Chronic superantigen exposure induces systemic inflammation, elevated bloodstream endotoxin, and abnormal glucose tolerance in rabbits: possible role in diabetes

Abstract

Excessive weight and obesity are associated with the development of diabetes mellitus type 2 (DMII) in humans. They also pose high risks of Staphylococcus aureus colonization and overt infections. S. aureus causes a wide range of severe illnesses in both healthy and immunocompromised individuals. Among S. aureus virulence factors, superantigens are essential for pathogenicity. In this study, we show that rabbits that are chronically exposed to S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1) experience impaired glucose tolerance, systemic inflammation, and elevated endotoxin levels in the bloodstream, all of which are common findings in DMII. Additionally, such DMII-associated findings are also seen through effects of TSST-1 on isolated adipocytes. Collectively, our findings suggest that chronic exposure to S. aureus superantigens facilitates the development of DMII, which may lead to therapeutic targeting of S. aureus and its superantigens.

Importance: Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo. Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes.

FIG 1

Chronic exposure to TSST-1 induces impaired glucose metabolism. (A) Blood glucose levels in two groups of rabbits (5/group) over time, as assayed weekly. Statistically significant differences were determined by two-way analysis of variance (ANOVA). (B) Relative pancreatic insulin transcript levels in the same two groups of animals, analyzed immediately after the glucose challenge test at the end of the 6-week experiment. Statistically significant differences were determined by Student’s t test. Error bars show standard deviations.

FIG 2

TSST-1 induces systemic inflammation and elevated circulating endotoxin levels in vivo. (A) Representative images of the spleens harvested from TSST-1 and PBS animals after 1 week and 6 weeks of treatment. (B) Average weights of spleens from TSST-1- and PBS-treated animals after 6 weeks of treatment. (C) TNF-α levels in plasma collected from the two groups of animals each week. (D) Average TNF-α levels in plasma collected from two groups of animals throughout the experiment. (E) Image shows representative results of the Limulus amebocyte lysate assay performed on collected plasma. (F) Average endotoxin (lipopolysaccharide [LPS]) levels in the circulation of two groups of animals after 6 weeks of treatment. Statistically significant differences were determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars show standard deviations.

FIG 3

TSST-1 induces liver damage. (A) Hematoxylin-and-eosin (H&E)-stained livers from TSST-1- and PBS-treated rabbits after 6 weeks of treatment. Circles indicate areas of microvesicle formation. (B) Frequencies of HepG2 cells labeled with annexin V antibody after treatment with TSST-1 (100 µg/ml) or PBS at various time points. (C) Percentages of HepG2 cells surviving after 24 and 48 h of treatment with TSST-1 (100, 50, and 10 µg/ml) or PBS. (D) Percentages of HepG2 cells surviving after 48 h of treatment with TSST-1 (50 µg/ml) alone, LPS (endotoxin; 10 and 100 ng/ml) alone, or TSST-1 and LPS together. Statistically significant differences were analyzed by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars show standard deviations.

FIG 4

TSST-1 induces inflammation in adipocytes. (A) IL-6 levels in human adipocyte supernatants after 24 and 48 h of treatment with TSST-1 (100, 50, 10, and 1 µg/ml) or PBS. (B) IL-6 levels in human adipocyte supernatants after 24 h of treatment with TSST-1 (50 µg/ml) alone, TNF-α (1 ng/ml) alone, or TSST-1 and TNF-α together. (C) IL-6 levels in human adipocyte supernatants after 6 h of treatment with TSST-1 (50 µg/ml) alone, LPS (endotoxin; 5 ng/ml) alone, or TSST-1 and LPS together. (D and E) IL-6, IL-8, TNF-α, MCP-1, and adiponectin transcript levels in adipocytes isolated from two groups of animals ex vivo after 1 week (D) or 6 weeks (E) of treatment. Statistically significant differences were determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars show standard deviations.

FIG 5

TSST-1 induces insulin resistance in adipocytes ex vivo. 2DG uptake ability of adipocytes isolated from two groups of animals after 1 week (A) and 6 weeks (B) of treatment ex vivo. Statistically significant differences were determined by Student’s t test (**, P < 0.01; ***, P < 0.001). Error bars show standard deviations.

FIG 6

TSST-1 induces lipolysis in adipocytes. (A) Glycerol levels in NDAD supernatants after 6 h of treatment with TSST-1 (20, 10, and 1 µg/ml) or PBS. (B) Relative expression levels of ATGL, HSL, and MGL genes in human adipocytes after 6 h of treatment with TSST-1 (20 µg/ml) or PBS. (C) HSL gene expression levels in ex vivo adipocytes isolated after 6 weeks of treatment with TSST-1 or PBS. Statistically significant differences were determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars show standard deviations.

FIG 7

Model of the possible role of SAgs in the development of DMII. As obesity progresses (42), the rates of S. aureus colonization and infection increase [1]. Since all pathogenic human S. aureus isolates produce SAgs, obese individuals have a high risk of frequent exposure to SAgs [2]. In vivo, SAgs induce proinflammatory cytokine (IL-6, IL-8, and TNF-α) production in adipocytes [3], which may contribute to the development of systemic inflammation [4]. By producing chemoattractants, such as MCP-1 and IL-8, adipocytes can further recruit immune cells to adipose tissue [5]. Once recruited, these immune cells can also be stimulated by SAgs to produce additional proinflammatory signals [6]. SAgs induce liver damage (hepatocyte apoptosis) [8], which leads to a reduction in the endotoxin clearance function of the liver, resulting in elevated circulating endotoxin (LPS) levels [9]. Together with the proinflammatory signals from the recruited immune cells, endotoxin in the circulation (perhaps spilling over from the intestinal microbiota) can further enhance and maintain ongoing systemic inflammation [7 and 10]; chronic systemic inflammation has previously been shown to have an important role in the development of peripheral insulin resistance [11]. TSST-1 induces lipolysis in adipocytes [12]. Free fatty acids can be taken up by skeletal muscle and subsequently converted into intramuscular lipids, which are strongly associated with the development of insulin resistance [13]. Chronic exposure to SAgs in vivo can lead to insulin resistance in adipocytes [14]. Altogether, chronic insulin resistance in multiple tissues results in impaired glucose tolerance, the hallmark of DMII [15]. Numbers in brackets indicate the representative order of the process.