Supplementary Materials for0.1 M carbonate (pH 9.5) buffer or Auramine-Rhodamine (AR) fluorescence...

www.sciencetranslationalmedicine.org/cgi/content/full/3/104/104ra102/DC1

Supplementary Materials for

Vitamin D Is Required for IFN- γγγγ–Mediated Antimicrobial Activity of Human Macrophages

Mario Fabri, Steffen Stenger, Dong-Min Shin, Jae-Min Yuk, Philip T. Liu, Susan

Realegeno, Hye-Mi Lee, Stephan R. Krutzik, Mirjam Schenk, Peter A. Sieling, Rosane Teles, Dennis Montoya, Shankar S. Iyer, Heiko Bruns, David M. Lewinsohn, Bruce W. Hollis, Martin Hewison, John S. Adams, Andreas Steinmeyer, Ulrich Zügel, Genhong

Cheng, Eun-Kyeong Jo, Barry R. Bloom, Robert L. Modlin*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 12 October 2011, Sci. Transl. Med. 3, 104ra102 (2011) DOI: 10.1126/scitranslmed.3003045

The PDF file includes:

Materials and Methods Fig. S1. Correlation between T cell–secreted IL-10 and IL-17 and induction of antimicrobial peptides. Fig. S2. IFN-γ induces genomic targets in human monocytes. Fig. S3. IFN-γ induces IL-15 mRNA and cell surface protein expression. Fig. S4. IFN-γ induces antimicrobial response in M. tuberculosis–infected monocytes. Fig. S5. Effect of serum concentration, heat inactivation, and type of serum on IFN-γ–induced antimicrobial response. Fig. S6. Effect of 25D supplementation of vitamin D–deficient serum on autophagy and autophagosome maturation in M. tuberculosis–infected macrophages. Fig. S7. Effect of 25D supplementation of vitamin D–deficient serum on antimicrobial activity of M. tuberculosis–infected macrophages. References

Supplementary Material, Fabri

Material and Methods

Reagents

Recombinant human IFN-! was purchased from BD Biosciences. TLR2/1L (19KD) is a

synthetic 19kDa M. tuberculosis derived lipopeptide (EMC Microcollections (S1)). IFN-! was

used at a concentration of 10 ng/ml and 19KD at a concentration of 10 !g/ml. 25D3 was

purchased (BioMol) and dissolved in ethanol at 10-4M in amber tubes and stored at -80ºC in

small aliquots. VAZ was from Bayer Schering AG, and used at 10-8M unless stated otherwise.

LysoTracker was purchased from Invitrogen, 3-MA, Wortmannin from Calbiochem/Merck

Biosciences and DAPI from Sigma. Monoclonal antibodies used were: anti-IFN-! and

corresponding isotype controls (BD Biosciences), unlabeled and PE-labelled anti-IL-15

monoclonal antibodies and corresponding isotype controls (R&D Systems), mouse or rabbit

anti-human LC3 antibodies (MBL International), anti TCR OKT3 antibody (Biolegend), anti-

LAMP1 (Santa Cruz Biotechnology) anti-rabbit-IgG-FITC, anti-rabbit-IgG-TRITC, and anti-

mouse IgG-CY2 (Jackson Immunoresearch). Tetanus toxoid was purchased from Hoffmann-La

Roche.

Ethics statement

This study was conducted according to the principles expressed in the Declaration of Helsinki.

The study was approved by the local Institutional Review Boards. All donors provided written

informed consent for the collection of peripheral blood and subsequent analysis.


Monocyte and macrophage cultures

Whole blood from healthy donors was obtained with informed consent. Peripheral blood

monoclear cells were prepared by Ficoll-hypaque density centrifugation and adherent

monocytes were isolated as described (S2). Monocytes, found to be 80-90% CD14+, were

cultured in 2% or 10% human serum or 10% fetal calf serum as indicated. We observed

differences in the kinetics of IFN-! mediated gene expression of cathelicidin and DEFB4 in

several donors. Therefore gene expression was analyzed in monocytes incubated for 20 and

24 hours in some experiments as indicated. In some donors, responses were not observed,

perhaps related to vitamin D levels, VDR polymorphisms or other factors. Human monocyte-

derived macrophages (MDMs) were prepared as described previously (S3), by culturing

peripheral blood monocytes for 4 days in the presence of 4 ng/ml human macrophage colony-

stimulating factor (Sigma).

T cell clones

M. tuberculosis-reactive Th1 and tetanus toxoid-reactive Th2 T cell clones were obtained as

follows. Clones BCD4.11, LCD4.15, B3 and 723C were derived from human T cells stimulated

with mycobacterial antigens, and clones AMG1, E7.10, E7100, and G7.100 with tetanus toxoid

by limiting dilution as described previously (S4). Clone 1024B was derived as previously

reported (S5). For supernatant production 1x105 T cell clones were stimulated with plate bound

OKT3 monoclonal antibody (2.5 !g/ml) as described earlier (S4). We purified T cell clones over

Ficoll-Paque (GE Healthcare) before use to remove residual antigen and feeder cells.

Supernatants were collected after 20h incubation and stored at -80°C. Cytokine levels for IL-4,

IL-10, GM-CSF and IL-17 were determined by cytokine bead array (BD Biosciences) following

the manufactures instructions. IFN-! was measure by cytokine bead array and ELISA (BD


Biosciences). Correlation between gene expressions and cytokine levels were calculated using

Microsoft Excel 2003 and GraphPad software.

PCR

mRNA was isolated from the cells using Trizol reagent (Invitrogen) according to the

manufacturers recommended protocol. cDNA was prepared and mRNA levels were assessed

and calculated with qPCR as previously described (1). Semiquantitative PCR was performed as

previously described (S3). Primer sequences for human cathelicidin, DEFB4, CYP27B1, VDR,

IL-15, CYP24A1, "-actin and h36B4 were previously reported (S1-3). Primer sequences for

human CD64 were: CD64Forward: TGGTTCTTGACAACTCTGCTC, CD64Reverse:

AGATGGAGCACCTCACAATG. Primer sequences for human iNOS were: iNOSForward: 5'-

GTT TGA CCA GAG GAC CCA G-3', iNOSReverse: 5'-ATC TCC TTT GTT ACC GCT TCC-3'

Primer sequences for mouse IL-15 and L32 were: IL-15Forward: 5’-

CACTTTTTAACTGAGGCTGGCATT-3’, IL-15Reverse: 5’-TCCAGTTGGCCTCTGTTTTAGG-3’,

L32Forward: 5’-AAGCGAAACTGGCGGAAAC-3' , L32Reverse: 5’-

TAACCGATGTTGGGCATCAG-3' .

Measurement of nitric oxide in culture supernatants

Human monocytes and macrophages stimulated with IFN-! or left untreated were cultured in

Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% HuS. After 24 hours

supernatants were collected and nitric oxide levels were measured by the Griess reaction (S6).

Mouse RAW macrophages stimulated with 19KD lipopeptide were used as positive control.

Measurement of 25D3 bioconversion


Cells were treated with IFN-! (10ng/ml) in 10%FCS overnight followed by incubation with

radiolabeled 3H-25D3 for 5 h in serum-free media. Measurement of 25D3 bioconversion to

1,25D3 or 24,25-dihydroxyvitamin D3 (24,25D3) was carried out as previously described (S2).

Vitamin sufficiency was defined as 25D serum levels "50 nmol/L, vitamin D deficiency as 25D

serum levels<50 nmol/L (S7).

IL-15 cell surface expression

Monocytes were stimulated for 24 hours with IFN-! or 19KD lipopeptide in 10% FCS. IL-15 cell

surface expression was assessed using flow cytometry as previously described (S2).

Serum collection and 25-hydroxyvitamin D (25D) quantification

Serum was collected and circulating concentrations of 25D were determined by

radioimmunoassay as previously described (S1). Serum was pooled from several donors; the

25D level in the pool used was approximately 98 nmol/L or 40 ng/ml unless state otherwise. For

heat-inactivation studies serum was incubated at 56°C for 30 min in a pre-heated water bath.

African-American and white donors were categorized by self-identification or visual

determination.

Stimulation of mouse bone-marrow derived macrophages (BMDMs)

Wild-type, MyD88-/-, and STAT1-/- mice were on a C57BL/6 background (The Jackson

Laboratory). All mice were maintained and bred at the UCLA Department of Laboratory Animal

Medicine mouse facility under specific pathogen-free conditions. Murine BMDMs were

generated by flushing bone marrow cells from the femurs and tibias of mice. These cells were

cultured for 7 days in DMEM (Invitrogen) containing 10% FBS, and 10% conditioned medium

from L929 cells over expressing M-CSF. Conditioned media was replaced on day 4 of


differentiation and every 2 days thereafter. BMDMs were serum starved in DMEM, 1% FBS, 1%

penicillin/streptomycin overnight prior to cytokine stimulation. BMDMs were stimulated with

recombinant murine IFN-! (Invitrogen), TLR2/1L (P3C; Alexis Biochemicals), or left untreated for

4 hours. Murine IFN-! was used at a concentration of 50 ng/ml and P3C at 100 ng/ml.

Primary monocytes siRNA transfection

siRNA transfection into primary human monocytes was accomplished using the Amaxa

Nucleofection System and the Human Monocyte Kit according to the manufacturer’s

recommendations as previous described (S8). siRNA constructs were used at 100 pmol per

transfection.

Mycobacterium tuberculosis growth and maintenance

M. tuberculosis (virulent strain H37Rv) was grown in suspension with constant, gentle rotation in

roller bottles (Corning) containing Middlebrook 7H9 broth (Becton Dickinson) supplemented with

1% glycerol, 0.05% Tween-80 (Sigma) and 10% Middlebrook OADC enrichment (Becton

Dickinson). Aliquots from logarithmically growing cultures were frozen in PBS containing 10%

glycerol and representative vials were thawed and enumerated for viable colony forming units

(CFU) on Middlebrook 7H11 plates. Bacterial viability was above 90% (BacLightTM, Invitrogen).

Infection of monocytes and MDMs, treatment with IFN-! and quantification of mycobacterial

growth

Monocytes or MDMs were infected for 4h or overnight with single cell suspensions of M.

tuberculosis at a multiplicity of infection (MOI) of five in a 6 well plate. Extracellular bacteria

were removed by vigorous washing. Adherent cells were detached by treatment with EDTA and

plated at a concentration of 5x105 cells/ml in a 24 well plate. The efficiency of infection, as


quantified by auramine rhodamine stain was in a range of 20-35%. Infected cells were treated

with IFN-! (10ng/ml), which was present throughout the 3 to 5 day-culture period in 10% vitamin

D-sufficient or deficient human serum. To determine the number of viable intracellular bacilli

infected cells were lysed with 0.3% saponin (Sigma). Lysates of infected cells were

resuspended vigorously, transferred into screw caps and sonicated in a preheated (37ºC) water

bath sonicator for 10 min. Aliquots of the lysates were diluted ten-fold in 7H9-medium. Four

dilutions of each sample were plated in duplicates on 7H11 agar plates and incubated at 37ºC

for 21 days. At all time points an aliquot of unlysed, infected cells was harvested and counted.

This allowed an exact quantification of cells as well as the determination of cellular viability by

trypan blue exclusion. Recovery of cells was >70% in all experiments, with cell viability regularly

exceeding 90% of total cells.

Confocal microscopy

Analysis of autophagy in monocytes and MDMs was assessed as described previously (S3).

For the quantification of autophagy, the percentages of the numbers of endogenous LC3

punctated cells were evaluated using fluorescence microscopy. Each experiment involved

scoring of at least 100 cells for each condition. For rapamycin treatment, cells were incubated

in medium that contained 500 nM rapamycin (Sigma) for 12 h. In some experiments, human

monocytes were loaded with LysoTracker, and then washed three times with PBS before

fixation for immunostaining. For silencing of hBeclin-1 or hAtg5 in primary cells, lentiviral

shRNAs were generated as described previously (S3). Briefly, lentiviruses were produced by

transient transfection using packaging plasmids (pMDLg/pRRE, pRSV-Rev, pMD2.VSV-G,

purchased from Addgene) with Lipofectamine 2000 mediated transient transfection into

HEK293T cells. Viral containing media were collected 72 h post-transfection and filtered.

Supernatant was used to infect cells in presence of 8 µg/ml Polybrene. For lentivirus infection,

human monocytes were infected with lentiviral vectors at a multiplicity of infection of 10 in a


presence of 8 µg/ml Polybrene. After 4 to 6 h, the medium was replaced with fresh medium,

and the cells were incubated for 3 days and then analyzed for gene specific knockdown.

Observation of the colocalization of lysosomes and M. tuberculosis-containing phagosomes was

performed using human monocyte-derived macrophages as described previously (S3). M.

tuberculosis H37Rv were fluorescently labeled with fluorescein isothiocyanate (FITC; Sigma) in

0.1 M carbonate (pH 9.5) buffer or Auramine-Rhodamine (AR) fluorescence (Sigma) and stored

at 4°C. Cells were prepared in 12-well culture dishes that contained 18 mm diameter round

glass coverslips (105 cells per well). The cells were then infected with labeled M. tuberculosis

(105 CFU) for 2 hr at 37°C, washed three times with PBS, and incubated for 30 h. The cells

were loaded with LysoTracker, or stained with anti-LAMP1 monoclonal antibody, or anti-LC3-

antibody, fixed and imaged by confocal microscopy. For quantification at least 100 internalized

mycobacteria were scored in seven random fields for each condition.

Statistics

P-values were calculated using Graphpad software for figure 1b and two-tailed Student’s t-tests

for all other figures.

Fig. S1 Supplementary Material, Fabri

Cath. mRNA

R=0.05

0

1

2

3

4

0 0.5 1 1.5 IL-10 (ng/ml)

Cath. mRNA

R=0.16

0

1

2

3

4

0 0.2 0.4 0.6 0.8 1 IL-17 (ng/ml)

DEFB4 mRNA

R=0.14

0 1 2 3 4 5 6 7

0 0.5 1 1.5 IL-10 (ng/ml)

DEFB4 mRNA

R=0.17

0 1 2 3 4 5 6 7

0 0.2 0.4 0.6 0.8 1 IL-17 (ng/ml)

Fold

cha

nge

Fold

cha

nge


Fig. S1. Correlation between T cell secreted IL-10 and IL-17 and induction of antimicrobial

peptides. Th1 and Th2 T cell clones were stimulated with plate bound anti-CD3 monoclonal

antibody and supernatants were collected after 18h. T cell supernatants were added to human

monocytes for 24h in 10% vitamin D-sufficient human serum and mRNA levels of genes

encoding antimicrobial peptides cathelicidin and DEFB4 were measured by qPCR. The

correlation between T cell supernatant cytokine levels (ng/ml) and monocyte antimicrobial gene

expression levels (mean fold change) were determined and represented as the correlation

coefficient, R.

*CYP24 mRNA

0

2

4

6

8

10

12

Fold

cha

nge



Fig. S2. IFN-! induces genomic targets in human monocytes. Primary human monocytes were

stimulated with recombinant human rIFN-! for 24h in 10% vitamin D-sufficient human serum.

CYP24 gene expression was assessed by qPCR (mean fold change, ± SEM, n=7). *p<0.05

!MFI= 30.69

!MFI= 17.48

!MFI= 9.73

IL-15

Media IFN-"# TLR2/1L

IL-15 mRNA

0 1 2 3 4 5 6 7

B

Fold

cha

nge

A

*



Fig. S3. IFN-! induces IL-15 mRNA and cell surface protein expression. Primary human

monocytes were stimulated with rIFN-! for 24h. A, IL-15 gene expression was assessed by

qPCR (mean fold change, ± SEM, n=3). B, Representative IL-15 cell surface flow cytometry

staining on monocytes stimulated with rIFN-! or TLR2/1 ligand at 24h (isotype control: solid line;

anti-IL-15 mAb: gray shaded area). *p<0.05

Cath.

DEFB4

!-actin

M. tb. -

Donor #1

Donor #2

Cath.

DEFB4

!-actin



Fig. S4. IFN-! induces antimicrobial response in M. tuberculosis infected monocytes. Primary

human monocytes were infected with M. tuberculosis H37Rv and cultured with medium or rIFN-

! in 10% vitamin D-sufficient human serum. Cathelicidin and DEFB4 gene expression was

assessed in infected cells and non-infected controls by PCR at 20h (two representative of three

donors shown). Densitometric analysis revealed an average inhibition of the induction of

cathelicidin gene expression by approximately 50% and DEFB4 gene expression by

approximately 60% in infected cells compared to uninfected cells.

DEFB4 mRNA

- 56°C; 30 min.

Cath. mRNA

Fold

cha

nge

* * ns ns 4! 4!

0!

1!

2!

3!

- 56°C; 30 min.

Cath. mRNA

10% HuS (25D=98 nmol/L)

10% FCS (25D=16nmol/L)

A

* ns

0

1

2

3

4

5

6

Fold

cha

nge

0!

1!

2!

3!

-

C

B DEFB4 mRNA Cath. mRNA

Fold

cha

nge

* * *

0!

1!

2!

3!

4!

5!

6!ns

0

1

2

3

10% HuS

(25D=98 nmol/L) 2% HuS

(25D=98 nmol/L) 10% HuS

(25D=98 nmol/L) 2% HuS

(25D=98 nmol/L)

10% HuS

(25D=98 nmol/L) 10% HuS

(25D=98 nmol/L)



Fig. S5. Effect of serum concentration, heat-inactivation and, type of serum on IFN-! induced

antimicrobial response. Monocytes were cultured in A, 2% or 10% untreated, or B, 10% heat-

inactivated (56°C; 30 min) vitamin D-sufficient (25D=98 nmol/L), or C, 10% fetal calf serum

(FCS, 25D=16 nmol/L) and stimulated with rIFN-! for 20 or 24h. Cathelicidin and DEFB4 gene

expression was determined by qPCR (mean fold change ± SEM, n=3-6). *p<0.05

LAMP-1 M. tb.-PE Merged

LC3 M. tb.-PE Merged

+ 25

D3

10%

HuS

(25D

=45

nmol

/L)

-

+25D3 -

10% HuS (25D=45 nmol/L)

+ 25

D3

10%

HuS

(25D

=45

nmol

/L)

-

A

C

B

D %

M. t

b./ly

soso

me

co-lo

caliz

atio

n %

M. t

b./a

utos

ome

co-lo

caliz

atio

n

IFN-!"

IFN-!"

+25D3 -

10% HuS (25D=45 nmol/L)

** ns

** ns

!

"!

#!

$!

%!

&!!

!

"!

#!

$!

%!

&!!



Fig. S6. Effect of 25D supplementation of vitamin D-deficient serum on autophagy and

autophagosome maturation in M. tuberculosis-infected macrophages. Human MDMs were

infected with Auramine-Rhodamine-labeled M. tuberculosis (red) for 4h, washed and stimulated

with rIFN-! for 30h in vitamin D-deficient serum (25D=45 nmol/L), with or without the addition of

25D3 to reach sufficient levels. A, Cells were fixed and immunolabeled with anti-LC3-FITC

antibody (green). Representative fluorescence-merged images are shown. B, Quantitative

analysis for A (mean ± SEM, n=3). C, Lysosomes were stained with anti-LAMP1-FITC antibody

(green) and cells were fixed. Representative fluorescence-merged images are shown. D,

Quantitative analysis for C (mean ± SEM, n=3). **p<0.01

+25D3 10% HuS (45 nmol/L)

CFU

(x 1

03)

Day 0 *

-

Day 3 ns

0

4

8

12

16

20



Fig. S7. Effect of 25D supplementation of vitamin D-deficient serum on antimicrobial activity of

M. tuberculosis-infected macrophages. Human MDMs were infected with M. tuberculosis

H37Rv and cultured with medium or rIFN-! in 10% vitamin D-deficient (25D=45 nmol/L) human

serum, with or without the addition of 25D3 to reach sufficient levels. Viable bacteria were

quantified by CFU assay after day 0 and 3 (mean ± SEM, n=3). *p<0.05.


References

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S2. S. R. Krutzik, M. Hewison, P. T. Liu, J. A. Robles, S. Stenger, J. S. Adams, R. L. Modlin, IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway. J. Immunol. 181, 7115 (2008).

S3. J. M. Yuk, D. M. Shin, H. M. Lee, C. S. Yang, H. S. Jin, K. K. Kim, Z. W. Lee, S. H. Lee, J. M. Kim, E. K. Jo, Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host. Microbe 6, 231 (2009).

S4. P. Salgame, J. S. Abrams, C. Clayberger, H. Goldstein, J. Convit, R. L. Modlin, B. R. Bloom, Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254, 279 (1991).

S5. P. A. Sieling, P. J. Hill, K. M. Dobos, K. Brookman, A. M. Kuhlman, M. Fabri, S. R. Krutzik, T. H. Rea, D. G. Heaslip, J. T. Belisle, R. L. Modlin, Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation. Journal of Immunology 180, 5833 (2008).

S6. S. Thoma-Uszynski, S. Stenger, O. Takeuchi, M. T. Ochoa, M. Engele, P. A. Sieling, P. F. Barnes, M. Rollinghoff, P. L. Bolcskei, M. Wagner, S. Akira, M. V. Norgard, J. T. Belisle, P. J. Godowski, B. R. Bloom, R. L. Modlin, Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291, 1544 (2001).

S7. A. C. Ross, J. E. Manson, S. A. Abrams, J. F. Aloia, P. M. Brannon, S. K. Clinton, R. A. Durazo-Arvizu, J. C. Gallagher, R. L. Gallo, G. Jones, C. S. Kovacs, S. T. Mayne, C. J. Rosen, S. A. Shapses, The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J. Clin. Endocrinol. Metab 96, 53 (2011).

S8. P. T. Liu, M. Schenk, V. P. Walker, P. W. Dempsey, M. Kanchanapoomi, M. Wheelwright, A. Vazirnia, X. Zhang, A. Steinmeyer, U. Zugel, B. W. Hollis, G. Cheng, R. L. Modlin, Convergence of IL-1beta and VDR activation pathways in human TLR2/1-induced antimicrobial responses. PLoS. ONE. 4, e5810 (2009).

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