Salmonella Typhimurium Infection Reduces the Ascorbic Acid Uptake in the Intestine

Abstract

Salmonella Typhimurium infection of the gastrointestinal tract leads to damage that compromises the integrity of the intestinal epithelium and results in enterocolitis and inflammation. Salmonella infection promotes the expression of inflammasome NLRP3, leading to activation and release of proinflammatory cytokines such as IL-1β, and the infected host often displays altered nutrient levels. To date, the effect of Salmonella infection and proinflammatory cytokine IL-1β on the intestinal uptake of ascorbic acid (AA) is unknown. Our results revealed a marked decrease in the rate of AA uptake in mouse jejunum infected with Salmonella wild type (WT). However, the nonpathogenic mutant (Δ invA Δ spiB) strain did not affect AA uptake. The decrease in AA uptake due to Salmonella WT infection is accompanied by significantly lower expression of mouse (m)SVCT1 protein, mRNA, and hnRNA levels. NLRP3 and IL-1β expression levels were markedly increased in Salmonella-infected mouse jejunum. IL-1β-exposed Caco-2 cells displayed marked inhibition in AA uptake and significantly decreased hSVCT1 expression at both protein and mRNA levels. Furthermore, the activity of the SLC23A1 promoter was significantly inhibited by IL-1β exposure. In addition, GRHPR (a known SVCT1 interactor) protein and mRNA expression levels were significantly reduced in Salmonella-infected mouse jejunum. These results indicate that Salmonella infection inhibits AA absorption in mouse jejunum and IL-1β-exposed Caco-2 cells. The observed inhibitory effect may partially be mediated through transcriptional mechanisms.

Figure 1

Effect of Salmonella infection on ¹⁴C-AA uptake in mouse jejunum. Streptomycin-pretreated mice were infected with Salmonella (WT) or mutant (Δ invA Δ spiB) (100 μl of 10¹⁰ bacteria/ml) by oral gavage. ¹⁴C-AA uptake was subsequently determined in mouse jejunum after 72 h following oral gavage. Values are mean ± SEM of at least 3 sets of animals. ^∗∗∗p < 0.001.

Figure 2

Salmonella infection decreases the level of expression of mSVCT1 in mouse jejunum. Western blot analysis was performed utilizing 60 μg of protein isolated from Salmonella-exposed mouse intestine to measure the mSVCT1 protein expression (a). The mSVCT1 mRNA (b) and hnRNA (c) expression levels in mouse intestinal mucosa were measured by RT-qPCR. Values are mean ± SEM of at least 3 sets of mice. ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

Figure 3

Effect of Salmonella infection on the expression of NLRP3 and IL-1β mRNA in mouse jejunum. The expression of NLRP3 (a) and IL-1β (b) mRNA levels in mouse jejunum by RT-qPCR. Values are mean ± SEM of at least 3 sets of animals. ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001.

Figure 4

Effect of IL-1β on AA uptake, hSVCT1 expression levels, and the activity of SLC23A1 promoter in Caco-2 cells. Concentration-dependent effect of IL-1β treatment on the hSVCT1 mRNA expression (a). Determined AA uptake in IL-1β (50 ng/ml)-exposed and control cells (b). Performed western blotting to quantify the expression of hSVCT1 protein in IL-1β-exposed and control cells (c). The activity of SLC23A1 promoter was determined in IL-1β-treated and control cells (d). Values are mean ± SEM of at least 3-4 independent experimental runs using different passage of cells. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

Salmonella infection decreases the expression of GRHPR in mouse jejunum. The GRHPR protein (a) and mRNA (b) expression levels in mouse jejunum were calculated from western blot and RT-qPCR data, respectively. Values are mean ± SEM of at least 3 sets of animal. ^∗∗p < 0.01.