Figure 1.
The baseline colonic expression of cytokines and IL-10 receptor subunits is developmentally regulated prior to weaning.
Cytokine and IL-10 receptor subunit expression was analyzed by real time PCR in whole colon from 1, 2 and 3 week-old C57BL/6J mice. The data represent mean fold change ± SEM, relative to 1 week-old mice (A, B). C. IL-10 cytokine levels in supernatants of whole colon incubated for 24 hours were assayed by ELISA and depicted as mean concentration ± SEM (*p<0.05, n = 5−7 mice per age group).
Figure 2.
2 week-old mice have higher colonic levels of proinflammatory and lower levels of anti-inflammatory mediators than adults.
Mice were raised under conventional conditions and sacrificed at 2 weeks or as adults (6–8 weeks of life) and the colon excised and processed for analysis of mRNA (A) of the proinflammatory cytokines MIP-2 and TNF-α. mRNA expression data is depicted as mean fold change ± SEM, relative to adult mice. B. Colonic mRNA expression of IL-10 and its receptor subunits is depicted for 2 week-old mice, relative to adult controls. C. MIP-2, TNF-α and IL-10 cytokine levels of colonic supernatants determined by ELISA are depicted as mean concentration ± SEM. (*p<0.05, **p<0.01, n = 5−7 per age group).
Figure 3.
LGG increases IL-10R2 receptor subunit expression and reduces MIP-2 and TNF-α expression in the colon of 2 week-old mice.
2 week-old mice were gavage fed 108 CFUs of LGG or equal volume of HBSS, then sacrificed 6 hours later. A. The colon was excised and the mRNA expression of IL-10, IL-10R1, and IL-10R2 was analyzed by real-time PCR. B. Colonic mRNA expression for MIP-2 and TNF-α was measured at 24 hours after LGG or vehicle control. Data are depicted as mean ± SEM (n = 6−7 mice per condition).
Figure 4.
LGG triggers IL-10 receptor-dependent phosphorylation of STAT3 and induces SOCS3 in the colon of 2 week-old mice.
2 week-old mice were treated by intraperitoneal injection with either 200 µg IL-10R Ab or an isotype-matched control immediately prior to gavage feeding with 108 CFUs LGG or vehicle alone. 6 hours after treatment, the mice were sacrificed and the colon was processed for western blot analysis of phospho- and total STAT3 (A). Similar analysis was performed for SOCS3 at 24 hours after treatment (B). Densitometric analysis was performed and the mean ratio ± SEM of detected phosphorylated STAT3 to total STAT3 was plotted for each condition (A). (n = 3 separate experiments, 5 mice per condition, *p<0.05, **p<0.01).
Figure 5.
LGG mediated suppression of MIP-2 and TNF-α expression in the developing murine colon is dependent on the IL-10 receptor.
2 week-old mice were treated by intraperitoneal injection with either 200 µg IL-10R Ab, or an isotype-matched control Ab, immediately prior to gavage feeding with 108 CFUs LGG or vehicle alone. 24 hours after treatment, colonic supernatants were collected and assayed for MIP-2 (A) and TNF-α (B) by ELISA. Data are depicted as mean ± SEM (n = 3 separate experiments, for a total of 7 mice per condition, *p<0.05).
Figure 6.
LGG protects against intestinal injury induced by PAF and LPS.
2 week-old mice were gavage-fed either LGG or PBS and then exposed to PAF/LPS 24 hours later. 2 hours after PAF/LPS exposure, mice were then sacrificed and colon was harvested for histologic analysis following H&E staining. PAF/LPS-treated mice that were pre-treated with LGG had lower intestinal injury scores than those pre-treated with vehicle alone (A), representative images are shown. Inflammatory cytokines MIP-2 and TNF-α were also measured by qRT-PCR after PAF/LPS and found to be decreased in the mice pre-treated with LGG (B). IL-10R2 and SOCS3 were significantly induced in the LGG pre-treated mice after PAF/LPS compared to vehicle-fed mice (C), while IL-10 was unchanged. Data are depicted as mean ± SEM (n = 5−7 mice per condition, *p<0.05, **p<0.01).