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-γ (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-γ and TNF being equivalent to WT controls (12). From these results, we hypothesized that NF-IL6 acts downstream from IFN-γ 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-γ and infected with L. monocytogenes ("LM + IFN-γ"). Total RNA was isolated from untreated and "LM + IFN-γ" 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-γ" 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-γ" 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-γ" 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.
Table 1. RNA samples, treatments, labelling, and microarrays. header descriptions
Biological experiment
Sample label
Sample name
Treatment
"+" refers to BMDMs simultaneously stimulated with IFN-gamma and infected with L. monocytogenes at a MOI of 10 bacilli:1 macrophage; "-" refers to untreated media controls
Cy-dye label
Cy5 (red) or Cy3 (green) fluorescent dyes
Slide barcode #
Hybridization to Slide with unique barcode #
Data table
Biological experiment
Sample label
Treatment
Cy-dye label
Slide barcode #
1
KO1treated
+
Cy3
12301884
1
KO1untreated
-
Cy5
12301884
1
WT1treated
+
Cy5
12301864
1
WT1untreated
-
Cy3
12301864
2
KO2treated
+
Cy5
12301883
2
KO2untreated
-
Cy3
12301883
2
WT2treated
+
Cy3
12301862
2
WT2untreated
-
Cy5
12301862
3
KO3treated
+
Cy3
12301878
3
KO3untreated
-
Cy5
12301878
3
WT3treated
+
Cy5
12301881
3
WT3untreated
-
Cy3
12301881
4
KO4treated
+
Cy5
12301882
4
KO4untreated
-
Cy3
12301882
4
WT4treated
+
Cy3
12301856
4
WT4untreated
-
Cy5
12301856
Total number of rows: 16
Table 2. Candidate genes used for further study header descriptions
ID_REF
unique identifier for each feature on the array
GAL ID
the Genbank accession ID number used in the GenePix gal file
GAL NAME
the gene symbol used in the GenePix gal file
VALUE_WT1
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143897
VALUE_WT2
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143901
VALUE_WT3
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143905
VALUE_WT4
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143912
VALUE_KO1
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143886
VALUE_KO2
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143893
VALUE_KO3
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143895
VALUE_KO4
log base 2 transformed normalized gene expression ratio representing treated vs. untreated for biological experiment GSM143896
WT_AVE
the average (arithmetic mean) of log base 2 transformed normalized gene expression ratio representing treated vs. untreated for all WT biological experiments (i.e. GSM143897, GSM143901, GSM143905, GSM143912)
KO_AVE
the average (arithmetic mean) of log base 2 transformed normalized gene expression ratio representing treated vs. untreated for all NF-IL6 KO biological experiments (i.e. GSM143886, GSM143893, GSM143895, GSM143896)
WT_STD_DEV
the standard deviation of log base 2 transformed normalized gene expression ratio representing treated vs. untreated for all WT biological experiments (i.e. GSM143897, GSM143901, GSM143905, GSM143912)
KO_STD_DEV
the standard deviation of log base 2 transformed normalized gene expression ratio representing treated vs. untreated for all NF-IL6 KO biological experiments (i.e. GSM143886, GSM143893, GSM143895, GSM143896)
P-VALUE
p-value calculated using a paired, 2-tailed t-test comparing the WT and KO gene expression values within each biological experiment = i.e. biological experiment 1 compares GSM143886 vs. GSM143897; biological experiment 2 compares GSM143893 vs. GSM143901; biological experiment 3 compares GSM143895 vs. GSM143905; biological experiment 4 compares GSM143896 vs. GSM143912.
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