   0070561)” was applied and quantified by GSVA in HCV patients in
   [111]GSE7123 with distinct treatment response. Interestingly, when
   changes in scores of VDR signaling pathway in PBMCs were compared, the
   increase in GSVA score was statistically significant in the marked
   responder group on days 7 (p = 0.03), 14 (p <0.001), and 28 (p <0.001)
   but not in the poor responder group ([112]Figure 5).

Figure 5.

   [113]Figure 5
   [114]Open in a new tab

   Gene expression profiles reveal an association between vitamin D
   receptor (VDR) signaling in peripheral blood mononuclear cells (PBMCs)
   and the viral response to interferon-based treatment. The gene set
   variation analysis (GSVA) scores of VDR signaling pathway in PBMCs from
   patients who have marked viral response to interferon-based treatment
   were significantly elevated on days 7, 14, and 28, but this was not the
   case for poor responders.*p < 0.05 in comparison to baseline, ***p <
   0.001 in comparison to baseline.

   Abbreviation: ns, not significant.

Discussion

   In this study, there was a relatively low prevalence of serum 25(OH)D
   deficiency, which was compared with the results of other studies that
   have mainly focused on naïve genotype 1 HCV-infected
   patients.[115]^41^,[116]^42 Furthermore, we demonstrate for the first
   time that an association between supplemental interferons and changes
   in 25(OH)D concentrations occurs in response to the interferon-based
   therapy in HCV-positive patients. Activations of VDR signaling in PBMCs
   were connected with increased 25(OH)D circulating levels ([117]Figure
   6).

Figure 6.

   [118]Figure 6
   [119]Open in a new tab

   A schematic model proposed for the association between vitamin D and
   interferon-α in chronic hepatitis C patients without cirrhosis. Dynamic
   changes of 25-hydroxyvitamin D (25[OH]D) concentrations during
   pegylated interferon-α based therapy in patients are stratified by
   sustained viral response (SVR) and non-SVR. (A) In the SVR cohort,
   pegylated interferon-α engages receptors on the hepatocyte cell-surface
   membrane, activating Janus-activated and tyrosine kinases (JAK) that
   phosphorylate the cytoplasmic signal transducers and activators of
   transcription (STAT) proteins. Then, STAT binds interferon regulatory
   factor 9 (IRF9), creating interferon-stimulated gene factor 3 (ISGF3),
   which is translocated into the nucleus where it attaches
   interferon-stimulated response elements to DNA. This engagement
   instigates transcription of multiple (>100) interferon-stimulated gene
   mRNAs to inhibit hepatitis C virus (HCV) replication. Moreover,
   carriers of the “A” allele of CYP2R1 rs10741657 have upsurges of
   25(OH)D concentrations following interferon-α therapies. In addition to
   25(OH)D suppressing HCV replication, the biologically active vitamin D
   metabolite 1,25‐dihydroxyvitamin D[3] (1,25[OH][2]D[3]) directly
   suppresses HCV-RNA replication. Furthermore, antiviral vitamin
   D–vitamin D receptor (VDR) signaling in peripheral blood mononuclear
   cells (PBMCs) is activated to improve the response to interferon-based
   therapy. (B) In the non-SVR cohort, pegylated interferon-α has a poor
   inhibitory effect on HCV replication through transient Jak/STAT
   activation alone in non-“A” allele carriers. Furthermore, VDR signaling
   in PBMCs is not activated, and PBMC proliferation is not inhibited.

   Liver biopsy has been used as the “gold standard” for assessment of
   hepatic fibrosis in patients with CHC, one of the limitations of biopsy
   is diagnostic inaccuracy secondary to sampling error, observer
   variation, and specimen size.[120]^43 Non-invasive assessment of liver
   fibrosis by elastography techniques has mostly been validated in the
   context of CHC. However, elastography is also unable to reliably
   differentiate between adjacent fibrosis stages, and there is
   considerable overlap in liver stiffness measure for intermediate stage
   disease. Moreover, different elastography devices may provide
   discordant results for defining liver fibrosis staging in CHC.[121]^43
   Furthermore, FIB-4 scores may have good diagnostic performances for
   fibrotic stage assessment compared with elastography, especially for
   cirrhosis.[122]^44 FIB‐4 scores are recommended by World Health
   Organization to assess hepatic fibrosis in resource‐limited setting
   rather than elastography.[123]^45 On the other hand, the clinical
   literature concerning the relationship between serum vitamin D and the
   severity of liver fibrosis is more controversial. Subjects with vitamin
   D deficiency were reported to be associated with more advanced fibrosis
   stage and higher necro-inflammatory grade.[124]^42^,[125]^46 In
   contrast, it has been demonstrated that there was no independent
   relationship between vitamin D status and fibrosis
   stage.[126]^47^,[127]^48 Given the limitations and variability of tests
   for assessment of liver fibrosis, our results did not find a possible
   existence of association between vitamin D status and hepatic fibrosis,
   and they further pointed toward a weak relationship between dynamic
   changes of vitamin D status and hepatic fibrosis.

   The relation between baseline vitamin D status and response to
   antiviral therapy is still a matter of debate. Some studies indicated
   that baseline 25(OH)D concentration was related to
   SVR,[128]^41^,[129]^42^,[130]^46 whereas other investigators discovered
   no relationship between vitamin D concentration and achievement of
   SVR.[131]^47^,[132]^49 In this study, we found that baseline 25(OH)D
   concentration was not independently connected with attainment of SVR.
   The discrepancy between these studies can probably be accounted for by
   the use of different measurement techniques, vitamin D cut-off values,
   and varied genotypes. On the other hand, the directionality of the
   association between viral response and dynamic changes of 25(OH)D
   levels can be measured with validity from our experimental design.
   Three scenarios could be proposed: (1) supplemental interferons
   modulate 25(OH)D concentrations with the disappearance of HCV; (2)
   supplemental interferons cannot modulate 25(OH)D levels with the
   existence of HCV; and (3) 25(OH)D levels are decreased after stopping
   supplemental interferons with the disappearance of HCV.

   IL-28B single nucleotide polymorphisms (SNPs) may play an important
   role in the management of HCV infection. However, the predictive role
   of IL-28B genotype is limited for HCV genotype 2 and 3 patients and for
   HCV patients with response-guided therapy.[133]^50 Moreover, SNPs in
   the IL-10 do not predict the treatment outcome of patients with HCV
   infection who are treated with pegylated interferon plus
   ribavirin.[134]^51 Furthermore, genetic polymorphisms in the inosine
   triphosphatase (ITPA) gene are associated with protection from
   ribavirin-associated anemia in patients; however, ITPA variants are not
   associated with the SVR.[135]^52 Accordingly, the association of SNPs
   with HCV infection is still ambiguous. On the other hand, circulating
   25(OH)D levels mirror not only vitamin D stores but also hepatic
   hydroxylation. Since the vitamin D metabolizing enzyme CYP2R1 encodes
   vitamin D 25-hydroxylase that transforms cholecalciferol to 25(OH)D,
   genetic variation in CYP2R1 is known to affect 25(OH)D
   concentrations.[136]^53 Indeed, carriers of the “A” allele of CYP2R1
   rs10741657 have statistically significantly greater serum 25(OH)D
   concentrations.[137]^54–57 Moreover, the treatment response following
   an interferon-based regimen has been previously reported to be
   associated with the carrier state of the “A” allele of CYP2R1
   rs10741657 and SVR.[138]^58 In addition, our findings indicate that
   interferon antiviral response, but not the existence of HCV and hepatic
   injury, is involved in altered 25(OH)D circulating levels. It is
   therefore tempting to suggest a genetic link between vitamin D
   biosynthetic process and interferon antiviral response.

   Our finding that vitamin D status is associated with an interferon
   modulation could be interpreted in several ways. In addition to the
   fact that pegylated interferon-α regulates hepatic gene expression
   through transient Janus-activated and tyrosine kinases (JAK)/signal
   transducers and activators of transcription (STAT) activation,[139]^59
   one explanation is that the inhibitory role of interferon-α on HCV is
   mediated through the antiviral effect of 25(OH)D, which is in agreement
   with experimental in vitro models.[140]^60^,[141]^61 Moreover, previous
   interventional and clinical studies[142]^62^,[143]^63 provide evidence
   that vitamin D3 supplementation raises rates of EVR or SVR following
   treatment with pegylated interferon-α-based therapy for CHC, although
   our study was observational and cannot directly prove causality.
   Another explanation is that the biologically active vitamin D
   metabolite, 1,25(OH)2D3, represses HCV-RNA reproduction in a
   dose-dependent way in cell culture, and interferons have a synergistic
   effect on reduced HCV secretion,[144]^64 which is in line with the in
   vitro study that indicates that synthetic calcitriol analogs inhibit
   HCV replication. This concept appears to be consistent with a role for
   1(OH) vitamin D3, which becomes 1,25(OH)2D3 in the liver and has been
   found to have an effect on improving the sensitivity of
   interferon-based therapy on HCV-infected hepatocytes.[145]^40

   Another major finding in our study was that there exists a more
   antiviral form of vitamin D-VDR signaling in PBMCs in CHC patients
   undergoing interferon-based treatment. VDR signaling in PBMCs may
   represent a novel predictor of the effectiveness of antiviral therapy
   in naïve HCV-infected patients. Indeed, it is reported that
   administration of vitamin D3 reduces PBMC proliferation and the
   cytokine (TNF-α, interferon-γ, and interferon γ-induced protein 10)
   production in PBMCs.[146]^39^,[147]^40 Based on our findings, we
   speculate that vitamin D3 may act directly on the VDR in PBMCs to
   inhibit PBMCs’ activation and affect the production of cytokines from
   PBMCs to improve the response to interferon-based therapy.

   The concept of the personal vitamin D response index (VDRI) portrays
   the efficacy of the molecular response to supplementation with vitamin
   D. Individuals can be classified as high, intermediate, and low
   responders to vitamin D. Variations in the VDRI may be partially on the
   basis of genetic variations in vitamin D metabolizing enzymes and
   genomic VDR binding and chromatin opening in PBMCs.[148]^65 Thus,
   changes in vitamin D sensitive molecular parameters, including the
   epigenetic condition and the corresponding transcription of genes of
   PBMCs during pegylated interferon-based therapy for CHC, may
   distinguish responders from non-responders to antiviral therapy.

   Our study has some major strengths. First, our study demonstrates for
   the first time that 25(OH)D levels positively correlate with
   supplemental interferons in humans, rather than over-relying on in
   vitro or animal models, although ours is not a randomized controlled
   trial. Second, our study was prospective and clearly established a
   homogenous cohort of patients with non-cirrhotic chronic HCV
   infections. Third, our study was physician- and researcher-blinded;
   physicians evaluating for viral response during our treatment periods
   were blinded to baseline serum vitamin D. Researchers assessing serum
   vitamin D levels were also blinded to clinical consequences. The
   de-identified serum samples were not matched to their corresponding
   clinical results until after the study period had closed. Fourth, our
   study recruited two independent cohorts to investigate pathway
   enrichment analysis, which allowed us to gain mechanistic insight into
   gene lists generated from genome-scale (omics) experiments.

   This study has many limitations that are worth attention. First, our
   sample size was small and only included CHC patients. This restrains
   the generalizability of our findings to only CHC patients rather than a
   chronic hepatitis B cohort. Second, we did not assess 1,25(OH)2D3
   levels, which may have clarified whether there are variances in the
   physiologically active form and dynamic changes in patients with CHC.
   Third, our study is observational and restrained by the incapability to
   explain possible unmeasured confounders. For example, physical activity
   and sunscreen use were not to be taken into consideration.

Conclusions

   In conclusion, several parameters influence vitamin D levels. As well as
   these factors, we posit that there is an interferon-α–vitamin D axis,
   at least in men. Extra studies will be required to investigate the
   mechanisms accountable to this interferon-α–vitamin D association.
   Therefore, a new project by the same team on a cohort of patients
   treated with direct-acting antiviral drugs is now in progress to
   further clarify the influence of interferons and liver inflammation on
   vitamin D levels in humans. In the meantime, results of the present
   study emphasize the relevance of interferon-α, circulating vitamin D
   levels, and VDR signaling in the circumstance of innate immunity
   concentrated in PBMCs.

Acknowledgments