Investigating immune metabolism and exhaustion in Type 2 Diabetes.

Abstract

Introduction In the era of rapid modernisation and urbanisation, the global incidence of non-communicable diseases such as type 2 diabetes (T2D) has significantly increased. This has been attributed to increased sedentary lifestyles and the adaptation of unhealthy diets, particularly in low-to-middle-income countries like South Africa. These changes promote the development of obesity, which is one of the major risk factors for T2D. Obesity, further plays a pivotal role in the pathoimmunological changes that are associated with outwards effects of poor glucose control and insulin resistance. Chronic inflammation and increased immune activation are a hallmark of T2D, and both these processes are partially mediated by T-cells. Interestingly, these pathological consequences are identified as early as in pre-diabetes before the onset of overt T2D. Upon activation, T-cells release cytokines that induce the activation of other immune cells and polarises T-cells towards the pro-inflammatory subset. This consequently leads to a pro-inflammatory milieu that alters T-cell function and predisposes individuals with pre-diabetes or patients with T2D to developing cardiovascular disease (CVD). Although few studies have implicated activated T-cells in mediating inflammation and altering myocardial function in poor glucose control, the underlying mechanisms and sequence of events remains scarce. Therefore, this study made use of a short-term high-fat diet (HFD)-induced mouse model of pre-diabetes to investigate inflammation and immune responses mediated by T-cells. Furthermore, it assessed and compared the modulatory effects of low-dose aspirin (LDA), metformin and fluvastatin (statin), well-acknowledged anti-inflammatory, anti-hyperglycaemic and cholesterol lowering drugs, respectively, on inflammation, T-cell activation, and cardiovascular risk. Methods This study involved the use of a diet-induced pre-diabetes and inflammation mouse model of glucose intolerance. Briefly, in phase one of the experiment, a total of 27 six-week-old male C57BL/6 mice were randomised into either a high-fat diet (HFD) (n=21) or low-fat diet (LFD) (n=6) groups for a total of 8 weeks. Phase two of the experiment subsequently initiated at week 9 whereby HFD-fed mice were randomised into a short-term treatment with either metformin, LDA or in combination with metformin (LDA+Met) or statin over an additional 6-week period (n=6/7group). Changes in body weights were monitored on a weekly basis. Glucose profiles, cholesterol levels, complete blood counts, T-cell associated cytokines, and the expression on T-cell markers were measured at the end of phase one (week 8) and phase two (week 14) of the experiments. The Kolmogorov-Smirnov test with Dallal-Wilkinson- Lillie was performed for normality testing. For parametric data, the mean differences between the LFDand HFD-fed groups were assessed using unpaired student t-test and were reported as mean ± standard error. The Man Whitney U test was used for non-parametric data and reported as the median and interquartile range [IQR]. Comparisons across the diet and treatments groups were assessed using a Two-way analysis of variance (ANOVA). A posthoc Tukey's multiple comparisons test was performed if the F-value reached statistical significance (p<0.05). The Kruskal-Wallis test, followed by a Dunn's posthoc test, was used for non-parametric data. A p-value of < 0.05 was considered statistically significant. The GraphPad Prism version 6 software (GraphPad Software Inc, CA, USA) was used for all statistical analysis. Results The HFD-fed group had significantly increased weight gain (29.17%) in comparison to the LFD-fed group (21.74%) after the 8-week period. Notably, HFD-feeding (HFF) was associated with impaired metabolic function marked by poor glucose control and a state of hypercholesterolemia. In particular, the HFD-fed group had increased fasting glucose (p<0.0001) and 2-hour postprandial area under curve (p=0.0029) when challenged with an oral glucose tolerance test in comparison to the LFD group. In addition, total cholesterol (Tc) (p=0.0039) and low-density lipoprotein (LDL)-c (p=0.0447) levels were higher in the HFD-group than LFD-group, whilst high-density lipoprotein (HDL)-c levels were comparable between the groups (p=0.1749). HFF was associated with enhanced levels of inflammation and generalised immune activation, marked by increased white cell count (WCC) (p=0.008) and elevated levels of interleukin (IL)-6 (p<0.0001), IL-2, tumour necrosis factor (TNF)-α (p=0.0312) and IL-17A (p<0.0001). Most importantly, HFF upregulated Fas (CD95) and downregulated CD69 (p=0.0009) expression on T-cells without altering the levels of programmed-cell death 1 (PD-1) (p=0.6408). The elevated levels of Fas were directly associated with body weight gain (r=0.93, p=0.0333). Short-term treatment with LDA+Met lowered insulin levels (p=0.0475) and fasting blood glucose (p<0.0001) when compared the untreated HFD-fed group. Although treatment with LDA monotherapy did not affect any cholesterol levels, metformin monotherapy and statin significantly lowered Tc and LDL-c when compared to the untreated HFD-fed group (p<0.05). Treatment with LDA+Met lowered WCC (p=0.0095), lymphocyte count (p=0.0264), IL-6 (p=0.0002), TNF-α (p=0.0465), IL-2 (p=0.0001) and IL-17A (p<0.0001), when compared to the untreated HFD-fed group. Lastly, LDA+Met (p=0.0010) but not LDA (p=0.147), upregulated the expression of CD69 on T-cells whilst both treatment groups had no impact on PD-1 levels. Treatment with fluvastatin had no effect on the levels of inflammation (p>0.05). Conclusion This study showed that T-cell dysfunction is congruent with a state of inflammation, hypercholesterolaemia and poor glucose control in the early stages of obesity. Notably, the altered Tcell function is partially mediated by the aberrant expression of Fas and CD69. The combinational treatment of LDA with metformin was more effective than the use of LDA only in improving glucose control, ameliorate inflammation, and moderate T-cell functions. These findings outline the pathological link between the development of inflammation, immune activation and altered lipid metabolism in a pre-diabetic state. More importantly, it highlights the cardiovascular risk properties of statins and enhanced anti-inflammatory efficacy of LDA when combined with metformin in poor glucose control.Therefore, alleviating inflammation and lowering glucose levels during the early development of T2D may be an effective strategy to attenuate T-cell remodeling in diet-driven metabolic disturbances.