Salmonella Typhimurium Infection Reduces the Ascorbic Acid Uptake in the Intestine

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.


Introduction
Vitamin C (ascorbic acid (AA)) is a powerful antioxidant that acts as a cofactor for several key biological reactions [1]. This micronutrient is not only important for the proper function of numerous enzymatic reactions, but also sustaining optimum vitamin C body homeostasis assists in the defense against certain diseases such as cataract formation, liver disease, cancer, osteoporosis, and heart disease [2][3][4][5]. The defense mechanisms can be accredited to vitamin C's antioxidant nature, which can mitigate the damaging effects of reactive oxygen species (ROS) and oxidative stress, which are generally observed in the above named diseases. Humans obtain vitamin C through dietary sources via intestinal absorption as they cannot synthesize this micronutrient de novo. Pronounced vitamin C deficiency is commonly observed in the elderly population, smokers, and alcoholics [6][7][8]. The cause of vitamin C deficiency in the conditions mentioned above is mainly attributed to the impaired absorption at the cellular level. Vitamin C deficiency is one of the contributing factors in the pathogenesis of inflammatory bowel disease (IBD, an intestinal disorder) [9][10][11][12][13]. Previous studies have shown that the administration of vitamin C to patients with sepsis displayed a considerable recuperation of their clinical symptoms [14][15][16].
Vitamin C is indispensable for normal cellular metabolic activities and immune function. In addition, humans have a limited capacity to store adequate levels of vitamin C in the body. With these factors considered, a severe and prolonged Salmonella infection may negatively impact vitamin C homeostasis and lead to disturbances in the nutrient levels of infected individuals. Currently, nothing is known about the consequence of Salmonella infection on vitamin C intestinal absorption. Therefore, in this study, we investigated whether Salmonella infection alters AA absorption and, if so, what are the precise molecular mechanisms involved in this process. Our findings revealed that Salmonella infection inhibits intestinal AA uptake, and this inhibitory effect is partially mediated through proinflammatory cytokines and the transcription of the SLC23A1 gene. The anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). In addition, western blot protocols utilized anti-rabbit IRDye-800 and anti-mouse IRDye-680 secondary antibodies sourced from LI-COR Biosciences (Lincoln, NE). The recombinant human IL-1β was acquired through R&D Systems, Inc. (Minneapolis, MN). Integrated DNA Technologies (San Diego, CA) supplied the individually synthesized custom oligonucleotide primers used in RT-qPCR analysis. All further chemicals, kits, and molecular biological reagents were obtained from reputable scientific manufacturers and stored appropriately to maintain substance integrity and stability.

Cell
Culture. Caco-2 cells from human colorectal adenocarcinoma cells (ATCC, Manassas, VA) served as an intestinal epithelial in vitro model. They were kept in EMEM containing 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) in a 37°C cell incubator with a 95% air-5% CO 2 and high-humidity atmosphere. Confluent monolayers of Caco-2 cells were used in these investigations to determine the effect of IL-1β on hSVCT1 protein and mRNA expression levels as well as to assess the impact of this proinflammatory cytokine on the rate of 14 C-AA uptake and the activity of the SLC23A1 promoter. For the IL-1β studies, Caco-2 cells were serum-starved to cause synchronization by maintaining cell cultures in EMEM with only 0.5% FBS overnight before treatment.

Bacterial Infection.
Both Salmonella Typhimurium (ATCC strain 14028 [55]) wild-type (WT) and nonpathogenic (Δ invA Δ spiB) mutant [49] were used in these investigations. LB (Luria Bertani) broth was the growth medium for overnight bacterial cultures and then PBS was used to    2.4. 14 C-Ascorbic Acid (AA) Uptake. Approximately 1 cm long sheets of jejunum with the submucosa intact were harvested from mice, which were then incubated in KR (Krebs-Ringer) buffer with or without unlabeled (1 mM) AA and in the presence of labeled (0.1 μCi) AA within glass test tubes immersed in a 37°C water bath for 7 min as described before [22,51]. Monolayers of Caco-2 cells were grown to a postconfluent phase before being treated with IL-1β (50 ng/ml) to perform 14 C-AA uptake [51]. Forty-eight hours later, KR buffer was used to incubate these adherent cells at 37°C with or without unlabeled (1 mM) AA and in the presence of AA (0.1 μCi) for 30 min. 1 N NaOH was then applied to the jejunum tissue or cells to cause lysis and then the samples were incubated at 80°C in a Fisher Scientific Isotemp 825F Incubator for 15 min and neutralized with 10 N HCl. A liquid scintillation counter (Beckman Coulter, Brea, CA) recorded the radiation levels of the individual uptake lysates.

Western Blot
Analysis. Caco-2 cells or mouse jejunal mucosa total protein samples were prepared in radioimmunoprecipitation (RIPA) buffer (Sigma) as described before [22,51]. NuPAGE 4-12% mini gels (Invitrogen) and buffers were used to separate each total protein lysate (60 μg) before the proteins were transferred onto Immobilon-FL PVDF    Figure 4: Effect of IL-1β on AA uptake, hSVCT1 expression levels, and the activity of SLC23A1 promoter in Caco-2 cells. Concentrationdependent 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.

Results and Discussion
3.1. Effect of Salmonella Infection on Uptake of AA and mSVCT1 Expression in Mouse Jejunum. Previous studies showed that Salmonella affects the intestinal mucosal physiology mainly through the immune/inflammatory response that is subsequently triggered [38,60]. Here, we used mice as an in vivo model for the investigations. Streptomycinpretreated mice were infected with Salmonella (WT) or nonpathogenic Δ invA Δ spiB mutant by oral gavage [49]. After 72 h of infection, we performed AA uptake in the jejunal sheets, which showed a significant (p < 0:001) decrease in the AA uptake in Salmonella-infected compared to uninfected control mouse jejunum (Figure 1). To further confirm that the effect of Salmonella infection in mice is mediated via its immune/inflammatory action, we used a nonpathogenic (avirulent) mutant (Δ invA Δ spiB) strain of Salmonella that does not cause intestinal inflammation. This mutant neither invades the intestinal mucosa nor replicates within the infected host. The nonpathogenic mutant-infected mice did not exhibit inhibition of the intestinal AA uptake (Figure 1). Therefore, we focused subsequent investigations on the inhibitory effect of the Salmonella (WT). Our evidence suggests that Salmonella infection inhibits intestinal AA uptake in mice mainly through the immune/inflammatory response that it induces [38,60]. The observed uptake inhibition was accompanied by a marked reduction in mSVCT1 protein (Figure 2(a)), mRNA (Figure 2(b)), and hnRNA expressions (hnRNA expression levels reflect changes in the transcription rate of a given gene [57]) ( Figure 2(c)) in Salmonella-infected compared to uninfected mouse jejunum. The latter indicates that the inhibition in SVCT1 expression in the Salmonella-infected mouse jejunum is partially mediated via altered transcription of the SLC23A1 gene. Future studies will confirm the observed decreased mSVCT1 expression levels in the isolated enterocytes.

Effect of Salmonella Infection on NLRP3 and IL-1β
Expression Levels in Mouse Jejunum. Previous studies have shown that Salmonella infection triggers inflammasome assembly through identification by cytoplasmic receptors such as NLRP3 to release proinflammatory cytokines such as IL-1β [44][45][46][47]. To test this, we determined NLRP3 mRNA expression levels in Salmonella-infected mouse jejunum. The results showed that the NLRP3 mRNA expression was significantly (p < 0:001) upregulated in Salmonella-infected mice jejunum (Figure 3(a)) suggesting an activated inflammatory response upon Salmonella infection in the intestine. To substantiate this finding, we also determined the expression level of IL-1β in Salmonella-infected mouse jejunum and found significantly (p < 0:0001) increased IL-1β mRNA expression in Salmonella-infected mouse jejunum compared to uninfected mice (Figure 3(b)).

3.3.
Effect of IL-1β on AA Uptake, hSVCT1 Expression, and the Activity of SLC23A1 Promoter in Intestinal Epithelial Caco-2 Cells. The effect of IL-1β on the uptake of AA in Caco-2 cells has never been investigated before. Therefore, Caco-2 cells were exposed to three different concentrations of IL-1β (10, 25, and 50 ng/ml) for 48 h, and hSVCT1 mRNA expression levels were determined. The results revealed a marked decrease in hSVCT1 mRNA expression  Mediators of Inflammation at all three IL-1β concentrations compared to control cells (Figure 4(a)). Subsequently, we examined the effect of IL-1β treatment (50 ng/ml for 48 h) on the uptake of AA in Caco-2 cells. The outcomes exhibited a marked inhibitory effect on the uptake of AA in cells exposed to IL-1β (Figure 4(b)). This inhibitory effect was accompanied by a marked reduction in the expression level of hSVCT1 protein (Figure 4(c)). Furthermore, to investigate the molecular mechanism(s) involved in AA uptake inhibition upon IL-1β exposure, we determined the effect of IL-1β treatment on the SLC23A1 promoter activity. The results displayed a marked decrease in the SLC23A1 promoter activity in IL-1β-exposed cells (Figure 4(d)). Collectively, these results indicate that the inhibition of AA uptake mediated by IL-1β exposure is partially facilitated through transcriptional mechanism(s) involving the SLC23A1 gene. The observed decreased expression of hSVCT1 caused by IL-1β is not a universal phenomenon among members of the solute carrier (SLC) superfamily of transporter proteins. Previous studies have shown that the PepT1 (peptide transporter 1, product of theSLC15A1 gene) was upregulated upon cytokine treatment [61,62]. Together, our findings suggest that Salmonella-triggered inflammasome assembly via recognition by NLRP3 induces proinflammatory cytokines such as IL-1β, which causes an inhibitory effect on the uptake of AA in intestinal epithelial cells.

Salmonella Infection
Reduces the Expression of GRHPR in Mouse Jejunum. Previously, we have identified GRHPR (glyoxalate reductase/hydroxypyruvate reductase) as an interacting partner for SVCT1, and this association upregulates AA uptake [63]. In this study, we have determined the role of GRHPR in Salmonella-infected AA uptake in mouse jejunum. Salmonella infection triggered a marked decrease in GRHPR protein and mRNA expression in mouse jejunum (Figures 5(a) and 5(b)). These findings show that GRHPR may also play a role in the observed inhibitory effect on AA uptake in mouse jejunum.
In conclusion, our investigations demonstrate that Salmonella infection decreases intestinal AA absorption, and this inhibitory effect is facilitated partially through proinflammatory cytokines which are induced as a result of Salmonella infection in aninfected host. In addition, the observed intestinal AA uptake inhibition caused by proinflammatory cytokines may provide a novel hypothesis to explain the suboptimal levels of vitamin C observed in IBD patients [9,12,13].

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
No potential conflict of interest was reported by the authors.

Authors' Contributions
T.T. and V.S.S. were responsible for the conceptualization. T.T. and V.S.S. were responsible for the formal analysis. T.T. and V.S.S. were responsible for the investigation. T.T., S.B.S., N.L., and V.S.S. wrote the original draft. T.T., S.B.S., N.L., and V.S.S. wrote, reviewed, and edited the manuscript. T.T., S.B.S., N.L., and V.S.S. were responsible for the visualization. All authors have read and approved the manuscript.