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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: J Immunol. 2018 Feb 16;200(7):2405–2417. doi: 10.4049/jimmunol.1701073

FIGURE 5.

FIGURE 5

Both p38-MAPK and STAT4 pathways appeared to be the selected IL-12+IL-18 co-signaling that led to inhibition of intracellular mycobacteria.

(A-B) show changes in mean Survival index of BCG bacilli in IL-12+IL-18-cotreated A549 cells(A) and hMDM (B) in the absence and presence of chemical inhibitors PDTC (10uM), U0126 (10uM), SB203580 (10uM) and DMSO in the 3-day culture. Data were pooled from 3 independent experiments using A549 cells (A) and 5 different experiments using hMDM (B) derived from 15 healthy uninfected donors. P values are calculated through ANOVA then Tukey’s multiple comparisons test, **** p < 0.0001. ns, not significant. (C) shows graph data of mean fold changes in expression levels for IL-18 receptor genes IL18R1 and IL18RAP in BCG-infected hMDM incubated for 24 hours with media, IL-12, IL-18 or IL-12+IL-18. Fold changes were calculated by expression levels of cytokine treatment versus those of media control. Data were generated using RT-qPCR and pooled from 3 independent experiments using hMDM from 12 healthy uninfected donors. P values are calculated through ANOVA then Dunnett’s test compared with control condition: treated with culture media, * p<0.05, ** p<0.01. (D-E) The inhibition of p38 renders IL-12+IL-18 co-signaling fails to induce enhanced expression of CAMP at mRNA levels in BCG-hMDM cells (D) and at protein levels in BCG-THP-1 cells (E). Data are derived from 3 independent assays using 9 healthy uninfected donors for D. The upper panel in E is a represented image from 4 replicates, the lower panel is pooled data for optical density analysis. P values are calculated using T test, * p< 0.05. (F) shows representative data demonstrating the knock-down for the transcription factor STAT4 in A549 cells stably transduced with the lentivirus construct LV-shSTAT4 in comparisons with the control. (G) compares mean Survival indexes for BCG bacilli in shSTAT4- and shControl-transduced A549 cells in the presence of media, IL-12, IL-18 or IL-12+IL-18. Note the reversion of IL-12+IL-18-mediated inhibition in the setting of STAT4 knock-down. Data were derived from 4 independent experiments. P values are calculated through ANOVA then Dunnett’s test compared with control condition: treated with culture media, **** p < 0.0001. (H). Representative western blot data shows that the shRNA knock-down of STAT4 in THP-1 cells in comparisons with the shRNA control. (I). Bar graph shows that the shRNA knock-down of STAT4 reverses the growth restriction of intracellular mycobacteria during IL-12+IL-18 treatment of BCG-infected THP-1 cells, compared to the shRNA control. Data are derived from 8 replicates in 2 independent experiments. P values are calculated through T test, **** p< 0.001. (J) Representative western blot shows shRNA knock-down of STAT4 reduced the CAMP protein expression during the IL-12+IL-18 treatment of BCG-infected A549 and THP-1 cells, respectively. The upper panel are representative western blot analysis from 4 replicates, the lower panel are quantitative analysis of CAMP protein levels by densitometry. P values are calculated through T test compared with control condition: treated with culture media, * p< 0.05, ** p< 0.01.