Dataset Information


Identification of genes involved in macrophage activation and effector functions against Listeria monocytogenes.

ABSTRACT: Listeria monocytogenes (LM) is a Gram-positive, facultative intracellular bacterium responsible for disseminated infections in immunocompromised individuals which can result in septicaemia and meningitis (1). Effective control of listeriosis requires both innate and adaptive immune responses with the principal mediators of bacterial killing being neutrophils and macrophages (2). Key cytokines driving the innate immune response are IFN-? (3) and TNF (4), which promote macrophage activation and drive the production of anti-microbial mediators such as reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Several studies using gene deficient mice demonstrated that ROI and RNI are required for optimal pathogen killing (5-8), but that macrophages also utilise an alternative, unknown mechanism besides ROI and RNI to efficiently control bacterial infection. Mice deficient for the transcription factor NF-IL6 are highly susceptible to a number of intracellular bacterial and fungal infections, including LM (9-12). NF-IL6-deficient mice infected with LM were unable to prevent bacterial dissemination, despite production of nitric oxide (NO), IFN-? and TNF being equivalent to WT controls (12). From these results, we hypothesized that NF-IL6 acts downstream from IFN-? and TNF during the activation of macrophage effector functions against LM (2). To facilitate the identification of putative effector genes in this unknown pathway, we compared gene expression profiles of WT and NF-IL6 deficient LM-infected macrophages by differential microarray. REFERENCES 1. Gellin, B. G. & Broome, C. V. (1989) Jama 261, 1313-1320. 2. Brombacher, F. & Kopf, M. (1996) Res Immunol 147, 505-511. 3. Buchmeier, N. A. & Schreiber, R. D. (1985) Proc Natl Acad Sci U S A 82, 7404-7408. 4. Havell, E. A. (1989) J Immunol 143, 2894-2899. 5. Dinauer, M. C., Deck, M. B., & Unanue, E. R. (1997) J Immunol 158, 5581-5583. 6. Endres, R., Luz, A., Schulze, H., Neubauer, H., Futterer, A., Holland, S. M., Wagner, H., & Pfeffer, K. (1997) Immunity 7, 419-432. 7. MacMicking, J. D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D. S., Trumbauer, M., Stevens, K., Xie, Q. W., Sokol, K., Hutchinson, N., et al. (1995) Cell 81, 641-650. 8. Shiloh, M. U., MacMicking, J. D., Nicholson, S., Brause, J. E., Potter, S., Marino, M., Fang, F., Dinauer, M., & Nathan, C. (1999) Immunity 10, 29-38. 9. Pizarro-Cerda, J., Desjardins, M., Moreno, E., Akira, S., & Gorvel, J. P. (1999) J Immunol 162, 3519-3526. 10. Screpanti, I., Romani, L., Musiani, P., Modesti, A., Fattori, E., Lazzaro, D., Sellitto, C., Scarpa, S., Bellavia, D., Lattanzio, G., et al. (1995) Embo J 14, 1932-1941. 11. Sugawara, I., Mizuno, S., Yamada, H., Matsumoto, M., & Akira, S. (2001) Am J Pathol 158, 361-366. 12. Tanaka, T., Akira, S., Yoshida, K., Umemoto, M., Yoneda, Y., Shirafuji, N., Fujiwara, H., Suematsu, S., Yoshida, N., & Kishimoto, T. (1995) Cell 80, 353-361. Keywords: bone marrow derived macrophages, interferon-gamma, macrophage activation, macrophage effector activity, NF-IL6, gene-deficient mouse model, Listeria monocytogenes, intracellular pathogen Overall design: A 2 x 2 factorial experimental design was used to compare the gene expression patterns of bone marrow derived macrophages from WT and NF-IL6-/- mice that were untreated or were simultaneously activated with IFN-? and infected with L. monocytogenes ("LM + IFN-?"). Total RNA was isolated from untreated and "LM + IFN-?" samples at 4 hours post-infection from 4 repeat infection experiments using TriReagent (Molecular Research Company, Cincinnati, USA). The total RNA was DNAseI treated and cleaned up using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) as per the manufacturer’s instructions. The mRNA was linearly amplified and labelled using the Amino Allyl MessageAmp™ II CyDye aRNA Amplification (Ambion, Austin, Texas, USA) as per the manufacturer’s instructions. The labelled cDNA was purified and hybridized to Mouse Exonic Evidence Based Oligonucleotide (Illumina, San Diego, CA, USA) microarrays printed at the Capar Microarray Facility (University of Cape Town, South Africa). For each infection experiment, labelled probes from the untreated and "LM + IFN-?" samples for the same mouse group was hybridized to the same microarray. Image and data analysis was done using Limma (46) and TIGR MeV (47). Data was normalized using print-tip loess and quantile normalization. DE genes were identified using a paired t-test with unequal variance, pairing the relative gene expression ratio of "LM + IFN-?" vs. untreated macrophages from WT and NF-IL6-/- from the same experiment. Top 10% of genes with the smallest p-values were selected further analysis (Table 2) and were functionally clustered. Due to budget limitations, biological replicates were done instead of technical repeats and a “balanced dye” approach used instead of dye-swaps to compensate for dye-labelling bias. “Dye balance” in this context specifies that each condition is measured equally often with the Cy3 dye as with the Cy5 dye. Therefore, the "LM + IFN-?" samples for each biological repeat was labelled with the opposite Cy-dye than in the previous biological repeat. Table 1 lists the "dye balance" labelling and hybridization reactions.The same principle was applied to “untreated” controls. There were 4 biological repeat experiments which were analysed over 8 individual arrays. None of the samples from the biological repeat experiments were pooled. Each biological infection experiment was performed independently of each other.

INSTRUMENT(S): UCT Brombacher lab Mus MEEBO 40K version1

SUBMITTER: Anita R Schwegmann  

PROVIDER: GSE6256 | GEO | 2007-09-12



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Protein kinase C delta is essential for optimal macrophage-mediated phagosomal containment of Listeria monocytogenes.

Schwegmann Anita A   Guler Reto R   Cutler Antony J AJ   Arendse Berenice B   Horsnell William G C WG   Flemming Alexandra A   Kottmann Andreas H AH   Ryan Gregory G   Hide Winston W   Leitges Michael M   Seoighe Cathal C   Brombacher Frank F  

Proceedings of the National Academy of Sciences of the United States of America 20071003 41

Activation of macrophages and subsequent "killing" effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-gamma and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the  ...[more]

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