BMM TB infection control

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:	9

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 name Referenced in (PMIDs) Classical Activation Nos2 1531844, 21441450, 15036034, 12215441, 10072066, 16920488 Tnf 15070757, 20059482,16920488, 21607943, 21240265, 20717022 CXCL9 20692533,16920488, 14734716, 19819674, 21607943, 20717022 CXCL10 1531844, 11907072,16920488, 14734716, 19105661, 19819674. CXCL11 20692533,19819674, 23029029, 22666284 CCL5 23223452,16920488, 20729857, 9822252, 18350541, 19841166 IL-6 16840796,15036034, 12215441, 10072066, 16920488, 11927645 Irf1 Ref in: 9822252, DOI:10.1016/j.cellimm.2013.01.010 Alternative activation Arginase-1 12098359;15036034, 12511873, 10072066, 12215441, 16920488 CCL22 23275605 ,15036034, 12511873, 10704248, 16920488, 14734716 CCL17 23275605,15036034, 12511873, 16920488, 14734716, 19105661 Ccl24 23275605, 20692533,16920488, 19105661, 20729857, 18350541 Relmα 21093321, 19029990, 12554797, 19105661; 17082649; 15142530 MYC 22067385 Mrc1 18250477,12401408, 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

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3561-170B1	macrophage, TB infection, non-stimulated BMDM, without Mtb	024hr	biol_rep1
3562-170C1	macrophage, TB infection, non-stimulated BMDM, without Mtb	028hr	biol_rep1
3563-170D1	macrophage, TB infection, non-stimulated BMDM, without Mtb	036hr	biol_rep1
3564-170E1	macrophage, TB infection, non-stimulated BMDM, without Mtb	048hr	biol_rep1
3565-170F1	macrophage, TB infection, non-stimulated BMDM, without Mtb	072hr	biol_rep1
3566-170G1	macrophage, TB infection, non-stimulated BMDM, without Mtb	120hr	biol_rep1
3607-170C6	macrophage, TB infection, non-stimulated BMDM, with Mtb	028hr(004h after stimulation)	biol_rep1
3608-170D6	macrophage, TB infection, non-stimulated BMDM, with Mtb	036hr(012h after stimulation)	biol_rep1
3609-170E6	macrophage, TB infection, non-stimulated BMDM, with Mtb	048hr(024h after stimulation)	biol_rep1
3610-170F6	macrophage, TB infection, non-stimulated BMDM, with Mtb	072hr(048h after stimulation)	biol_rep1
3611-170G6	macrophage, TB infection, non-stimulated BMDM, with Mtb	120hr(096h after stimulation)	biol_rep1
3633-171B1	macrophage, TB infection, non-stimulated BMDM, without Mtb	024hr	biol_rep2
3634-171C1	macrophage, TB infection, non-stimulated BMDM, without Mtb	028hr	biol_rep2
3635-171D1	macrophage, TB infection, non-stimulated BMDM, without Mtb	036hr	biol_rep2
3636-171E1	macrophage, TB infection, non-stimulated BMDM, without Mtb	048hr	biol_rep2
3637-171F1	macrophage, TB infection, non-stimulated BMDM, without Mtb	072hr	biol_rep2
3638-171G1	macrophage, TB infection, non-stimulated BMDM, without Mtb	120hr	biol_rep2
3679-171C6	macrophage, TB infection, non-stimulated BMDM, with Mtb	028hr(004h after stimulation)	biol_rep2
3680-171D6	macrophage, TB infection, non-stimulated BMDM, with Mtb	036hr(012h after stimulation)	biol_rep2
3681-171E6	macrophage, TB infection, non-stimulated BMDM, with Mtb	048hr(024h after stimulation)	biol_rep2
3683-171G6	macrophage, TB infection, non-stimulated BMDM, with Mtb	120hr(096h after stimulation)	biol_rep2
3705-172B1	macrophage, TB infection, non-stimulated BMDM, without Mtb	004hr	biol_rep3
3706-172C1	macrophage, TB infection, non-stimulated BMDM, without Mtb	006hr	biol_rep3
3707-172D1	macrophage, TB infection, non-stimulated BMDM, without Mtb	012hr	biol_rep3
3708-172E1	macrophage, TB infection, non-stimulated BMDM, without Mtb	024hr	biol_rep3
3709-172F1	macrophage, TB infection, non-stimulated BMDM, without Mtb	028hr	biol_rep3
3710-172G1	macrophage, TB infection, non-stimulated BMDM, without Mtb	036hr	biol_rep3
3711-172H1	macrophage, TB infection, non-stimulated BMDM, without Mtb	048hr	biol_rep3
3712-172I1	macrophage, TB infection, non-stimulated BMDM, without Mtb	072hr	biol_rep3
3713-172A2	macrophage, TB infection, non-stimulated BMDM, without Mtb	120hr	biol_rep3
3967-173D4	macrophage, TB infection, non-stimulated BMDM, without Mtb	024hr	biol_rep4
3972-173I4	macrophage, TB infection, non-stimulated BMDM, with Mtb	028hr(004h after stimulation)	biol_rep4
3973-173A5	macrophage, TB infection, non-stimulated BMDM, with Mtb	036hr(012h after stimulation)	biol_rep4
3974-173B5	macrophage, TB infection, non-stimulated BMDM, with Mtb	048hr(024h after stimulation)	biol_rep4
3975-173C5	macrophage, TB infection, non-stimulated BMDM, with Mtb	072hr(048h after stimulation)	biol_rep4
3976-173D5	macrophage, TB infection, non-stimulated BMDM, with Mtb	120hr(096h after stimulation)	biol_rep4