Regulation of Staphylococcal Enterotoxin-Induced Inflammation in Spleen Cells from Diabetic Mice by Polyphenols

Abstract

Patients with diabetes are known to be more susceptible to infections following the establishment of Staphylococcus aureus in their nasal passages and on their skin. The present study evaluated the effects of staphylococcal enterotoxin A (SEA) on the immune responses of spleen cells derived from diabetic mice, and examined the effects of polyphenols, catechins, and nobiletin on inflammation-related gene expression associated with the immune response. (-)-Epigallocatechin gallate (EGCG), possessing hydroxyl groups, interacted with SEA, whereas nobiletin, possessing methyl groups, did not interact with SEA. The exposure of spleen cells derived from diabetic mice to SEA enhanced the expression of interferon gamma, suppressor of cytokine signaling 1, signal transducer and activator of transcription 3, interferon-induced transmembrane protein 3, Janus kinase 2, and interferon regulatory factor 3, suggesting that SEA sensitivity is variable in the development of diabetes. Both EGCG and nobiletin changed the expression of genes related to SEA-induced inflammation in spleen cells, suggesting that they inhibit inflammation through different mechanisms. These results may lead to a better understanding of the SEA-induced inflammatory response during diabetogenesis, and the establishment of methods to control these effects with polyphenols.

Keywords: Staphylococcus aureus; diabetes; inflammation; polyphenols; staphylococcal enterotoxin A.

Figure 1

Polyphenols used in this study. (A) Structure of catechin. (B) Structure of nobiletin. G: galloyl group.

Figure 2

Interactions between SEA and polyphenols. (A) Melting profile of SEA in the presence of polyphenols. The images show the profile of the derivative of fluorescence emission as a function of temperature [d(Fluorescence)/dT]. SEA was used as the protein, and each polyphenol served as the ligand. The compounds were reacted at 37 °C for 2 h before the interaction was analyzed using the protein thermal shift assay. (B) Melting temperature of SEA in the presence of polyphenols. * p < 0.05 compared to the control. The data are presented as the mean ± standard deviation of four independent experiments.

Figure 3

The effect of pH on the interaction between SEA and polyphenols. All samples were prepared at a final concentration of 3.0 mM. Each test sample was mixed with SEA (5.0 μg/mL) in 100 μL of Milli-Q water, PBS (pH 7.2), or McIlvaine buffer (pH 4.0, 6.0, or 8.0) and incubated at 37 °C for 24 h. Following centrifugation (4000× g, 5 min), the supernatant was applied to SDS-PAGE and visualized by Western blotting. Milli-Q water was used as a positive control. * p < 0.05 vs. the control. The values represent the mean ± standard deviation of three independent experiments.

Figure 4

Comparison of SEA-induced JAK/STAT pathway-related gene expression in spleen cells from normal C57BL/6J and ICR mice. (A) IFN-γ, (B) SOCS1, (C) STAT3, (D) IFITM3, (E) IRF3, (F) JAK2. Spleen cells from normal mice (C57BL/6J and ICR mice) were exposed to SEA (50 ng/mL), and the expression of JAK/STAT pathway-related genes was examined by real-time RT-PCR. Gene expression was normalized to Hprt gene expression. The fold change was determined relative to the control without SEA. * p < 0.05 compared to the control. The values represent the mean ± standard deviation of three independent experiments.

Figure 5

Comparison of SEA-induced JAK/STAT pathway-related gene expression in spleen cells from normal mice and diabetic mice (DM). (A) IFN-γ, (B) SOCS1, (C) STAT3, (D) IFITM3, (E) IRF3, (F) JAK2. Spleen cells from normal mice (control) and DM were exposed to SEA (50 ng/mL), and the expression of JAK/STAT pathway-related genes was examined by real-time RT-PCR. Gene expression was normalized to that of the Hprt gene. The fold change was determined relative to the control without SEA. * p < 0.05 compared to (−) SEA, † p < 0.05 compared to the control. The values represent the mean ± standard deviation of three independent experiments.

Figure 6

The effect of polyphenols on the SEA-induced expression of JAK/STAT pathway-related genes in spleen cells from normal mice and diabetic mice (DM). (A) IFN-γ, (B) SOCS1, (C) STAT3, (D) IFITM3, (E) IRF3, (F) JAK2. Spleen cells from normal mice (control) and DM were exposed to different combinations of SEA (50 ng/mL), EGCG (0.05 mM), and nobiletin (0.05 mM), and the expression of JAK/STAT pathway-related genes was examined by real-time RT-PCR. Gene expression was normalized to that of the Hprt gene. The fold change was determined relative to the control without SEA. * p < 0.05 compared to normal—SEA, † p < 0.05 compared to DM—SEA, # p < 0.05 compared to normal. The values represent the mean ± standard deviation of three independent experiments.

Figure 7

The effect of EGCG and nobiletin on SEA-induced changes in JAK/STAT pathway-related gene expression. Spleen cells from normal mice and diabetic mice (DM) were exposed to SEA, EGCG, and nobiletin. The results for C57BL/6J mice (normal) are shown only for the group treated with EGCG. The fold change was determined relative to the control without SEA treatment. Differential expression of mRNA is shown by the intensity of green (upregulation) versus red (downregulation).