Figure 2.
In Vivo LPS Stimulation Promotes Pro-inflammatory Cytokine Production and Inhibits C/EBPα Expression
(A) A schematic depicting the treatment of LPS-stimulated humanized NOD/SCID/IL2rγnull (hu-NSG) mice. After 10–12 weeks of CD34+ HSC engraftment, humanization of NSG mice was confirmed by flow cytometry. Different doses of LPS were injected intraperitoneally into hu-NSG mice. Peripheral blood (PB) was collected for analysis at various time points. (B and C) LPS stimulated the expression of pro-inflammatory cytokines in humanized NSG mice. Total RNA was isolated from PB for qRT-PCR analysis of cytokine (B) TNF-α and (C) IL-6 mRNAs. The lower dose of LPS (12.5 μg) and shorter stimulation time (3 h) led to more pronounced cytokine expression in mouse PB. (D–G) LPS upregulated the mRNA level of several inflammatory mediators: (D) TIMP1, (E) MMP1, (F) MMP8, and (G) MMP9. (H and I) In vivo LPS stimulation inhibited the expression of (H) C/EBPα mRNA and (I) its downstream gene p21 in humanized NSG mice. As described above, total RNA was isolated from PB for qRT-PCR analysis of the desired genes. Each determination analysis was performed in triplicate. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significant difference. Analysis by two-tailed Student’s t test.