Vitamin D deficiency is common in HIV/hepatitis C virus (HCV) coinfected patients . In HIV-infected patients, vitamin D status has been associated with non-AIDS-related outcomes, AIDS-related outcomes, and chronic inflammation . In HCV infection, vitamin D deficiency has been associated with chronic inflammation, liver fibrosis progression and chronic hepatitis C (CHC)-related outcomes [3,4].
Vitamin D is an important modulator of immune response. Vitamin D receptor (VDR) is widely expressed in most adaptive immune system cells and it has been described as a critical role of vitamin D/VDR system in the control of T-cells activation and cytokine secretion . Intestinal epithelial VDR has also demonstrated to be implicated in maintaining the integrity of the intestinal mucosal barrier  and to play a protective role in the mucosal injury .
The disruption of gut epithelial integrity, related to HIV infection and cirrhosis, increases mucosal translocation of bacteria and bacterial products (bacterial DNA and endotoxin) from the intestinal lumen to the systemic circulation . Different markers of bacterial translocation have been evaluated, such as plasma levels of the DNA sequences encoding the well conserved 16S rRNA subunit (16S ribosomal DNA), common to most bacteria . The bacterial translocation is a major cause of systemic immune activation in HIV infection and CHC, and accelerates the course of liver damage in HIV/HCV coinfected patients . Given the evidence for a protective role of vitamin D in the digestive mucosal integrity, it is reasonable to hypothesize that vitamin D may reduce the markers of bacterial translocation. Similarly, an interventional study on vitamin D supplementation in patients with chronic kidney disease demonstrated reduced blood endotoxin activity .
Our major aim was to analyze the association between plasma levels of 25-hydroxy-vitamin D [25(OH)D, the main circulating vitamin D metabolite] and bacterial 16S ribosomal DNA (bactDNA) in HIV/HCV coinfected patients. We also analyzed the relationship between plasma levels of 25(OH)D and inflammation biomarkers, as a minor objective.
Patients and methods
A cross-sectional study was performed in 120 HIV/HCV coinfected patients who underwent a liver biopsy between 2000 and 2008. Liver biopsies were performed on patients who were potential candidates for anti-HCV therapy and had not received previous interferon therapy.
The study was approved by the Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) and it was conducted in accordance with the Declaration of Helsinki.
Clinical and laboratory data
Clinical and epidemiological data were obtained from medical records on the day of sample collection. Consumption of more than 50 g of alcohol per day for at least 12 months was considered as a high intake. Biochemistry panel was measured using an autoanalyzer Hitachi 912 (Boehringer Mannheim, Mannheim, Germany), while patients were fasting.
The seasonality was stratified as first semester (winter/spring) vs. second semester (summer/autumn).
Liver biopsies were performed as we previously described . Liver fibrosis and necroinflammatory activity were estimated according to Metavir score as follows : F0, no fibrosis; F1, mild fibrosis; F2, significant fibrosis; F3, advanced fibrosis; and F4, definite cirrhosis. The degree of necroinflammation (activity grade) was scored as follows: A0, no activity; A1, mild activity; A2, moderate activity; A3, severe activity.
In addition, several plasma biomarkers were assayed: Chemokines CXCL1, CXCL5, CXCL9, CXCL10, CCL2, CCL7 and CCL11 were measured using LINCOplex kit (LINCO Research, St. Charles, Missouri 63304, USA) in a Luminex 100 analyzer (Luminex Corporation, Austin, Texas, USA) according to the manufacturer's specifications. Basic fibroblast growth factor (bFGF) was measured via enzyme-linked immunosorbent assay of Invitrogen Corporation (Camarillo, California, USA).
25-hydroxy vitamin D measurement
Plasma samples were obtained and stored at –80°C at the same time of the liver biopsy. Plasma 25-hydroxy vitamin D [25(OH)D] was quantified by enzyme immunoassay according to the manufacturer[Combining Acute Accent]s instructions for the test [25-Hydroxy Vitamin D EIA kit, Immunodiagnostic Systems Ltd (IDS Ltd), Boldon, UK]. Vitamin D deficiency was defined as a 25(OH)D plasma level below 25 nmol/l (<10 ng/ml), vitamin D insufficiency as 25(OH)D plasma level from 25 nmol/l to 74 nmol/l (10–30 ng/ml), and vitamin D sufficiency as a 25(OH)D plasma level above 75 nmol/l (>30 ng/ml) .
Quantitative real-time PCR for bacterial 16S rDNA detection
DNA was extracted from 200 μl of plasma using the QIAamp Mini-Elute Virus Spin Kit (Qiagen, Hilden, Germany) in a QIAcube automated extractor (Qiagen), as recommended by the manufacturer. The PCR was performed in a LightCycler Instrument version 1.5 (Roche Molecular Biochemicals, Mannheim, Germany) to amplify the DNA sequence encoding the well conserved 16S rRNA subunit (16S rDNA) and the conditions for the amplification reaction were as previously described . Several statistical cutoffs were selected for 16S rDNA plasma levels: low [<46 copies/μl (25th percentile, p25th)], moderate [46 copies/μl (p25th) to 78 copies/μl (p50th)], high [78 copies/μl (p50th) to 159 copies/μl (p75th)], and very high [>159 copies/μl (p75th)].
The statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) 19.0 (SPSS INC, Chicago, Illinois, USA). Values were expressed as absolute number (percentage) and median (p25th; p75th). Statistical significance was defined as P < 0.05. All P values were two-tailed.
Categorical data and proportions were analyzed using the χ2 test or Fisher[Combining Acute Accent]s exact test. Mann–Whitney U test or Kruskal–Wallis test were used to compare data among independent groups. We also performed adjusted logistic regression analyses to evaluate the association between the vitamin D status (factor) and plasma bactDNA levels (outcome). We included the vitamin D status with the Enter algorithm (Forced Entry) and the covariables with the Stepwise algorithm (at each step, factors are considered for removal or entry: a P value for entry and exit of 0.15 and 0.20, respectively). Thus, each logistic regression test was only adjusted by the most significant covariables associated with each one of the outcome variables, avoiding the over-fitting of the regression. The covariables used were age, sex, seasonality (winter/spring vs. summer/autumn), intravenous drug users (IVDU), high alcohol intake, body mass index, nadir CD4+, combination antiretroviral therapy (cART), undetectable HIV-RNA (<50 copies/ml), CD4+ cell count, prior AIDS-defining conditions, HCV genotype 1, Metavir fibrosis stage F not less than 2 and HCV-RNA at least 500 000 IU/ml.
This study included 120 HIV/HCV coinfected patients, whose characteristics at the time of liver biopsy are shown in Table 1. The median age was 39.9 years, 71.7% were male, and 40.8% showed prior AIDS-defining conditions. On the date of the sample collection, CD4+ cell count was 449 cells/μl, 69.7% had an HIV-RNA below 50 copies/ml, 83.3% patients were on cART, 51.7% had HCV genotype 1, and 75% had an HCV-RNA above 500 000 UI/ml. Furthermore, 63.3% of the patients showed significant fibrosis (F≥2) and 64.2% a moderate activity grade (A≥2).
The median of plasma 25(OH)D level was 48.6 nmol/l (p25th: 41.3; p75th: 56). Moreover, 18 (15%) patients had vitamin D deficiency, 93 (77.5%) patients had vitamin D insufficiency and nine (7.5%) patients had normal values of vitamin D. When patients were stratified according to vitamin D status (Table 1), we found that patients with 25(OH)D deficiency had higher percentage of prior AIDS-defining conditions (prior-AIDS; P = 0.047) and significant fibrosis (F≥2; P = 0.045). No relationship was detected between bactDNA levels and liver fibrosis (data not shown).
Vitamin D status and bacterial translocation
Figure 1 shows the relationship between plasma 25(OH)D level and plasma bactDNA levels in HIV/HCV coinfected patients. The median plasma bactDNA level was 78.6 copies/μl (p25th: 46.3; p75th: 159.2). Plasma bactDNA levels were significantly lower in patients with optimal 25(OH)D status (≥75 nmol/l) [37 copies/μl (p25th:25.4; p75th:103.6)] than patients with insufficient 25(OH)D status (25–74 nmol/l) [84.2 copies/μl (p25th: 52; p75th: 162.1)] (P = 0.042), and nearly significant compared with patients who had 25(OH)D deficiency (<25 nmol/l) [77.3 copies/μl (p25th: 45; p75th: 143.5)] (P = 0.060) (Fig. 1a). Conversely, low bactDNA levels (<p25th) were found in 66.7% of the patients with optimal 25(OH)D status, whereas plasma bactDNA levels between p25th and p50th, p50th and p75th, and above p75th were found in 11.1% of the patients with optimal 25(OH)D status in each range (P = 0.029) (Fig. 1b).
Furthermore, adjusted logistic regression analyses showed that plasma 25(OH)D not less than 75 nmol/l was associated with low plasma bactDNA levels (<p25th) [adjusted OR = 8.13 (95% confidence interval, CI = 1.82; 36.67); (P = 0.006)].
Vitamin D status and plasma inflammation markers
Plasma inflammation markers were available in 63 of 120 HIV/HCV coinfected patients. The patients with optimal vitamin D status [25(OH)D ≥75 nmol/l] had lower levels of all the markers studied, being this difference significant for CCL7 (P = 0.047) and bFGF (P = 0.042), and nearly significant for CXCL1 (P = 0.082), CXCL9 (P = 0.055) and CCL11 (P = 0.059) (See Supplemental Table 1, http://links.lww.com/QAD/A860).
The major result of our study was the significant association between optimal vitamin D plasma levels and low plasma levels of bactDNA and inflammation biomarkers. To our knowledge, this is the first study that correlates plasma vitamin D status with bacterial translocation in HIV/HCV coinfected patients.
The findings of our study are supported by previous pieces of evidence reported about the link between vitamin D status and mucosal barrier integrity [6,7]. In addition, vitamin D deficiency accelerates the development of experimental colitis and other inflammatory diseases, whereas vitamin D treatment protects against colitis and intestinal inflammation [16–18]. Although the mechanism by which vitamin D affects the intestinal barrier is incompletely understood, there are supporting data that vitamin D regulates intestinal barrier function through the epithelial vitamin D/VDR signaling system [6,19], which preserves the mucosal barrier integrity and epithelial barrier permeability by regulating the tight junction . Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased intestinal permeability.
Bacterial translocation is a phenomenon that happens frequently in HIV-infected patients. The HIV infection-related depletion of mucosal CD4+ lymphocytes has been linked to disruption of gut epithelial integrity and increased bacterial translocation, and consequently with a persistent systemic inflammation . There are contradictory results regarding the association between vitamin D status and plasma biomarkers. Although a deficient vitamin D status has been correlated with increased levels of plasma inflammatory markers by Ansemant et al., this relationship among vitamin D status, microbial translocation and inflammation could not be demonstrated in another cohort of HIV-infected patients . In our study, we found that optimal vitamin D plasma levels were linked to low plasma levels of bactDNA and inflammation biomarkers. As Missailidis et al. discussed, a possible explanation to this observed disparity could be the differences in study populations.
The systemic immune activation in HIV/HCV coinfection linked to bacterial translocation accelerates the course of liver damage in these patients . Furthermore, hepatic macrophages or Kupffer cells are responsible for clearing bacterial translocation products; but these cells may be infected by HIV and it might result in their impaired ability to clear these bacterial translocation products . Moreover, bacterial translocation is a mechanism that contributes to liver diseases [15,24]. There is some evidence that bacterial translocation promotes hepatic fibrogenesis  and the presence of bactDNA in patients with cirrhosis is associated with hemodynamic consequences . As bacterial translocation is an event which has consequences in HCV liver disease progression, it is of interest to assess the benefit for liver function of preventing intestinal bacterial translocation.
In this study, plasma vitamin D status also correlated with significant fibrosis, as we described in a previous article . However, no association was detected between bactDNA levels and liver fibrosis; in contrast to a previous article in which we found such association probably due to the use of a larger number of HIV/HCV coinfected patients .
In conclusion, the optimal vitamin D status was associated with low bacterial translocation and inflammation in HIV/HCV coinfected patients, suggesting that vitamin D treatment could be a useful therapeutic target in the management of bacterial translocation related to HIV/HCV coinfection.
Funding/Support: This work has been supported by grants given by Fondo de Investigación de Sanidad en España (FIS), Spanish Health Founds for Research (grant numbers PI11/01556, PI14/01094, PI11/00245, PI14CIII/00011), Red Española de Investigación en SIDA (RIS), AIDS Research Network (grant numbers RD12/0017/0024 and RD12/0017/0004) and ‘Fundación para la Investigación y la Prevención del Sida en España’ (FIPSE) (grant number 361020/10).
This work has been (partially) funded by the RD12/0017/00XX project as part of the Plan Nacional R + D + I and cofinanced by ISCIII – Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER).
J.B. is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS), Refs INT10/009 and INT12/154. M.G.A. and M.A.J.S. are supported by ‘Instituto de Salud Carlos III’ (grant numbers CD12/00442, CD13/0001, respectively).
Author's contributions: study concept and design: M.G.A., J.B., and S.R. Acquisition of data: J.B., J.C.L., A.C., and T.A. Analysis and interpretation of data: M.G.A. and S.R. Administrative, technical, or material support: M.G.A., M.A.J.S., M.G.R., and P.G.B. Drafting of the manuscript: M.G.A., M.A.J.S., and S.R. Critical revision of the manuscript for important intellectual content: J.B. Statistical analysis: M.G.A. and S.R. Study supervision: S.R.
Conflicts of interest
The authors have no commercial or other association that might pose conflicts of interest.
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