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. 2011 May 27;286(30):26873–26887. doi: 10.1074/jbc.M111.238287

FIGURE 4.

FIGURE 4.

SOCS1 inhibits response to but not the secretion of IL-12, does not hamper responses to IFN-γ, and regulates the global transcriptional responses to infection of macrophages with M. tuberculosis. Total RNA was extracted from SOCS1−/− and WT BMM at the indicated time points after infection with M. tuberculosis H37Rv (A, B, E, and F) or after stimulation with 20 ng/ml recombinant IL-12 p70 (C). The accumulation of IL-12 p40 (A), IL-12 p35 (B), IFN-γ (C and F), IL-12Rβ1 (E), and Hprt mRNA was measured by real time PCR. The mean -fold accumulation of the transcripts of triplicate cultures per time point in relation to Hprt ± S.E. (error bars) is depicted. The level of IFN-γ in the supernatant of triplicate cultures of IL-12-stimulated SOCS1−/− or WT BMM was measured by ELISA (D). *, differences with control BMM are significant (p < 0.05, Student's t test). The levels of IFN-γ mRNA in SOCS1−/− and WT BMM incubated or not with 10 μg/ml anti-p40/p70 IL-12-neutralizing antibodies (BD Biosciences) 1 h before infection with M. tuberculosis (F) were determined as described above. Twenty-four h after infection with M. tuberculosis, SOCS1−/− or WT BMM were co-incubated with 100 units of recombinant IFN-γ or left untreated. The number of cfu ± S.E. in lysates from triplicate cultures at each time point after infection is depicted (G). SOCS1−/− and WT BMM were treated with IFN-γ 24 h after infection with M. tuberculosis. Infected and mock controls were harvested at the indicated time points after infection. The accumulation of CXCL10 (G) and Hprt mRNA was measured by real-time PCR. The mean -fold accumulation of CXCL10 mRNA of triplicate cultures per time point in relation to Hprt ± S.E. is depicted (H). Differences between IFN-γ-treated and untreated cells are significant (p < 0.05, Student's t test). To obtain the microarray data, RNA was isolated from M. tuberculosis-infected or uninfected, WT or SOCS1−/− BMM cultures, with four independent samples in each group. The Venn diagram illustrates the number of genes altered by pathogen exposure (independently of the genotype) or differentially expressed by SOCS1−/− and WT BMM (independently of infection) (I). The statistical significance of differentially expressed probes was identified by two-way analysis of variance (p < 0.05), including the Benjamini and Hochberg false discovery rate correction (5%).