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BETA-BACT Report Summary

Project ID: 708193
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - BETA-BACT (Beta-cell inflammation and dysfunction induced by bacterial translocation)

Reporting period: 2016-06-01 to 2017-05-31

Summary of the context and overall objectives of the project

An obesity-driven type 2 diabetes (T2D) epidemic is ongoing with an estimated 600 million patients affected by 2035. T2D induces significant morbidity and mortality through the development of micro- and macrovascular complications and is projected to be one of the leading causes of death in 2030, making the need for better treatment essential. Failure of pancreatic beta cells is the cause for development and progression of the disease. Thus, therapeutic aims to reverse or stabilize beta-cell function in T2D are critical to slow the course of this devastating disease.
The cause for beta-cell loss is unknown but seems related to a chronic inflammatory process driven by cytokines, macrophages and the innate immune system. The triggers involved in this process, however, are incompletely understood. However, in order to specifically target beta-cell inflammation, the processes that initiate and drive the influx of these immune cells into pancreatic beta cells need to be unraveled.
Recent research indicates that intestinal microbiota composition is associated with development of T2D in large epidemiological studies. Notably, bacterial-derived endotoxins, in particular lipopolysaccharides (LPS) derived from intestinal Gram-negative bacteria, show a transient rise following a meal that is limited in lean control subjects but pronounced in metabolically impaired individuals. The prevailing hypothesis is that high energy/fat intake leads to gut microbiota dysbiosis and alterations in circulating bacteria and endotoxins through leakage. Thus, direct exposure of beta cells to Gram-negative bacteria as well as their toxins might trigger beta-cell inflammation in T2D.
A critical receptor in this respect may be the Toll-like receptor (TLR), predominantly subtype 4 (TLR4). As such, LPS binds to TLR-4, which is expressed on many tissues including the beta cell. Many studies have linked exposure to LPS to impaired glucose and lipid metabolism through TLR4-related mechanisms. Specifically, for beta cells, infusion of LPS in mice was recently shown to impair glucose-stimulated insulin secretion (GSIS) and insulin production through TLR-4 related pathways. In addition, in TLR4-/- mice, both LPS and a high-fat diet were unable to elicit an inflammatory response in pancreatic islets. In particular, macrophage infiltration was absent as well as production of IL-1β. Moreover, the detrimental effects of inflammation on insulin production and secretion were also mitigated.
Taken together, these data suggest that altered intestinal microbiota, likely secondary to the consumption of a high-fat diet, and enhanced translocation of bacteria and their toxins could serve as an important trigger of TLR-4 mediated low-grade inflammation in the pancreas resulting in beta-cell dysfunction. Thus modulation of microbiota composition/translocation could potentially halt the decline in beta-cell function characterized of type 2 diabetes mellitus.
Overview of the action:
1. To determine whether increased translocation of (Gram-negative) bacteria and concomitant TLR4 upregulation occurs in the pancreas of humans with T2D patients, and to identify the specific microorganisms involved.
2a. To determine the effect of the identified, most abundant pathogenic bacteria (from Objective 1) on glucose metabolism, beta-cell function and inflammation, by introducing the cultured microorganisms to mice.
2b. To determine the role of TLR4 in glucose dysregulation, beta-cell dysfunction and inflammation, induced by the most abundant pathogenic bacteria (from Objective 1) in mice.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Interim results

Objective 1.
- The presence of bacterial DNA was observed in the pancreas of diabetic and non-diabetic subjects, with higher amount of bacteria in diabetic subjects.
- using metagenomics sequencing in the first 10 subjects, mainly Proteobacteria were identified (90.79% ± 6.57%). On the class level, mostly Gammaproteobacteria (60.92% ± 19.92%) were identified. In a number of T2DM patients, the most dominant species was the Enterobacter.

After literature search we decided to do further tests with Enterobacter cloacae (ATCC 13047), which is Gram-negative, LPS producing, facultative anaerobe, belonging to the taxa of gammaproteobacteria and, although a commensal bacterium, considered an opportunistic potentially pathogenic strain.

Objective 2a: In objective 2a, we aimed to assess the effect of the bacterium obtained in objective 1, on pancreatic beta-cell function.
- Heat-inactivated E. Cloacae impaired beta-cell function (glucose-stimulated insulin secretion) in freshly isolated rodent islets. Inoculation of the clonal beta-cell line INS1E did not show any effect on the insulin secretion (data not shown), suggesting that immune cells present in the freshly isolated islets are mediating the effect of the inoculated bacteria.
- In vivo, gavaging viable bacteria into C57BL/6 mice resulted in diarrhea in many of the treated mice, resulting in lower body weight as well as lower fasting glucose levels and improved glucose tolerance during the OGTT. Based on these observations, we concluded that oral gavage of E. Cloacae is not a suited model to assess the effects of this bacterium on pancreatic beta-cell function
- Given the challenges met during the gavage studies, we decided to introduce HI bacteria directly into the pancreas of C57BL/6 mice to observe their pancreatic effects. To do so, in a recently conducted pilot study with 4 mice per group, 6-week old mice were put on a high-fat diet or low-fat diet for 4 weeks and E. Cloacae was injected in the pancreas via the common bile duct. We observed that HI-bacteria (compared to sham surgery) had higher fasting glucose levels and impaired insulin response.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

These preliminary data indicate that DNA of G-bacteria is present in the pancreas of patients with T2DM and that one of the observed bacteria, E. Cloacae, is able to modulate beta-cell function both in vitro and in vivo.

Further work is planned during the second year of the fellow hip:
- further detailing of microbiota in pancreatic biopsies from T2D patients and controls (sequencing)
- investigating the mechanisms of E. Cloacae induced beta-cell dysfunction with appropriate controls
- Assessment of the effects of different metabolites produced by proteobacteria on beta-cell function
- Sequencing of tissue from performed gavage studies to monitor bacterial translocation
- Extend intraductal injection studies to assess direct in vivo effects of E. Cloacae at the level of the pancreatic beta-cell
- Further investigate the role TLR4 by studying effects of E.Cloacae in TLR4 pancreas specific k.o. mice

With these results I expect to be able to detail the role of the G-negative bacterium E. Cloacae on beta-cell inflammation and whether modulation of this bacterium-interaction with the immune system is a viable way of protecting pancreatic islets.
The socio-economic and societal implications will be addressed at the final report and are dependent on the results.
Clearly, if the G-negative bacteria from the gut or the products they secrete are a driver in beta-cell failure, they form a clear target for therapy, as beta-cell failure is driving force in the development and progression of diabetes.

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