Project Title

Evaluating the Impact of Metformin on Mycobacterium tuberculosis Growth in Macrophages (EIMT-GM)

Project Objectives

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB); although a treatable disease TB remains a leading cause of morbidity and mortality globally. This in large is driven by the unparalleled increase in anti-mycobacterial drug resistance. Ten million people fell ill with TB in 2018, where 558 000 were new rifampicin resistant cases and 82% of rifampicin cases were multi-drug resistant. Treatment regimens for TB consist of combinations of anti-mycobacterial drugs aimed at eradicating infection while preventing the development of resistance and recurrent infection. Anti-TB regimen is lengthy and associated with high toxicity; driving poor treatment adherence and development of multi- and extensively drug-resistant TB. Current treatment regimens are pathogen-targeted strategies, which can be severely compromised by the development of drug resistance. Host-directed therapies provide a largely unexploited approach as adjunctive anti-TB therapy. The current search for novel therapeutics has focused on the use of repurposed drugs aimed at optimizing the host’s response against the mycobacterium. Host-directed therapy has been proposed as adjuvant therapy for TB, to improve the efficacy of current treatment outcomes. Targeting the host rather than the pathogen offers one probable solution to the challenge of antibiotic resistance and killing non-replicating bacilli, because it depends neither on bacterial division nor on the bacterial susceptibility to drugs. Metformin is an FDA approved drug, well-known for its glucose lowering effect on type 2 diabetic individuals. A group of studies have reported the potential role of metformin as adjunctive therapy for TB. However, relatively little is known on how metformin modulates the cellular interaction between Mtb and macrophages. This project seeks to assess the additive effect of metformin on the ability of macrophages to control intracellular Mtb in the presence and absence of isoniazid. Understanding t

Host Organisation

EDCTP Project Call

Career Development Fellowship (CDF)

Study Design

Approach: Follows experimental procedure in Aim 1. Multi-plex ELISA technology will be used to evaluate cytokine concentrations using commercially available ELISA kits and commercially available Luminex MAP® kits according to the manufacturers’ directions (Figure 5). Cells will undergo a 6h incubation (3h infection, one wash with PBS, 3h incubation). This will be followed by treatments as per the plate layout. Supernatant will be collected, filtered with 0.2um syringe and store at -80. The assay will be run as per instruction booklet. Assays will be run in triplicate according to the manufacturers’ protocol. Data will be collected using a dual-laser flow-based detection instrument. Statistical analysis A five-parameter regression formula will be used to calculate the sample concentrations from the standard curves. Correlations between data sets will be evaluated using Pearson’s correlation coefficient (r). The figures will be generated using Graph Pad Prism (Graph Pad Software, San Diego, CA) software programs with statistical analyses performed using SAS software (SAS Institute Inc., Cary, NC). Evaluation of potential outliers within the triplicate determinations for each sample will be performed using the Grubbs formula at the 5% critical Z-value. RNA sequence technology will be used to evaluate changes in signaling pathways under each treatment condition. A systems biology approach using bio-informatics analysis combining soluble cytokine and RNA data will be used to identify biologic mechanisms underpinning results observed in Aim 1 (Figure 6). Library preparation The steps to prepare cDNA library for sequencing are described below: RNA Isolation, RNA selection/depletion and cDNA synthesis. Single-cell RNA sequencing (scRNA-Seq): scRNA-Seq will provide the expression profiles of individual cells. Patterns of gene expression will be identified through gene clustering analyses. This can uncover the existence of rare cell types within a cell population that may n

Project Summary

Whilst the incidence of TB has shown a slight decline, the burden of multi- and extensively drug-resistant TB remains a threat to the global control of the epidemic. Anti-TB regimen is lengthy and associated with high toxicity, driving poor treatment adherence and development of multi- and extensively drug-resistant TB. Current treatment regimens are pathogen-targeted strategies, which can be severely compromised by the development of drug resistance. Drugs targeting host processes may largely avoid the development of bacterial antibiotic resistance, a major public health concern for TB. However, these drugs may also have generally increased risk for side effects on the host. Host-directed therapies provide a largely unexploited approach as adjunctive anti-TB therapy, either to directly increase the ability of the host immune system to effectively eliminate mycobacteria or to limit collateral tissue damage associated with infection that can result in morbidity and mortality. The current search for novel therapeutics has focused on the use of repurposed drugs aimed at optimizing the host’s response against the mycobacterium. New attention has focused on exploring HDTs, and there has been a spike in reviews articulating fundamental principles; each aspect of the host response can conceivably be modulated in a way that maximizes bacterial killing while minimizing inflammatory tissue damage. These approaches are still speculative, and there are only a few actively used host-directed drugs in the field. However, there is growing sentiment that alternative approaches are needed and that manipulation of host immunity coupled to a greater understanding of biological mechanisms hold new promise for anti-TB therapy. Thus far, most HDTs are still theoretical. Knowing the basic biology of mycobacterial pathogenesis, as heterogeneous as that might be, may guide the search for more effective and specific HDTs. Metformin (MET) is an FDA approved drug, well-known for its glucose lowering effect on type 2 diabetic individuals. However, relatively little is known on how MET modulates the cellular interaction between Mtb and macrophages. We propose to bridge this gap in our understanding of biologic mechanisms underpinning MET effect by adapting and applying a recently-described experimental model published by the Sigal group at the AHRI. This model focused on live cell imaging to track Mtb infection in primary human macrophages. This project seeks to apply this model among other experimental assays to assess the additive effect of MET on the ability of macrophages to control intracellular Mtb in the presence and absence of INH and the standard four drug anti-TB regimen. The overall objective of this research is to characterize whether MET modulates Mtb growth within macrophages. MET use as HDT could potentially shorten TB treatment, improve TB treatment outcomes and improve early host eradication of TB infection, and reduce TB transmission. But it is important to note that all studies and/or trials heretofore published are retrospective in nature. Hence, an established experimental role of MET HDT would initiate well-designed prospective randomized in vitro, in vivo and human studies in well-defined populations. We provide critical evidence that advances current knowledge on the biologic mechanism of action of MET in TB therapy. Our data suggests metformin exerts its host directed therapeutic effects via maintaining cell viability, allowing MET to enhance macrophage mediated mycobacterium tuberculosis killing, reduce bacilli growth and mediate host pro-inflammatory responses. Furthermore, our novel finding on the synergistic effect of MET in potentiating the pro-inflammatory response of isoniazid should be considered to re-evaluate the current standard four-drug tuberculosis treatment regimen. During this reporting period the experimental work for aims 1 (Measure intracellular Mtb growth rate and Mtb expansion within infected macrophages in the presence and absence of MET, MET and INH, INH only, and the standard four drug anti-TB therapy with and without MET) and 2 (Identify soluble proteins and signalling pathways associated with enhanced Mtb control in the presence of MET) were complete. The analysis and reporting of the data-set from aims 1 and 2 are currently underway with the intention of submitting the manuscript to PNAS. https://www.caprisa.org/Pages/EDCTP-funded%20studies

Current Organisation

CAPRISA

Current Job Title

Research Associate

Biography