Journal of Infectious Diseases Advance Access published January 22, 2015 1
BIMODAL INFLUENCE OF VITAMIN D IN HOST RESPONSE TO SYSTEMIC CANDIDA INFECTION – VITAMIN D DOSE MATTERS
cr ipt
Joan Hui Juan Lim1,*, Sharada Ravikumar1,*, Yan-Ming Wang2, Thomas Paulraj Thamboo3, Lizhen Ong4, Jinmiao Chen5, Jessamine Geraldine Goh1, Sen Hee Tay6, Lufei Chengchen7, Mar Soe Win1,7, Winnie Leong1, Titus Lau8, Roger Foo9, Haris Mirza10, Kevin Shyong Wei Tan10, Sunil Sethi4, Ai Leng Khoo11, Wee Joo Chng7,12,13, Osato Motomi7, Mihai G. Netea14, Yue Wang2, Louis Yi Ann Chai1,# 1
us
2
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore Department of Pathology, National University Health System, Singapore
4
Department of Laboratory Medicine, National University Health System, Singapore
an
3
5
M
Singapore Immunology Network, Agency for Science, Technology and Research, Singapore
6
Division of Rheumatology, University Medicine Cluster, National University Health System, Singapore
7
8
pt ed
Cancer Science Institute, Yong Loo Lin School of Medicine, National University of Singapore Division of Nephrology, University Medicine Cluster, National University Health System, Singapore
9
Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
ce
10
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 11
Ac
Pharmacy and Therapeutic Office, Group Corporate Development, National Healthcare Group, Singapore 12
Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore 13
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
Division of Infectious Diseases, University Medicine Cluster, National University Health System, Singapore
2 14
Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands #
*
These authors contributed equally to the manuscript
Vitamin D levels are linked to susceptibility to infections, but its relevance in
an
candidaemia is unknown. We aimed to investigate the in-vivo sequelae of vitamin D3 supplementation in systemic Candida infection.
Implicating the role of vitamin D in Candida infections, we showed that candidemia
M
patients had significantly lower 25-OHD concentrations. Candida-infected mice treated with low dose 1,25(OH)2D3 had reduced fungal burden and better survival
pt ed
relative to untreated mice. Conversely, higher 1,25(OH)2D3 doses led to poor outcomes. Mechanistically, low dose 1,25(OH)2D3 induced proinflammatory immune responses. This was mediated through suppression of SOCS3 and induction of VDR binding with the vitamin D response elements in the IFN- promoter. These beneficial
ce
effects were negated with higher vitamin D3 doses. While the anti-inflammatory effects of vitamin D3 are well-described, we found that
Ac
lower doses conversely conferred proinflammatory benefits in Candida infection. Our
study highlights caution against extreme deviations of vitamin D levels in infections.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
Abstract
cr ipt
Corresponding author: Louis Yi Ann Chai, MD, MRCP(UK), PhD, Division of Infectious Diseases, University Medicine Cluster, National University Health System, NUHS Tower Block, 1E Kent Ridge Road, Singapore 119228, Singapore, Tel: +65-67795555, Fax: +65-6-8724130, Email: [email protected]
3
Introduction Beyond its classical role in calcium and bone metabolism, Vitamin D3 is known to
cr ipt
exert a protean influence on the immune system [1, 2]. One such effect described is the propensity of Vitamin D3 to skew host cytokine response away from a
proinflammatory profile towards a type 2 T helper cell (Th2) and regulatory T cell
(Treg) response [3-5]. It also limits the antigen presenting capacity of macrophages
phenotype, inducing heightened interleukin-10 (IL-10) levels [6, 7]. With the innate
an
and bridging adaptive immune system forming the critical frontline defense protecting the host against pathogens, it is compelling to speculate whether vitamin D may influence host susceptibility to infections. Candidemia is the 4th most common
M
bloodstream infection (BSI) in hospitals worldwide [8]. The options of anti-fungal agents available are limited, and patients with Candida BSI and invasive candidiasis
pt ed
have mortality rates of up to 40% [9]. This is partly due to the host’s inability to mount an effective immune response against the invading fungi [10].
We had earlier investigated the role of 1,25(OH)2D3 on in vitro cytokine production induced by Candida stimulation of human peripheral blood mononuclear cells
ce
(PBMC). We found that 1,25(OH)2D3 skewed cytokine response towards an antiinflammatory profile with down-regulation of IL-6, tumour necrosis factor alpha (TNF-
Ac
α) and interferon gamma (IFN-) [11]. However the biological relevance of these
effects was not known. Hence we aim to investigate and validate the sequelae of the immunomodulatory properties of vitamin D3 in-vivo.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
and directs the differentiation of dendritic cells towards a more tolerogenic
4
Methods
cr ipt
Stimuli and reagents C. albicans cells (SC5314) were grown overnight at 30°C in yeast extract-peptone-
dextrose (YPD) broth. Cells were washed twice in phosphate-buffered saline (PBS) and diluted to 5×106 microorganisms/ml. Heat-killed C. albicans blastoconidia was
from Fluka Biochemika, Sigma-Aldrich (Missouri, MI) and dissolved in absolute ethanol. The pharmaceutical preparation of 1,25(OH)2D3, Calcijex, (1 µg/ml
an
ampoule) was from Abbott Laboratories (Quebec, Canada).
M
Mice
Eight weeks old Balb/c mice, weighing 20-24 g, were infected with 2.5x105 live C.
pt ed
albicans (SC5314) via tail vein injection. One hundred microliters of vitamin D3 (Calcijex) was administered intra-peritoneally from day 2 post-infection at the doses as stipulated (0.001 µg/ml, 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml or PBS as control) for 3 days. Daily weight and survival were charted, and fungal burden in the kidneys were assessed 6 days after infection. The number of viable Candida organism was
ce
determined by plating serial dilutions on Sabouraud dextrose agar plates and the colony forming units (CFUs) were counted after 24 h incubation at 37oC. For the
Ac
survival studies, following Candida infection (on Day 0), the mice were treated from
Day 2 onwards with the stipulated daily doses of Calcijex for either 3 days or continuously.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
used at a concentration of 1×106 microorganisms/ml. 1,25(OH)2D3 was purchased
5
PBMC isolation Separation and stimulation of PBMC from healthy volunteers was performed as
cr ipt
previously described [12]. Blood was drawn into sodium heparin tubes (BD Vacutainer® Franklin Lakes, NJ) after informed consent. PBMC were isolated by
density centrifugation on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden).
Cells were washed, counted and adjusted to 5x106 cells/ml in culture medium (RPMI
Stimulation assays were performed in 96-well round-bottom plates using 100 µl of PBMC with C. albicans and the respective fractions to a total volume of 200 µl per
an
well. After 24h or 48h of incubation in humidified atmosphere (5% CO2) at 37oC, the
Vitamin D measurement
M
supernatants were collected and stored at –20°C until further assay.
Vitamin D levels were measured using de-identified serum of patients with Candida
pt ed
bloodstream infection (BSI) in our hospital against anonymized serum of hospitalized and healthy subjects archived in our hospital’s clinical laboratory. The Roche Elecsys Vitamin D commercial assay was used to determine 25-hydroxyvitamin D in human serum, whereby more than 95% of the vitamin D (25-OHD) measured in serum was
ce
vitamin D3 [13]. This electrochemiluminescence-based immunoassay was read on a Cobas® e411 analyzer.
Ac
Recruitment and phagocytosis assay Neutrophil and macrophage recruitment was assessed following IP injection of mice with inactivated Candida 1x106 microorganisms/ml at 4 h and 72 h respectively. The peritoneal cells were extracted by injecting 4ml of ice-cold PBS-heparin into the
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
1640 DM supplemented with gentamicin, L-glutamine, and sodium pyruvate).
6
peritoneal cavity. After washing, cytospin of the sample extract followed by enumeration of the cell types was performed under microscopy. To ascertain
cr ipt
phagocytosis and killing, peritoneal phagocytes as obtained were washed and counted in a counting chamber. The process of phagocytosis and intracellular killing were studied in an adherent monolayer of phagocytes [14].
The mouse kidneys were studied by light microscopy with haematoxylin and eosin and Gomori Methanamine Silver staining after being fixed in 4% formalin, processed
an
and embedded in paraffin, and sectioned at 3 µm. Sections were assessed via light microscopy for the presence of Candida and the locations in the kidney where the
M
organisms were present (in the glomeruli, cortex, medulla, pelvicalyces).
Stimulation and cytokine measurement
pt ed
Spleen cells (5 x 105/ml) from mice on day 6 of infection were stimulated with heatkilled C. albicans (1x106/ml) in 96-well microtitre plates at 37oC. After 24 or 48 h, supernatant was collected and cytokine measurements were made using ELISA according to the instructions of the manufacturer (eBioscience, San Diego, CA).
ce
Detection limits were 20 pg/ml (IL-1b, TNF-α and IFN-) and 10pg/ml (for IL-17 and
Ac
IL-10) respectively.
RNA isolation and quantitative PCR Splenocytes were harvested from mice pre-treated with 3 days of vitamin D3 (or PBS as control) while human PBMC were pre-incubated with vitamin D3 (or PBS as
control) for 30 min. The effects of both low dose (0.001 µg/ml) and high dose (1
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
Histology
7
µg/ml) 1,25(OH)2D3 were studied. The cells were then stimulated with heatinactivated C. albicans for 4 h. RNA was extracted from the splenocytes and PBMC
cr ipt
using 500 µl TRIzol reagent (Sigma, St. Louis, MO). Subsequently, 200 μl chloroform and 500 μl 2-propanol were used to separate the RNA from DNA and proteins. After washing with 75% ethanol, the dry RNA was dissolved in 50 μl of
diethylpyrocarbonate water. To obtain cDNA, we reverse-transcribed 1 μg DNase-
mixture with a total volume of 20 μl. Quantitative PCR was performed using the ABI Prism 7000 Thermocycler and SYBR Green. The primers used were as listed in
an
Supplementary Appendix 1. Quantification of the PCR signals for each sample was performed by comparing the cycle threshold values, in duplicate, for the gene of
M
interest with the cycle threshold values for the B2M as housekeeping gene. Mean relative mRNA expression was calculated using the comparative Ct method. Values
pt ed
are expressed as a ratio of fold increase to mRNA levels of unprimed cells.
Chromatin Immunoprecipitation (CHIP)
To investigate VDR binding to the IFN- promotor, PBMC from healthy volunteers were either treated or not treated with Vitamin D3 for 30 min followed by stimulation
ce
with C. albicans for 4 hrs. Nuclear proteins were cross-linked to DNA with 1% formaldehyde and incubated at room temperature for 10 min on a rocking platform. Cross-linking was stopped by adding glycine to a final concentration of 0.2M for 5
Ac
min. The cells were collected by centrifugation and washed twice with ice cold PBS. The cell pellets were resuspended in 500 µl of SDS lysis buffer and the lysates were sonicated to yield DNA fragments of 200-500 bp. Cell debris was removed by centrifugation. 5% of the sonicated chromatin was set aside for input control and the
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
treated total RNA with oligo(dT) primers (0.01 μg/ml) in a reverse transcription-PCR
8
rest was divided equally into 2 tubes, one for anti-VDR antibody and the other for the control IgG. The lysates were diluted 1:10 in ChIP dilution buffer. 1 µg of anti-VDR
cr ipt
antibody (Santa Cruz, sc-1008) or non-specific IgG (Santa Cruz, sc-2027) were added to the respective tubes and incubated overnight at 4oC on a rotating platform. The immunocomplexes were collected using Recombinant Protein A-Sepharose beads (Invitrogen). The beads were washed sequentially for 10 min at 4oC with
finally washed twice with TE buffer and the immune complexes were eluted using 300 µl of elution buffer at RT for 20 min with rotation. The immune complexes and
an
the input samples were reverse cross linked at 65oC overnight in the presence of proteinase K (Invitrogen). DNA was extracted with phenol chloroform extraction and
M
ethanol precipitation.
Real-time PCR reactions were performed using Sybr Green qPCR master mix
pt ed
(Applied Biosystems) with genomic primers for the BED promoter regions [15] of the IFN- gene (see Supplementary Appendix 1) using ABI Prism 7000 apparatus. The results were normalized with respect to the input. Non-specific IgG results were subtracted using the formula 2-∆Ct*100 (VDR ab) – 2-∆Ct*100 (IgG), where ∆Ct is Ct (ChIP and Ct is the cycle number.
ce
DNA)-Ct (Input)
Flow cytometry
Ac
Splenocytes were harvested and washed twice with 1x PBS supplemented with 0.2% (w/v) BSA and stained with CD4-APC/CD25-PE flurochromes, then fixed and permeabilized using FoxP3 staining kit (Biolegend, 320018, San Diego, CA). The cells were then intracellularly stained using Foxp3-Alexa Fluor 488 or isotype control.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
rotation using low salt buffer, high salt buffer and LiCl wash buffer. The beads were
9
The stained cells were then analyzed on the BDTM LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and data analysis was performed using Flow Jo
cr ipt
software (version 7.6.5) (Tree Star, Ashland, OR).
Statistics
Results were pooled from at least 3 sets of experiments (unless otherwise stated)
performed using Mann-Whitney U test or paired Wilcoxon Signed Rank test where
an
relevant. P value < 0.05 was considered significant.
Ethics Statement
M
Institutional review board approval (from the Domain Specific Review Boards, National Healthcare Group, Singapore) was obtained to perform the studies involving human subjects. Ethics approval for conduct of the animal study had been attained
pt ed
through the Biological Resource Centre (BRC), Singapore, Institutional Animal Care and Use Committee (IACUC).
ce
Results
Candidemia patients have lower Vitamin D levels To underline the role of vitamin D in candidemia in the clinical setting, we measured
Ac
25-OHD levels in sera obtained from 28 hospitalized patients with Candida BSI compared against anonymized sera of 75 hospitalized patients in our hospital. As shown in Figure 1, patients with Candida BSI had significantly lower 25-OHD levels (12.31+1.69 ng/ml versus 17.04+1.28 ng/ml, p=0.045). The 25-OHD level measured
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
and presented as means + standard error of the mean. Statistical analysis was
10
in a cohort of 30 healthy volunteers was 20.88+1.51 ng/ml and this was significantly higher than in candidemia patients (p
BIMODAL INFLUENCE OF VITAMIN D IN HOST RESPONSE TO SYSTEMIC CANDIDA INFECTION – VITAMIN D DOSE MATTERS
cr ipt
Joan Hui Juan Lim1,*, Sharada Ravikumar1,*, Yan-Ming Wang2, Thomas Paulraj Thamboo3, Lizhen Ong4, Jinmiao Chen5, Jessamine Geraldine Goh1, Sen Hee Tay6, Lufei Chengchen7, Mar Soe Win1,7, Winnie Leong1, Titus Lau8, Roger Foo9, Haris Mirza10, Kevin Shyong Wei Tan10, Sunil Sethi4, Ai Leng Khoo11, Wee Joo Chng7,12,13, Osato Motomi7, Mihai G. Netea14, Yue Wang2, Louis Yi Ann Chai1,# 1
us
2
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore Department of Pathology, National University Health System, Singapore
4
Department of Laboratory Medicine, National University Health System, Singapore
an
3
5
M
Singapore Immunology Network, Agency for Science, Technology and Research, Singapore
6
Division of Rheumatology, University Medicine Cluster, National University Health System, Singapore
7
8
pt ed
Cancer Science Institute, Yong Loo Lin School of Medicine, National University of Singapore Division of Nephrology, University Medicine Cluster, National University Health System, Singapore
9
Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
ce
10
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 11
Ac
Pharmacy and Therapeutic Office, Group Corporate Development, National Healthcare Group, Singapore 12
Department of Haematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore 13
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
Division of Infectious Diseases, University Medicine Cluster, National University Health System, Singapore
2 14
Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands #
*
These authors contributed equally to the manuscript
Vitamin D levels are linked to susceptibility to infections, but its relevance in
an
candidaemia is unknown. We aimed to investigate the in-vivo sequelae of vitamin D3 supplementation in systemic Candida infection.
Implicating the role of vitamin D in Candida infections, we showed that candidemia
M
patients had significantly lower 25-OHD concentrations. Candida-infected mice treated with low dose 1,25(OH)2D3 had reduced fungal burden and better survival
pt ed
relative to untreated mice. Conversely, higher 1,25(OH)2D3 doses led to poor outcomes. Mechanistically, low dose 1,25(OH)2D3 induced proinflammatory immune responses. This was mediated through suppression of SOCS3 and induction of VDR binding with the vitamin D response elements in the IFN- promoter. These beneficial
ce
effects were negated with higher vitamin D3 doses. While the anti-inflammatory effects of vitamin D3 are well-described, we found that
Ac
lower doses conversely conferred proinflammatory benefits in Candida infection. Our
study highlights caution against extreme deviations of vitamin D levels in infections.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
Abstract
cr ipt
Corresponding author: Louis Yi Ann Chai, MD, MRCP(UK), PhD, Division of Infectious Diseases, University Medicine Cluster, National University Health System, NUHS Tower Block, 1E Kent Ridge Road, Singapore 119228, Singapore, Tel: +65-67795555, Fax: +65-6-8724130, Email: [email protected]
3
Introduction Beyond its classical role in calcium and bone metabolism, Vitamin D3 is known to
cr ipt
exert a protean influence on the immune system [1, 2]. One such effect described is the propensity of Vitamin D3 to skew host cytokine response away from a
proinflammatory profile towards a type 2 T helper cell (Th2) and regulatory T cell
(Treg) response [3-5]. It also limits the antigen presenting capacity of macrophages
phenotype, inducing heightened interleukin-10 (IL-10) levels [6, 7]. With the innate
an
and bridging adaptive immune system forming the critical frontline defense protecting the host against pathogens, it is compelling to speculate whether vitamin D may influence host susceptibility to infections. Candidemia is the 4th most common
M
bloodstream infection (BSI) in hospitals worldwide [8]. The options of anti-fungal agents available are limited, and patients with Candida BSI and invasive candidiasis
pt ed
have mortality rates of up to 40% [9]. This is partly due to the host’s inability to mount an effective immune response against the invading fungi [10].
We had earlier investigated the role of 1,25(OH)2D3 on in vitro cytokine production induced by Candida stimulation of human peripheral blood mononuclear cells
ce
(PBMC). We found that 1,25(OH)2D3 skewed cytokine response towards an antiinflammatory profile with down-regulation of IL-6, tumour necrosis factor alpha (TNF-
Ac
α) and interferon gamma (IFN-) [11]. However the biological relevance of these
effects was not known. Hence we aim to investigate and validate the sequelae of the immunomodulatory properties of vitamin D3 in-vivo.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
and directs the differentiation of dendritic cells towards a more tolerogenic
4
Methods
cr ipt
Stimuli and reagents C. albicans cells (SC5314) were grown overnight at 30°C in yeast extract-peptone-
dextrose (YPD) broth. Cells were washed twice in phosphate-buffered saline (PBS) and diluted to 5×106 microorganisms/ml. Heat-killed C. albicans blastoconidia was
from Fluka Biochemika, Sigma-Aldrich (Missouri, MI) and dissolved in absolute ethanol. The pharmaceutical preparation of 1,25(OH)2D3, Calcijex, (1 µg/ml
an
ampoule) was from Abbott Laboratories (Quebec, Canada).
M
Mice
Eight weeks old Balb/c mice, weighing 20-24 g, were infected with 2.5x105 live C.
pt ed
albicans (SC5314) via tail vein injection. One hundred microliters of vitamin D3 (Calcijex) was administered intra-peritoneally from day 2 post-infection at the doses as stipulated (0.001 µg/ml, 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml or PBS as control) for 3 days. Daily weight and survival were charted, and fungal burden in the kidneys were assessed 6 days after infection. The number of viable Candida organism was
ce
determined by plating serial dilutions on Sabouraud dextrose agar plates and the colony forming units (CFUs) were counted after 24 h incubation at 37oC. For the
Ac
survival studies, following Candida infection (on Day 0), the mice were treated from
Day 2 onwards with the stipulated daily doses of Calcijex for either 3 days or continuously.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
used at a concentration of 1×106 microorganisms/ml. 1,25(OH)2D3 was purchased
5
PBMC isolation Separation and stimulation of PBMC from healthy volunteers was performed as
cr ipt
previously described [12]. Blood was drawn into sodium heparin tubes (BD Vacutainer® Franklin Lakes, NJ) after informed consent. PBMC were isolated by
density centrifugation on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden).
Cells were washed, counted and adjusted to 5x106 cells/ml in culture medium (RPMI
Stimulation assays were performed in 96-well round-bottom plates using 100 µl of PBMC with C. albicans and the respective fractions to a total volume of 200 µl per
an
well. After 24h or 48h of incubation in humidified atmosphere (5% CO2) at 37oC, the
Vitamin D measurement
M
supernatants were collected and stored at –20°C until further assay.
Vitamin D levels were measured using de-identified serum of patients with Candida
pt ed
bloodstream infection (BSI) in our hospital against anonymized serum of hospitalized and healthy subjects archived in our hospital’s clinical laboratory. The Roche Elecsys Vitamin D commercial assay was used to determine 25-hydroxyvitamin D in human serum, whereby more than 95% of the vitamin D (25-OHD) measured in serum was
ce
vitamin D3 [13]. This electrochemiluminescence-based immunoassay was read on a Cobas® e411 analyzer.
Ac
Recruitment and phagocytosis assay Neutrophil and macrophage recruitment was assessed following IP injection of mice with inactivated Candida 1x106 microorganisms/ml at 4 h and 72 h respectively. The peritoneal cells were extracted by injecting 4ml of ice-cold PBS-heparin into the
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
1640 DM supplemented with gentamicin, L-glutamine, and sodium pyruvate).
6
peritoneal cavity. After washing, cytospin of the sample extract followed by enumeration of the cell types was performed under microscopy. To ascertain
cr ipt
phagocytosis and killing, peritoneal phagocytes as obtained were washed and counted in a counting chamber. The process of phagocytosis and intracellular killing were studied in an adherent monolayer of phagocytes [14].
The mouse kidneys were studied by light microscopy with haematoxylin and eosin and Gomori Methanamine Silver staining after being fixed in 4% formalin, processed
an
and embedded in paraffin, and sectioned at 3 µm. Sections were assessed via light microscopy for the presence of Candida and the locations in the kidney where the
M
organisms were present (in the glomeruli, cortex, medulla, pelvicalyces).
Stimulation and cytokine measurement
pt ed
Spleen cells (5 x 105/ml) from mice on day 6 of infection were stimulated with heatkilled C. albicans (1x106/ml) in 96-well microtitre plates at 37oC. After 24 or 48 h, supernatant was collected and cytokine measurements were made using ELISA according to the instructions of the manufacturer (eBioscience, San Diego, CA).
ce
Detection limits were 20 pg/ml (IL-1b, TNF-α and IFN-) and 10pg/ml (for IL-17 and
Ac
IL-10) respectively.
RNA isolation and quantitative PCR Splenocytes were harvested from mice pre-treated with 3 days of vitamin D3 (or PBS as control) while human PBMC were pre-incubated with vitamin D3 (or PBS as
control) for 30 min. The effects of both low dose (0.001 µg/ml) and high dose (1
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
Histology
7
µg/ml) 1,25(OH)2D3 were studied. The cells were then stimulated with heatinactivated C. albicans for 4 h. RNA was extracted from the splenocytes and PBMC
cr ipt
using 500 µl TRIzol reagent (Sigma, St. Louis, MO). Subsequently, 200 μl chloroform and 500 μl 2-propanol were used to separate the RNA from DNA and proteins. After washing with 75% ethanol, the dry RNA was dissolved in 50 μl of
diethylpyrocarbonate water. To obtain cDNA, we reverse-transcribed 1 μg DNase-
mixture with a total volume of 20 μl. Quantitative PCR was performed using the ABI Prism 7000 Thermocycler and SYBR Green. The primers used were as listed in
an
Supplementary Appendix 1. Quantification of the PCR signals for each sample was performed by comparing the cycle threshold values, in duplicate, for the gene of
M
interest with the cycle threshold values for the B2M as housekeeping gene. Mean relative mRNA expression was calculated using the comparative Ct method. Values
pt ed
are expressed as a ratio of fold increase to mRNA levels of unprimed cells.
Chromatin Immunoprecipitation (CHIP)
To investigate VDR binding to the IFN- promotor, PBMC from healthy volunteers were either treated or not treated with Vitamin D3 for 30 min followed by stimulation
ce
with C. albicans for 4 hrs. Nuclear proteins were cross-linked to DNA with 1% formaldehyde and incubated at room temperature for 10 min on a rocking platform. Cross-linking was stopped by adding glycine to a final concentration of 0.2M for 5
Ac
min. The cells were collected by centrifugation and washed twice with ice cold PBS. The cell pellets were resuspended in 500 µl of SDS lysis buffer and the lysates were sonicated to yield DNA fragments of 200-500 bp. Cell debris was removed by centrifugation. 5% of the sonicated chromatin was set aside for input control and the
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
treated total RNA with oligo(dT) primers (0.01 μg/ml) in a reverse transcription-PCR
8
rest was divided equally into 2 tubes, one for anti-VDR antibody and the other for the control IgG. The lysates were diluted 1:10 in ChIP dilution buffer. 1 µg of anti-VDR
cr ipt
antibody (Santa Cruz, sc-1008) or non-specific IgG (Santa Cruz, sc-2027) were added to the respective tubes and incubated overnight at 4oC on a rotating platform. The immunocomplexes were collected using Recombinant Protein A-Sepharose beads (Invitrogen). The beads were washed sequentially for 10 min at 4oC with
finally washed twice with TE buffer and the immune complexes were eluted using 300 µl of elution buffer at RT for 20 min with rotation. The immune complexes and
an
the input samples were reverse cross linked at 65oC overnight in the presence of proteinase K (Invitrogen). DNA was extracted with phenol chloroform extraction and
M
ethanol precipitation.
Real-time PCR reactions were performed using Sybr Green qPCR master mix
pt ed
(Applied Biosystems) with genomic primers for the BED promoter regions [15] of the IFN- gene (see Supplementary Appendix 1) using ABI Prism 7000 apparatus. The results were normalized with respect to the input. Non-specific IgG results were subtracted using the formula 2-∆Ct*100 (VDR ab) – 2-∆Ct*100 (IgG), where ∆Ct is Ct (ChIP and Ct is the cycle number.
ce
DNA)-Ct (Input)
Flow cytometry
Ac
Splenocytes were harvested and washed twice with 1x PBS supplemented with 0.2% (w/v) BSA and stained with CD4-APC/CD25-PE flurochromes, then fixed and permeabilized using FoxP3 staining kit (Biolegend, 320018, San Diego, CA). The cells were then intracellularly stained using Foxp3-Alexa Fluor 488 or isotype control.
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
rotation using low salt buffer, high salt buffer and LiCl wash buffer. The beads were
9
The stained cells were then analyzed on the BDTM LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and data analysis was performed using Flow Jo
cr ipt
software (version 7.6.5) (Tree Star, Ashland, OR).
Statistics
Results were pooled from at least 3 sets of experiments (unless otherwise stated)
performed using Mann-Whitney U test or paired Wilcoxon Signed Rank test where
an
relevant. P value < 0.05 was considered significant.
Ethics Statement
M
Institutional review board approval (from the Domain Specific Review Boards, National Healthcare Group, Singapore) was obtained to perform the studies involving human subjects. Ethics approval for conduct of the animal study had been attained
pt ed
through the Biological Resource Centre (BRC), Singapore, Institutional Animal Care and Use Committee (IACUC).
ce
Results
Candidemia patients have lower Vitamin D levels To underline the role of vitamin D in candidemia in the clinical setting, we measured
Ac
25-OHD levels in sera obtained from 28 hospitalized patients with Candida BSI compared against anonymized sera of 75 hospitalized patients in our hospital. As shown in Figure 1, patients with Candida BSI had significantly lower 25-OHD levels (12.31+1.69 ng/ml versus 17.04+1.28 ng/ml, p=0.045). The 25-OHD level measured
Downloaded from http://jid.oxfordjournals.org/ at Lakehead University on March 18, 2015
us
and presented as means + standard error of the mean. Statistical analysis was
10
in a cohort of 30 healthy volunteers was 20.88+1.51 ng/ml and this was significantly higher than in candidemia patients (p
