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|TCSample_description='''Experimental Design'''<br><br>The experiment was designed across 11 time points: 0, 2, 4, 6, 12, 24, 28, 36, 48, 72, 120 hours after macrophage stimulation and 4, 12, 24, 48, 72 hours after infection. Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice (n = 5-10 ). After euthanasia, the tibias and femurs aseptically removed and the bone marrow was flushed out with cold DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin. Bone marrow cells from several animals (n=5-10 ) were pooled and were cultured for 10 days at 37°C under 5% CO2 in PLUTNIK differentiation medium (DMEM supplemented with 10% FCS, 5% Horse serum, 100U/ml penicillin G, 100μg/ml streptomycin 2mML-glutamine, 1mM sodium poyruvate, 50 μM mercaptoethanol and 30% L929 conditioned medium as a source of M-CSF. On day 10, cells (now mature BMDM) were harvested and plated in 6-well TC grade dishes. Each well was seeded with 5x10e6 BMDMs in DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin in the presence or absence activators (100U/ml IL-4 and/or 100U/ml IL-13 or 100U/ml IFNg). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) HN878 at a MOI 5:1 (bacilli:macrophage). Thereafter at specific time points the culture supernatants was removed , aliquoted and stored at -80 degree C until further analysis (.e.g. measure production of cytokines, chemokines and iNOS). The BMDMs were lyzed in situ in the well in 1 ml of Qiazol Lysis Solution (Qiagen) and the lysates stored at -80 degree C until further extraction and analysis at RIKEN. The experiment was done in parallel using 96-well TC plates where 1x105 BMDMs were plated in triplicate and treated as above for the 6-well plates. At the specific time points, the culture supernatants was collected and used for measuring the producton iNOS. The BMDMs were lyzed in 0.1% Triton X100 and cell lysate used for measuring (i) the Arginase 1 activity and (ii) CFU load in the cell lysates. Once all the samples were collected, the Qiazol cell lysates were sent to RIKEN, where the total RNA was isolated using the miRNeasy Mini kit (QIAGEN) and fully analysed. The total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop, USA). The quality and concentration of total RNA was confirmed using the BioAnalyzer (Agilent 2100 BioAnalyzer). The sample quality was checked by performing quatitative RT-PCR for some of the important marker genes of classically activated macrophages (iNOS, IL-1b, TNFa) and alternatively activated macrophages (Arg1, Ym1, IL-10). Three biological replicates were performed.<br><br><html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/Experimental_design.jpg' onclick='javascript:window.open("https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/Experimental_design.jpg", "imgwindow", "width=960,height=720");' style='width:700px;cursor:pointer'/></html>'''Figure 1. Schematic of experimental design'''<br><br>Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice, harvested and left alone or stimulated with activators to drive polarization to caMphs (100U/ml IFNg) or aaMphs (100U/ml IL-4 and/or 100U/ml IL-13). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) Beijing strain HN878 at a MOI 5:1 (bacilli : macrophage). Thereafter at specific time points, culture supernatants were analyzed to quantify the production of cytokines, chemokines and iNOS, and the BMDMs were lyzed in 1 ml of Qiazol Lysis Solution (Qiagen) and the DNA and total RNA (including microRNAs, long non-coding RNAs etc) extracted. The epigenomic footprint of the cells will be studied by analysing the genomic DNA methylation patterns by bisulifte sequencing; and the RNA will be used for RNAseq (microRNA and non-coding long RNA), CAGE analysis (promoter and gene expression) and qRT-PCR (confirmation).<br>
|TCSample_description='''Experimental Design'''<br><br>The experiment was designed across 11 time points: 0, 2, 4, 6, 12, 24, 28, 36, 48, 72, 120 hours after macrophage stimulation and 4, 12, 24, 48, 72 hours after infection. Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice (n = 5-10 ). After euthanasia, the tibias and femurs aseptically removed and the bone marrow was flushed out with cold DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin. Bone marrow cells from several animals (n=5-10 ) were pooled and were cultured for 10 days at 37°C under 5% CO2 in PLUTNIK differentiation medium (DMEM supplemented with 10% FCS, 5% Horse serum, 100U/ml penicillin G, 100μg/ml streptomycin 2mML-glutamine, 1mM sodium poyruvate, 50 μM mercaptoethanol and 30% L929 conditioned medium as a source of M-CSF. On day 10, cells (now mature BMDM) were harvested and plated in 6-well TC grade dishes. Each well was seeded with 5x10e6 BMDMs in DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin in the presence or absence activators (100U/ml IL-4 and/or 100U/ml IL-13 or 100U/ml IFNg). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) HN878 at a MOI 5:1 (bacilli:macrophage). Thereafter at specific time points the culture supernatants was removed , aliquoted and stored at -80 degree C until further analysis (.e.g. measure production of cytokines, chemokines and iNOS). The BMDMs were lyzed in situ in the well in 1 ml of Qiazol Lysis Solution (Qiagen) and the lysates stored at -80 degree C until further extraction and analysis at RIKEN. The experiment was done in parallel using 96-well TC plates where 1x105 BMDMs were plated in triplicate and treated as above for the 6-well plates. At the specific time points, the culture supernatants was collected and used for measuring the producton iNOS. The BMDMs were lyzed in 0.1% Triton X100 and cell lysate used for measuring (i) the Arginase 1 activity and (ii) CFU load in the cell lysates. Once all the samples were collected, the Qiazol cell lysates were sent to RIKEN, where the total RNA was isolated using the miRNeasy Mini kit (QIAGEN) and fully analysed. The total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop, USA). The quality and concentration of total RNA was confirmed using the BioAnalyzer (Agilent 2100 BioAnalyzer). The sample quality was checked by performing quatitative RT-PCR for some of the important marker genes of classically activated macrophages (iNOS, IL-1b, TNFa) and alternatively activated macrophages (Arg1, Ym1, IL-10). Three biological replicates were performed.<br><br><html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/Experimental_design.jpg' onclick='javascript:window.open("https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/Experimental_design.jpg", "imgwindow", "width=960,height=720");' style='width:700px;cursor:pointer'/></html>'''Figure 1. Schematic of experimental design'''<br><br>Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice, harvested and left alone or stimulated with activators to drive polarization to caMphs (100U/ml IFNg) or aaMphs (100U/ml IL-4 and/or 100U/ml IL-13). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) Beijing strain HN878 at a MOI 5:1 (bacilli : macrophage). Thereafter at specific time points, culture supernatants were analyzed to quantify the production of cytokines, chemokines and iNOS, and the BMDMs were lyzed in 1 ml of Qiazol Lysis Solution (Qiagen) and the DNA and total RNA (including microRNAs, long non-coding RNAs etc) extracted. The epigenomic footprint of the cells will be studied by analysing the genomic DNA methylation patterns by bisulifte sequencing; and the RNA will be used for RNAseq (microRNA and non-coding long RNA), CAGE analysis (promoter and gene expression) and qRT-PCR (confirmation).<br>
|Time_Course=
|Time_Course=
|category_treatment=activation
|category_treatment=Activation
|collaborators=Frank Brombacher
|collaborators=Frank Brombacher
|description=mouse_macrophage_TB_infection_IL13
|description=mouse_macrophage_TB_infection_IL13
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|time_points=
|time_points=
|time_span=120 hours
|time_span=120 hours
|timepoint_design=activation
|timepoint_design=Activation
|tissue_cell_type=macrophage
|tissue_cell_type=Macrophage
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=liBz9sBAEBOhWIa4uygS1B
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=liBz9sBAEBOhWIa4uygS1B
}}
}}

Revision as of 12:25, 4 February 2015

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:FILE
Link to TET:[{{{tet_file}}} TET]
Sample providers :Frank Brombacher
Germ layer:mesoderm
Primary cells or cell line:primary cells
Time span:120 hours
Number of time points:11


Overview

The present study was designed uncover the macrophage transcriptom during IFN-g, IL-4 or IL-13 macrophage activation (classical versus alternative), to discriminate differences between IL-4 and IL-13-induced alternative macrophage activation and to uncover the transcriptom dynamics during infection with Mycobacterium tuberculosis HN878 by high throughput transcriptome analysis method, CAGE (cap analysis of gene expression). Helicos CAGE analysis will enable us to construct promoter-based networks of transcriptional regulation in a time course during macrophage activation and during infection in classical and alternative activated macrophages.

Sample description

Experimental Design

The experiment was designed across 11 time points: 0, 2, 4, 6, 12, 24, 28, 36, 48, 72, 120 hours after macrophage stimulation and 4, 12, 24, 48, 72 hours after infection. Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice (n = 5-10 ). After euthanasia, the tibias and femurs aseptically removed and the bone marrow was flushed out with cold DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin. Bone marrow cells from several animals (n=5-10 ) were pooled and were cultured for 10 days at 37°C under 5% CO2 in PLUTNIK differentiation medium (DMEM supplemented with 10% FCS, 5% Horse serum, 100U/ml penicillin G, 100μg/ml streptomycin 2mML-glutamine, 1mM sodium poyruvate, 50 μM mercaptoethanol and 30% L929 conditioned medium as a source of M-CSF. On day 10, cells (now mature BMDM) were harvested and plated in 6-well TC grade dishes. Each well was seeded with 5x10e6 BMDMs in DMEM supplemented with 10% FCS and 100U/ml penicillin G, 100μg/ml streptomycin in the presence or absence activators (100U/ml IL-4 and/or 100U/ml IL-13 or 100U/ml IFNg). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) HN878 at a MOI 5:1 (bacilli:macrophage). Thereafter at specific time points the culture supernatants was removed , aliquoted and stored at -80 degree C until further analysis (.e.g. measure production of cytokines, chemokines and iNOS). The BMDMs were lyzed in situ in the well in 1 ml of Qiazol Lysis Solution (Qiagen) and the lysates stored at -80 degree C until further extraction and analysis at RIKEN. The experiment was done in parallel using 96-well TC plates where 1x105 BMDMs were plated in triplicate and treated as above for the 6-well plates. At the specific time points, the culture supernatants was collected and used for measuring the producton iNOS. The BMDMs were lyzed in 0.1% Triton X100 and cell lysate used for measuring (i) the Arginase 1 activity and (ii) CFU load in the cell lysates. Once all the samples were collected, the Qiazol cell lysates were sent to RIKEN, where the total RNA was isolated using the miRNeasy Mini kit (QIAGEN) and fully analysed. The total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop, USA). The quality and concentration of total RNA was confirmed using the BioAnalyzer (Agilent 2100 BioAnalyzer). The sample quality was checked by performing quatitative RT-PCR for some of the important marker genes of classically activated macrophages (iNOS, IL-1b, TNFa) and alternatively activated macrophages (Arg1, Ym1, IL-10). Three biological replicates were performed.

Figure 1. Schematic of experimental design

Bone marrow derived macrophages were generated from 8-12 week old BALB/c male mice, harvested and left alone or stimulated with activators to drive polarization to caMphs (100U/ml IFNg) or aaMphs (100U/ml IL-4 and/or 100U/ml IL-13). After 24 hours of stimulation, the BMDMs were left alone or infected with live logarithmic phase Mycobacterium tuberculosis (MTB) Beijing strain HN878 at a MOI 5:1 (bacilli : macrophage). Thereafter at specific time points, culture supernatants were analyzed to quantify the production of cytokines, chemokines and iNOS, and the BMDMs were lyzed in 1 ml of Qiazol Lysis Solution (Qiagen) and the DNA and total RNA (including microRNAs, long non-coding RNAs etc) extracted. The epigenomic footprint of the cells will be studied by analysing the genomic DNA methylation patterns by bisulifte sequencing; and the RNA will be used for RNAseq (microRNA and non-coding long RNA), CAGE analysis (promoter and gene expression) and qRT-PCR (confirmation).

Quality control
These are the well established marker of classical and alternative activation. The role of these genes were evaluated in human and mouse model of macrophage activation.

Marker gene Expression from CAGE data

''Gene nameReferenced in (PMIDs)
Classical ActivationNos21531844, 21441450, 15036034, 12215441, 10072066, 16920488
Tnf15070757, 20059482,16920488, 21607943, 21240265, 20717022
CXCL920692533,16920488, 14734716, 19819674, 21607943, 20717022
CXCL101531844, 11907072,16920488, 14734716, 19105661, 19819674.
CXCL1120692533,19819674, 23029029, 22666284
CCL523223452,16920488, 20729857, 9822252, 18350541, 19841166
IL-616840796,15036034, 12215441, 10072066, 16920488, 11927645
Irf1Ref in: 9822252, DOI:10.1016/j.cellimm.2013.01.010
Alternative activationArginase-112098359;15036034, 12511873, 10072066, 12215441, 16920488
CCL2223275605 ,15036034, 12511873, 10704248, 16920488, 14734716
CCL1723275605,15036034, 12511873, 16920488, 14734716, 19105661
Ccl2423275605, 20692533,16920488, 19105661, 20729857, 18350541
Relmα21093321, 19029990, 12554797, 19105661; 17082649; 15142530
MYC22067385
Mrc118250477,12401408, 15530839, 19029990, 10072066, 19105661

References:
[1] Bronte, V. and P. Zanovello (2005). "Regulation of immune responses by L-arginine metabolism." Nat Rev Immunol 5(8): 641-54.
[2] Chacon-Salinas, R., J. Serafin-Lopez, et al. (2005). "Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes." Clin Exp Immunol 140(3): 443-9.
[3] Davis, A. S., I. Vergne, et al. (2007). "Mechanism of inducible nitric oxide synthase exclusion from mycobacterial phagosomes." PLoS Pathog 3(12): e186.
[4] Ehrt, S., D. Schnappinger, et al. (2001). "Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase." J Exp Med 194(8): 1123-40.
[5] El Kasmi, K. C., J. E. Qualls, et al. (2008). "Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens." Nat Immunol 9(12): 1399-406.
[6] Raju, B., Y. Hoshino, et al. (2008). "Gene expression profiles of bronchoalveolar cells in pulmonary TB." Tuberculosis (Edinb) 88(1): 39-51.
[7] Varin, A., S. Mukhopadhyay, et al. "Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion." Blood 115(2): 353-62.

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples


3560-170A1macrophage, bone marrow derived000hrpool1
3577-170I2macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb002hrbiol_rep1
3578-170A3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb004hrbiol_rep1
3579-170B3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb006hrbiol_rep1
3581-170D3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb024hrbiol_rep1
3582-170E3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb028hrbiol_rep1
3583-170F3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb036hrbiol_rep1
3584-170G3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb048hrbiol_rep1
3585-170H3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb072hrbiol_rep1
3586-170I3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb120hrbiol_rep1
3617-170D7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb028hr(004h after stimulation)biol_rep1
3618-170E7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb036hr(012h after stimulation)biol_rep1
3619-170F7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb048hr(024h after stimulation)biol_rep1
3620-170G7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb072hr(048h after stimulation)biol_rep1
3621-170H7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb120hr(096h after stimulation)biol_rep1
3632-171A1macrophage, bone marrow derived000hrpool2
3649-171I2macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb002hrbiol_rep2
3650-171A3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb004hrbiol_rep2
3651-171B3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb006hrbiol_rep2
3652-171C3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb012hrbiol_rep2
3653-171D3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb024hrbiol_rep2
3654-171E3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb028hrbiol_rep2
3655-171F3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb036hrbiol_rep2
3656-171G3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb048hrbiol_rep2
3658-171I3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb120hrbiol_rep2
3689-171D7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb028hr(004h after stimulaton)biol_rep2
3690-171E7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb036hr(012h after stimulation)biol_rep2
3691-171F7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb048hr(024h after stimulation)biol_rep2
3692-171G7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb072hr(048h after stimulation)biol_rep2
3693-171H7macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb120hr(096h after stimulation)biol_rep2
3704-172A1macrophage, bone marrow derived000hrpool3
3724-172C3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb002hrbiol_rep3
3725-172D3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb004hrbiol_rep3
3726-172E3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb006hrbiol_rep3
3727-172F3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb012hrbiol_rep3
3728-172G3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb024hrbiol_rep3
3729-172H3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb028hrbiol_rep3
3730-172I3macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb036hrbiol_rep3
3731-172A4macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb048hrbiol_rep3
3732-172B4macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb072hrbiol_rep3
3733-172C4macrophage, TB infection, stimulated BMDM+IL-13(aaMph), without Mtb120hrbiol_rep3
3982-173A6macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb028hr(004h after stimulation)biol_rep4
3985-173D6macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb072hr(048h after stimulation)biol_rep4
3986-173E6macrophage, TB infection, stimulated BMDM+IL-13(aaMph), with Mtb120hr(096h after stimulation)biol_rep4
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