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Microbes and Infection xx (2014) 1e7 www.elsevier.com/locate/micinf

Original article

Vitamin D supplementation promotes macrophages anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression Q4

Nallely Lopez-Lopez a, Irma Gonzalez-Curiel a, Julio Casta~ neda-Delgado a, Alejandra Montoya-Rosales a, Benjamin Gandara-Jasso a,b, Jose Antonio Enciso-Moreno a, Bruno Rivas-Santiago a,* a

Medical Research Unit Zacatecas, Mexican Institute for Social Security, Zacatecas, Mexico b Medical Research Coordination SSA-Zacatecas, Zacatecas, Mexico Received 2 April 2014; accepted 24 June 2014

Abstract The increasing number of people with type 2 diabetes (DM2) is alarming and if it is taken into account that the relative odds of developing tuberculosis in diabetic patients ranges from 2.44 to 8.33 compared with non-diabetic patients, thus in developing countries where these two diseases are encountering face to face, there is a need for prophylaxis strategies. The role of vitamin D has been widely implicated in growth control of Mycobacterium tuberculosis (Mtb) during primary infection mainly through the induction of certain antimicrobial peptides (AMPs). In this study we evaluated the vitamin D serum levels, CYP27B1-hydroxylase enzyme, vitamin D receptor (VDR) and AMPs gene expression in Healthy donors, DM2 and TB patients. Results showed that DM2 group has lower VDR and AMPs expression levels. When Monocytes Derived Macrophages (MDM) from DM2 patients with low VDR expression were supplemented with vitamin D, MDMs eliminate efficiently M. tuberculosis. This preliminary study suggests the use of vitamin D as prophylaxis for tuberculosis in high DM2 endemic countries. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Q1

Keywords: Diabetes; Vitamin D; Antimicrobial peptides; Tuberculosis; Vitamin D receptor (VDR); CYP27B1-hydroxylase enzyme; Cathelicidin; Defensin

1. Introduction Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a public health problem, indeed causes 1.8 million deaths annually according World Health Organization bulleting. Although a wide proportion of the population in developing countries is exposed to the bacillus only a minority is infected and develops the progressive disease, however, infection spreads faster within susceptible groups such as in HIV-infected individuals and type 2 diabetes mellitus (DM2)

* Corresponding author. Medical Research Unit Zacatecas, IMSS. Interior de la Alameda #45, col. Centro, Zacatecas, Cp.98000, Mexico. E-mail address: [email protected] (B. Rivas-Santiago).

patients [1,2]. Certainly, the incidence of TB is two to eight times higher in patients with diabetes than in those individuals without it, which is quite comparable to the odds seen with the HIV-infected patients [3,4]. With rising rates of obesity and diabetes, particularly in low-income countries where high rates of TB remain higher, there is concern that DM2 poses a threat to global TB control. Recently, several researching groups have proposed vitamin D as an adjuvant for tuberculosis treatment. Certainly, some remarkable studies have shown that stimulation of TLR2/1 increases the expression of these receptors as well as the CYP27B1-hydroxylase enzyme, which catalyzes the conversion to the active form of vitamin D which finally leads to the induction of antimicrobial peptide cathelicidin LL-37 thus increasing the intracellular killing of mycobacteria [5].

http://dx.doi.org/10.1016/j.micinf.2014.06.010 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Lopez-Lopez N, et al., Vitamin D supplementation promotes macrophages anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.06.010

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N. Lopez-Lopez et al. / Microbes and Infection xx (2014) 1e7

Summarizing, cathelicidin is required for the 1,25D3triggered antimicrobial activity against intracellular Mtb. Indeed, it has been demonstrated that reduced serum levels of this vitamin are associated with active TB [5,6]. Thus, Mily et al. performed a dose-finding study and determined that 5000 IU once-daily of vitamin D3 is the optimal dose for the induction of LL-37 on macrophages with efficient M. tuberculosis intracellular killing by macrophages, and the effect is synergically increased when vitamin D3 is co-administered with 500 mg of phenyl butyrate (PB) twice-daily [7]. In the present study, we sought to determine serological levels of vitamin D, VDR and CYP27B1-hydroxylase mRNA levels in DM2 patients and whether 1,25D3 supplementation to Mtb infected macrophages reduces CFUs. This information may offer an opportunity to reduce the rate of TB in this vulnerable group.

informed consent was obtained from all study participants according to the guidelines of the National Committee of Ethics at IMSS according to Helsinki's declaration.

2. Material and methods

Peripheral blood was divided into three portions one was drawn directly in 2e3 PAXgene™ tubes (PreAnalytix, Hombrechtikon, Switzerland) for gene expression, other part was used for serum and plasma separation and the third part was used for further peripheral blood mononuclear cells separation as described below. Total RNA was extracted and purified using PAXgene™ Blood RNA kit, and contaminating DNA was removed by oncolumn DNase digestion according the manufacturer's directions (PreAnalytix, Hombrechtikon, Switzerland). Total RNA was enriched and concentrated by eliminating of tRNA, 5S rRNA and 5.8 rRNA molecules using RNeasy Mini Elute Clean Up System (QIAGEN, Inc., USA). Then, 250 ng of globin transcripts-free RNA was reverse transcribed using oligo dT primers and Super Script II Reverse Transcriptase (Invitrogen, Carslbard, California).

2.1. Individuals recruitment and clinical criteria 33 adults of both genders, aging from 22 to 65 years old were enrolled in this study under strict clinical and laboratory criteria. Subjects were stratified mainly in three groups. The first belonging to those patients diagnosed with type 2 diabetes mellitus (DM2). These patients were recruited according with fasting plasma glucose levels of at least 150 mg/dl, and random plasma glucose of at least 200 mg/dl and 2-h plasma glucose of at least 200 mg/dl during an oral glucose tolerance test. All individuals also had no evidence of another specific type of diabetes and all patients were non-insulin dependent. Only patients with known disease duration of over 1 year according with clinical files and recent HbA1c  7.0% were enrolled [8]. The second group was recorded as TB patients. These patients were examined clinically, and a chest radiograph was obtained. To determine progressive tuberculosis, sputum samples were collected for acid-fast bacilli (AFB) smear and Mtb culture tests, worthwhile to mention that samples were taken immediately after diagnosis and before submission to any antimicrobial therapy. The third group was recorded as healthy donors. Donors that were clinically healthy were submitted to coetaneous test for tuberculin (TST) though the Mantoux method, using Tuberculin Purified Protein Derivative TUBERSOL® (5 TU/0.1 ml) (Aventis, Pasteur, Canada), and the in vitro T-cell interferon gamma (IFN-g) release assay QuantiFERON® TB-Gold test (Cellestis, Victoria, Australia) were used in accordance to manufacturers guideline. For the anergy test, Candidine (1:1000, 0.1 ml) was administered by intradermic injection into the foreside of the forearm. Healthy individuals were enrolled if they do not showed any clinical disease manifestation, negative TST, negative QuantiFERON® TB-Gold test, positive candidine test and without diagnostic for DM2 and no DM2 familiar antecedents. Approval to perform venipunctures was given by the Institutional boards at the Mexican Institute for Social Security (IMSS) and the General Hospital #1 of Zacatecas. Written

2.2. 25-(OH) vitamin D serum levels determination Vitamin D status is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D (25(OH)D) because of its stability. Its measurement is important as a clinical indicator of nutritional vitamin D deficiency. Normal concentrations are among 30e50 ng/ml [9]. Thus we evaluate the 25-(OH) vitamin D concentration through direct ELISA (ALPCO immunoassays, Salem NH, USA). 2.3. Sample collection, RNA isolation and reverse transcription

2.4. Gene expression analysis determined by real time PCR Real-time PCR was performed using the Light Cycler 2.0 (Roche, Germany), Light Cycler Taqman mastermix, and the specific probe for each gene selected from the Universal Probe Library (Roche, Germany). All primers were designed with Universal Probe Library software from Roche using a specific probe (Table 1). The relative expression of each sample was calculated using human mRNA HPRT as reference gene in all experiments and the DDCT method as described previously [10]. This method is based on the expression levels of a target gene versus one reference gene (HPRT ) comparing between control group and target group. The comparative threshold cycle method was used to assess relative changes in mRNA levels between healthy individuals (control) and the TB and DM2 patients reflected in fold changes. Thus healthy controls were normalized to one uniformly (Fig. 2A and B). 2.5. Cell culture, infection and 1,25D3 supplementation To isolate monocytes, heparinized blood was obtained from each donor PBMCs were isolated by Ficoll-Hypaque

Please cite this article in press as: Lopez-Lopez N, et al., Vitamin D supplementation promotes macrophages anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.06.010

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Table 1 Primers sequences. Gene

Protein

Forward sequence

Reverse sequence

Probe sequence

HPRT VDR CYP27B1 CAMP DEFB4 DEB103A

HPRT VDR CYP27B1 LL-37 HBD-2 HBD-3

tcccctgttgactggtcatt aggctgcaaaggcttcttc cttgcggactgctcactg tcggatgctaacctctaccg gtctccctccaacaaaatgc gagcacttgccgatctgttc

tgccgaagatgcgatgaagg atgtccacacagcgtttgag cgcagactacgttgttcagg gtctgggtccccatccat gagggagcccttttctgaatc cagaaatattattgcagagtc agagg

gctgagga catcacca ctcctcct tccaggtc tgtggctg ctgccttc

(Nycomed Pharma AS, Oslo, Norway). PBMCs were cultured in 962-mm2 polystyrene dishes (Costar, Ontario, Canada). After 2 h, non-adherent cells were removed. The remaining adherent cells were incubated for 1 h in Hanks' solution (BioWhittaker, Walkersville, MD), removed with a cell scraper, counted, and allowed to re-adhere in 24-well plates (Costar, Corning, NY), cells were incubated for seven days with high glucose DMEM (Gibco, Gand Island NY, USA) containing 10% of human AB serum (SigmaeAldrich, Saint Louis MO, USA), penicillin and L-glutamine in 5% CO2 at 37  C. 5  105 Monocytes Derived Macrophages (MDMs) were used for infection. Cytospin preparations were prepared from adherent uninfected cells to allow evaluation of the nuclear and cellular morphology by Wright's staining. Purity of MDMs was assessed by flow cytometry (>90%). Culture medium was then carefully removed, and cells infected with Mtb H37Rv strain at a MOI 5:1 in high Glucose DMEM supplemented with 10% of human AB serum for 2 h. Mtb that were not phagocytosed were eliminated by washing thoroughly with Hank's solutions supplemented with kanamycin. To eliminate possible residues of kanamycin, cells were re-washed three times with non-supplemented PBS. Cells were first stimulated with 108 M of 1,25D3 after 18 h of infection then stimulation was done every 24 h per 3 days [11]. Subsequently, cells were lysed with SDS 0.01% for 10 min

3

and reaction was stopped with bovine serum albumin. Released Mtb were plated onto 7H10 agar (Difco, Detroit, MI, USA) supplemented with OADC (Beckton Dickinson, MD, USA). Plates were incubated at 37  C for 21 days, after that time colony forming units were counted. 2.6. Statistics Normality of the data was analyzed using a KolmogoroveSmirnov normality test for each data set. For nonparametric data multiple comparison test of KruskaleWallis was used to identify differences among groups. For parametric data the ANOVA test was performed. According with the multiple comparison test a post-hoc test was performed (Dunn's or Tukey test). For the analysis of the clinical characteristics of the individuals, normality was verified using the same test after which a non-parametric multiple comparison test of KruskaleWallis was used to identify differences among groups. Two-sided p values of

Vitamin D supplementation promotes macrophages' anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression.

The increasing number of people with type 2 diabetes (DM2) is alarming and if it is taken into account that the relative odds of developing tuberculos...
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