Persistent hyperglycemia modulates gut immune function and microbiota in rats

Abstract

Background: Since hyperglycemia-induced cellular dysfunction could be associated with alterations of the immune system, we tested the hypothesis that hyperglycemia augments the aberrant immune responses such as inflammation and differentiation of CD4(+) T lymphocytes in the mesenteric lymph nodes (MLNs), and induces alterations of microbiota both under physiological and pathological conditions.

Methods: Male Wistar rats were randomly allocated into 4 groups: Control and Endotoxemia (lipopolysaccharide, LPS 1 mg/kg) with or without hyperglycemia. The hyperglycemia groups were administered glucose solution (10-40 %), while the normoglycemia groups were administered saline. Alterations of the mRNA expressions of inflammatory cytokines and CD4(+) T lymphocyte transcriptional factor expressions in the MLNs, and those of the intestinal microbiota were analyzed at 24 hr.

Results: Hyperglycemia was kept approximately 250-350 mg/dL during the 24 hr study period. At the end of 24 hr, hyperglycemia augmented the mRNA expressions of interleukin (IL)-1β and IL-6 in the MLNs, while both the helper T (Th) 2 and regulatory-T (Treg) transcriptional factors were simultaneously up-regulated under non-endotoxemic condition. LPS injection significantly modulated the obligate anaerobe bacterial populations of the Bacteroidetes class, and altered the population sizes of the Clostridium perfringens and the Bacteroides fragilis subgroup. Hyperglycemia did not enhance these alterations of the microbiota evoked by LPS, although it did modify the bacterial populations of the L. reuteri subgroup and staphylococci in healthy condition without endotoxemia.

Conclusions: The present study indicates that both gut immune function and microbiota are significantly modulated by persistent hyperglycemia.

Keywords: CD4+ T lymphocyte subsets; Gut barrier function; Mesenteric lymph nodes; Pro-inflammatory cytokine.

Fig. 1

Changes of blood glucose concentrations during 24 hr study period. Comparison between four groups was performed by mixed-effects analysis of variance (ANOVA) followed by Tukey’s post hoc test. Abbreviation; CN: control with normoglycemia group, CH: control with hyperglycemia group, EN: endotoxemia with normoglycemia group, EH: endotoxemia with hyperglycemia group. † P < 0.01: CH versus CN, ‡: P < 0.01 EH versus EN. Data are expressed as mean ± SD

Fig. 2

Changes of TNF − α, IL-1β and IL-6 mRNA expression in MLNs. a) Expression of TNF-α mRNA at 24 hr study periods in MLNs. b) Expression of IL-1β mRNA at 24 hr study periods in MLNs. c) Expression of IL-6 mRNA at 24 hr study periods in MLNs. Inflammatory cytokines were analyzed by two-way ANOVA for interaction between factor A (control versus endotoxemia) and factor B (normoglycemia versus hyperglycemia). Ninety five percent confidence intervals for each four factors were also presented. If the interaction between two factors didn’t exist, independent effects of these two factors were confirmed. If the interaction existed, comparison of two groups were performed using Student’s t-test separately in the Control group (CN vs CH) and the Endotoxemia group (EN vs EH). Abbreviation; CN: control with normoglycemia group, CH: control with hyperglycemia group, EN: endotoxemia with normoglycemia group, EH: endotoxemia with hyperglycemia group. Data are expressed as mean ± SD. White and black bars indicate control and endotoxemia group, respectively

Fig. 3

Changes of Tbx21, Gata3, Rorc and Foxp3 mRNA expression in MLNs. a) Expression of Tbx21 mRNA at 24 hr study period in MLNs. b) Expression of Gata3 mRNA at 24 hr study period in MLNs. c) Expression of Rorc mRNA at 24 hr study period in MLNs. d) Expression of Foxp3 mRNA at 24 hr study period in MLNs. Four types of transcriptional factors for CD4+ T lymphocytes were analyzed by two-way ANOVA for interaction between factor A (control versus endotoxemia) and factor B (normoglycemia versus hyperglycemia). Ninety five percent confidence intervals for each four factors were also presented. If the interaction between two factors didn’t exist, independent effects of these two factors were confirmed. If the interaction existed, comparison of two groups were performed using Student’s t-test separately in the Control group (CN vs CH) and the Endotoxemia group (EN vs EH). Abbreviation; CN: control with normoglycemia group, CH: control with hyperglycemia group, EN: endotoxemia with normoglycemia group, EH: endotoxemia with hyperglycemia group. Data are expressed as mean ± SD. White and black bars indicate control and endotoxemia group, respectively. P values described in the figure indicates those obtained by post hoc test

Fig. 4

Alterations of obligate and facultative anaerobes, individual bacteria classes in colon contents. Alterations of obligate and facultative anaerobes, individual bacteria classes in colon contents were analyzed by two-way ANOVA for interaction between factor A (control versus endotoxemia) and factor B (normoglycemia versus hyperglycemia). Ninety five percent confidence intervals for each four factors were also presented. If the interaction between two factors didn’t exist, independent effects of these two factors were confirmed. If the interaction existed, comparison of two groups were performed using Student’s t-test separately in the Control group (CN vs CH) and the Endotoxemia group (EN vs EH). The main effect of endotoxemic insult was found only in class Bcteroidetes. Abbreviation; CN: control with normoglycemia group, CH: control with hyperglycemia group, EN: endotoxemia with normoglycemia group, EH: endotoxemia with hyperglycemia group. Data are expressed as mean ± SD (log10 cells/g of colon contents). White and black bars indicate control and endotoxemia group, respectively