Characterisation of immune responses to mycobacterial infections in a murine model of type 2 diabetes

Alim, Md Abdul (2018) Characterisation of immune responses to mycobacterial infections in a murine model of type 2 diabetes. PhD thesis, James Cook University.

[img]
Preview
PDF (Thesis)
Download (17MB) | Preview
View at Publisher Website: https://doi.org/10.25903/5bda83954eaec
 
19


Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains one of the major global health threats. The disease is responsible for significant morbidity and mortality, with an estimated one third of the world's population having latent TB infections (LTBI). Reactivation of LTBI occurs in immunocompromised individuals. Diabetes mellitus (DM) is a condition that impairs host defence, leading to higher morbidity and mortality in individuals with TB. The global burden of DM is unprecedented with 425 million people living with diabetes in 2017 and 4 million diabetes-associated deaths. Type 2 diabetes (T2D) comprises more than 90% of the global diabetes incidence and is attributed to genetic, environment and life style factors. Obesity, caused mostly by a combination of sedentary life style and consumption of an energy-dense diet rich in refined carbohydrate and fat, is considered a leading cause of the global increase of T2D. The resurgent interest in TB-T2D co-morbidity is due to the slow reduction in global TB incidence and the rapid escalation of T2D particularly in TB endemic areas. Epidemiological studies have found a strong correlation of T2D with increased TB susceptibility, reactivation of LTBI and treatment failure, however the underlying mechanisms for this are unclear. Non-tuberculous mycobacterial (NTM) infections are also increasing in individuals with diabetes in comparison to the general population. Among the NTM species, Mycobacterium fortuitum is a leading cause of skin and soft tissue infections and capable of causing severe pulmonary infections. There is no data available on T2D-M. fortuitum co-morbid conditions.

A few studies have investigated the reason for increased TB susceptibility in diabetics although the findings are often contradictory. Animal model studies designed to investigate immune dysregulation in diabetes/mycobacterium co-morbidity have typically used streptozotocin induced type 1 diabetic murine models rather than models of T2D. Hence, characterisation of a T2D murine model that incorporates the Western diet and accurately models the clinical features of human T2D is highly desirable. Such a model is also required to investigate the immunological dysfunction in host-mycobacterial co-morbid infections and thus allow the development of therapeutic and preventative strategies.

The first Aim of this study was to characterise a diet-induced diabetic mouse model of T2D for subsequent mycobacterial infection studies. Male C57BL/6 mice were used for all experiments with groups receiving either an energy-dense diet (EDD) for a period of 30 weeks to induce diabetes or a standard rodent diet (SRD) for the same time period. The metabolic and biochemical parameters including body weight, fasting blood glucose (FBG) level, glucose tolerance test (GTT), glycated haemoglobin (HbA1c) level, urine albumin, creatinine and albumin creatinine ratio (ACR) were assessed after 25 and 30 weeks. Visceral adipose tissue, liver, pancreas and kidney from mice were analysed using a variety of histological stains and image analysis techniques. Body weight gain was increased in EDD fed mice compared to SRD fed control animals. After both 25 and 30 weeks, EDD fed mice had a significantly higher FBG levels, glucose intolerance and HbA1c levels compared to controls. Analysis of urine biochemical parameters demonstrated higher microalbumin, lower creatinine and higher ACR in EDD fed mice compared to controls. Histological examination of visceral adipose tissue, liver and pancreas revealed adipocyte hypertrophy, hepatic steatosis and a compensatory pancreatic islet hyperplasia, respectively in mice fed the EDD compared to controls. Mesangial matrix thickening within the glomeruli, thickening of the Bowman's capsules and glomerular size were increased in the kidneys of EDD fed mice compared to controls. These results suggested that feeding an EDD for 30 weeks can induce a T2D phenotype in mice. The metabolic, biochemical and histological findings demonstrated a chronic hyperglycaemic state due to insulin resistance. The urine biochemical parameters together with histological observations also confirmed renal damage which is one of the common complications of diabetes in humans.

The second Aim of this research was to investigate macrophage phagocytic function in vitro. In initial experiments, peritoneal exudate macrophages (PEM), resident peritoneal macrophages (RPM) and alveolar macrophages (AM) isolated from diabetic and control mice were assessed for the respective abilities to phagocytose mycolic acid coated beads and induce cytokines. Subsequently, the phagocytic uptake, bacterial killing capacity and cytokine secretion of these macrophages was assessed by co-culture with M. fortuitum, M. bovis (BCG) and M. tuberculosis (H37Rv). Results demonstrated that uptake of the mycolic acid coated beads was reduced significantly in PEM, RPM and AM from diabetic mice compared to controls. There were no significant differences in the production of cytokines from these cells following co-culture with the beads. However, there was a significant reduction in mycobacterial uptake and killing in both RPM and AM from the diabetic mice compared to controls. Furthermore, there was a reduction in TNF-α, MCP-1, IL-6 and IL-1β production by both RPM and AM from diabetic mice compared to controls during co-culture. These findings indicated that the uptake, killing and cytokine production were impaired in macrophages from diabetic mice compared to controls.

The final Aim of this study was to investigate the susceptibility of diabetic and control mice to M. fortuitum, M. bovis (BCG) and M. tuberculosis (H37Rv) infections by evaluating animal survival, organ bacterial loads, tissue pathology and organ cytokine levels during the course of high- or low-dose intravenous infections. Diabetic mice showed higher mortality compared to controls following challenge with a high-dose of all mycobacteria. Following low-dose challenge with all mycobacteria species, the bacillary burden was higher in spleen, liver and lungs of diabetic mice compared to controls at 14 and 30/35 days post infection. Estimation of the number and size of inflammatory granuloma-like lesions in the liver, revealed increased inflammation, with higher loads of acid-fast bacilli in diabetic mice compared to controls. Furthermore, the total area of inflammation observed in the lungs of diabetic mice was significantly higher than controls. Assessment of tissue cytokines demonstrated an overall lower production of TNF-α, MCP-1, IL-6 and IL-1β in the liver and lungs of diabetic mice compared to controls although an opposite trend was observed in the spleen. The overall production of IL-12, IL-2 and IFN-γ was reduced in the spleen, liver and lungs of diabetic mice compared to controls.

In summary, this thesis describes the influence of an EDD on the induction of diabetes in a mouse model and the increased susceptibility of these diabetic animals to mycobacterial infection. It was found that the EDD has the potential to induce overt clinical features of T2D in mice that closely mirror the pathology observed in human T2D patients. Investigation of antimycobacterial immune responses (in vitro and in vivo) showed that the uptake, killing and cytokine production (TNF-α, MCP-1, IL-6 and IL-1β) by macrophages was impaired in T2D. A higher bacterial load and high numbers of inflammatory lesions were observed in the organs of diabetic mice which is also indicative of impairment of macrophage phagocytic function. Decreased production of pro-inflammatory cytokines including IL-12, IL-2 and IFN-γ in diabetic mice indicated that T helper 1 cells mediated responses in mycobacterial infections maybe delayed or impaired. This immune defect in diabetics may ultimately lead to delayed or defective granuloma formation. Our results also suggest the possibility of a breakdown of the granuloma due to the persistent hyperglycaemia that occurs in diabetes. The current study has demonstrated that antimycobacterial immunity was dysregulated in T2D mice which is one of the key reasons for increased mycobacterial susceptibility. Moreover, the diet-induced murine model of T2D that we have developed can be used for future in depth studies of host-mycobacterial susceptibility. The model can also be used to investigate other infections that are exacerbated by diabetes.

Item ID: 56004
Item Type: Thesis (PhD)
Keywords: murine model, macrophage, tuberculosis, type 2 diabetes, phagocytosis
Related URLs:
Copyright Information: Copyright © 2018 Md Abdul Alim
Additional Information:

Publications arising from this thesis are available from the Related URLs field. The publications are:

Alim, Md Abdul, Sikder, Suchandan, Bridson, Tahnee L., Rush, Catherine M., Govan, Brenda L., and Ketheesan, Natkunam (2017) Anti-mycobacterial function of macrophages is impaired in a diet induced model of type 2 diabetes. Tuberculosis, 102. pp. 47-54.

Date Deposited: 01 Nov 2018 04:58
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110802 Medical Infection Agents (incl Prions) @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1117 Public Health and Health Services > 111706 Epidemiology @ 50%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920104 Diabetes @ 100%
Downloads: Total: 19
Last 12 Months: 9
More Statistics

Actions (Repository Staff Only)

Item Control Page Item Control Page