The Pivotal Role of Aryl Hydrocarbon Receptor-Regulated Tight Junction Proteins and Innate Immunity on the Synergistic Effects of Postbiotic Butyrate and Active Vitamin D3 to Defense against Microbial Invasion in Salmonella Colitis

Abstract

Our recent report illustrated the unitedly advantageous effects of postbiotic butyrate on active vitamin D3 (VD3)-orchestrated innate immunity in Salmonella colitis. There is growing awareness that aryl hydrocarbon receptor (AhR) can regulate intestinal immunity and barrier function, through modulating cecal inflammation and junction proteins expression. Hence, we researched the participation of AhR-regulated tight junction functions on the united effects of butyrate and VD3 on intestinal defense to Salmonella infection. Salmonella colitis model were elicited by oral gavage with 1 × 10⁸ CFU of a S. typhimurium wild-type strain SL1344 in C57BL/6 mice. Before and after the colitis generation, mice were fed with butyrate and/or VD3 by oral gavage in the absence or presence of intraperitoneal injection of AhR inhibitor for 4 and 7 days, respectively. We observed that butyrate and VD3 could concert together to reduce the invasion of Salmonella in colitis mice by enhancing cecal cytokines and antimicrobial peptides expression and reducing zonulin and claudin-2 protein expressions in mucosal stain, compared to single treatment, which were counteracted by AhR inhibitor. It implies that AhR is involved in the united effects of butyrate and VD3 on the intestinal defense to Salmonella infection in colitis mice. This study discloses the promising alternative therapy of combining postbiotic and VD3 for invasive Salmonellosis and the pivotal role of AhR pathway.

Keywords: Salmonella colitis; active vitamin D3; acyl hydrocarbon receptor; innate immunity; postbiotics; tight junction.

Figure 1

The involvement of AhR in the synergistic effects of admixture of VD3 and butyrate attenuates the severity of Salmonella colitis in mice. Mice were bred and housed under the technical regulation of the animal facility of the Center for Cellular and Biomolecular Research, Kaohsiung, Taiwan. The 6–8-week-old female C57BL/6 mice (Charles River, Wilmington, MA, USA) were treated or infected as described in the material and methods and divided into the following groups: Control (Open control), ST (S.Tm infected), VD (VD3 and S.Tm infected), BU (butyrate 100 mg/kg mice and S.Tm infected), VD+BU (combination of VD3 and butyrate plus S.Tm infected), and VD+BU+AHRi (combination of VD3 and butyrate plus S.Tm infected and AhR inhibitor). Diarrhea situation scores (a) and loss of body weight (b) of mice were recoded daily. Segments of cecum were harvested, fixed in formaldehyde, and stained with hematoxylin and eosin. Representative histological images (×20 and ×50 magnification) of cecum from the different experimental groups were shown in (c) and the analyzed pathological scores for colitis in (d) The data shown are means ± SEM (n = 7 mice/group). *, p < 0.05.

Figure 2

The involvement of AhR in the synergistic effects of BU on the VD3-regulated cecal proinflammatory cytokines and AMPs in Salmonella colitis mice. Mice were treated or infected as described in the material and methods. Before and after the colitis induction, mice were oral gavaged with BU or VD3 or combination of both in the absence or presence of intraperitoneal injection of AhR inhibitor (AHRi) for 4 and 7 days, respectively. Total RNA was extracted from the cecal tissues. IL-17A (a), IL-22 (b), LL-37 (CRAMP) (c), mIL-6 (d), mTNF-α, (e), mIL-1β (f), and mBD-3 (g) expressions were analyzed using quantitative RT-PCR. Values are measured as fold increase compared to the level of control mice. The data shown are means ± the SEM (n = 7 mice/group). * p < 0.05, ** p < 0.01.

Figure 2

The involvement of AhR in the synergistic effects of BU on the VD3-regulated cecal proinflammatory cytokines and AMPs in Salmonella colitis mice. Mice were treated or infected as described in the material and methods. Before and after the colitis induction, mice were oral gavaged with BU or VD3 or combination of both in the absence or presence of intraperitoneal injection of AhR inhibitor (AHRi) for 4 and 7 days, respectively. Total RNA was extracted from the cecal tissues. IL-17A (a), IL-22 (b), LL-37 (CRAMP) (c), mIL-6 (d), mTNF-α, (e), mIL-1β (f), and mBD-3 (g) expressions were analyzed using quantitative RT-PCR. Values are measured as fold increase compared to the level of control mice. The data shown are means ± the SEM (n = 7 mice/group). * p < 0.05, ** p < 0.01.

Figure 3

The involvement of AhR in the combined effects of VD3 and butyrate on attenuating systemic bacterial translocation of Salmonella colitis mice. Mice were bred and housed under the technical regulation of the animal facility of the Center for Cellular and Biomolecular Research, Kaohsiung, Taiwan. The 6–8-week-old female C57BL/6 mice (Charles River, USA) were treated or infected as described in the material and methods. Before and after the colitis induction, mice were oral gavage with BU or VD3 or combination of both in the absence or presence of intraperitoneal injection of AhR inhibitor (AHRi) for 4 and 7 days, respectively. The number of bacteria was counted from liver (a) and spleen (b) homogenates of different groups, as shown in the material and methods. The data shown are represented as the means ± the SEM of the bacterial load in the liver and spleen (n = 7). * p < 0.05.

Figure 4

The involvement of AhR in the combined effect of VD3 and butyrate on reducing the colon epithelial zonulin and claudin-2 proteins expression following Salmonella colitis in mice. Mice were treated or infected as described in the material and methods and divided into the following groups: Control (Open control), ST (S.Tm infected), VD (VD3 and S.Tm infected), BU (butyrate 100 mg/kg mice and S.Tm infected), VD+BU (combination of VD3 and butyrate plus S.Tm infected), and VD+BU+AHRi (combination of VD3 and butyrate plus S.Tm infected and AhR inhibitor). (a) Zonulin and claudin-2 proteins expression in these groups were detected by immunohistochemistry staining (original magnification, ×400; scale bar, 25 µm; n = 3). The levels of zonulin (b) and claudin-2 (c) immunohistochemistry staining were analyzed and calculated by image J. * p < 0.05, *** p < 0.001.