human monocyte derived macrophages SUPPORTING...

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Bridgette M. Cumming1, Kelvin Addicott1, John H. Adamson1, Adrie J.C. Steyn1,21Africa Health Research Institute, Durban, South Africa;

2Department of Microbiology, UAB Center for AIDS Research, University of Alabama at Birmingham, USA

Mycobacterium tuberculosis infection decelerates bioenergetic metabolism and alters mitochondrial substrate preferences in the macrophage host

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• About 1/3 of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb)1.

• Infection with Mtb does not confer immunity against future infections demonstrating Mtb hasdefinitive strategies to manipulate the immune response and prevent complete eradication.

• At the onset of tuberculosis infection, macrophages phagocytose Mtb as the first line of defence.Several studies have indicated that Mtb infection of murine bone marrow derived macrophagesresults in a shift to aerobic glycolysis that induces the production of pro-inflammatory cytokines2,3.

• To further our understanding of Mtb modulation of the macrophage to consider host-directedadjunctive therapy as a novel intervention against TB, we investigated how Mtb tempers thebioenergetic metabolism of human monocyte derived macrophages using extracellular flux andmetabolite analyses.

Introduction

• Human monocyte derived macrophages (hMDMs) were infected with Mtb H37Rv, M. bovis BCG(BCG) or heat killed Mtb (Dead Mtb) at multiplicities of infection (MOI) of 1, 2.5 or 5 (as indicated)for 24 hrs.

• The infected macrophages were analysed for their:• Overall bioenergetic status using extracellular flux technology (Agilent Seahorse XF96).• Metabolite levels and isotopomers after incubation with [U-13C]glucose using LC-MS/MS and

13C-tracing.

Methods

Mtb infection modulates the bioenergetic phenotype of

macrophages distinct to that of BCG and heat killed Mtb

infections

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Figure 1. Increasing MOI of Mtb depresses mitochondrial respiration in hMDM in contrast to BCG infection,

which increases the spare respiratory capacity of the hMDMs. (A-C) Cell Mito Stress Test (CMST) of hMDM

infected with increasing MOI of Mtb, BCG and heat-killed Mtb for 24 hrs. (D-F) Respiratory parameters of the

macrophages calculated from the CMST. (G-I) Phenograms demonstrating a shift in Mtb infected hMDMs towards an

energy phenotype reminiscent of quiescence at an MOI of 5.

Figure 2. Unlike BCG infection, Mtb does not increase the rate of glycolysis in hMDMs. (A-C) Glycolytic Rate

Assay of hMDM infected with increasing MOI of Mtb, BCG and heat-killed Mtb for 24 hrs. (D-F) Basal Glycolysis and

compensatory glycolysis of the macrophages expressed as proton efflux rate (PER, pmol/min) as determined from the

Glycolytic Rate Assay on the XF96. The glycolytic proton efflux rate gives a direct measure of the acidification rate due

to glycolysis without the acidification contribution from mitochondrial respiration.

Figure 3. Mtb infection decelerates bioenergetic metabolism in hMDMs.13C-tracing of metabolites

extracted from hMDM infected with Mtb, BCG or heat-killed Mtb at an MOI of 4 for 24 hours. Less 13C

incorporation is observed in the metabolites of glycolysis, TCA cycle and the pentose phosphate pathway

of the Mtb infected hMDMs in comparison to the uninfected hMDMs and those that are infected with BCG

and heat-killed Mtb. Although the levels of the glycolytic intermediates are decreased in all of the infected

hMDMs, the levels of the TCA cycle intermediates from Mtb infected hMDMs are higher possibly due to

lack of cycling through the TCA cycle.

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Figure. 4. Inhibition of glycolysis (2-deoxyglucose) or lactate production (oxamate and

dichloroacetic acid) do not redirect the bioenergetic metabolism of Mtb infected macrophages

towards OXPHOS. Phenograms of uninfected and Mtb (1 or 5: MOI) hMDM cells treated with (A)

oxamate (B) 2-deoxyglucose (C) and RAW264.7 cells (Mtb, MOI 5) treated with dichloroacetic acid.

Circles: uninfected macrophages, Squares: Mtb infected macrophages.

Inhibition of glycolysis in Mtb infected macrophages does not redirect energy metabolism to OXPHOS

Mtb infection alters mitochondrial substrate preference of the macrophage

Mtb infection decelerates bioenergetic metabolism of the macrophage

Figure 5. Mtb infection decreases the mitochondrial

dependency on glucose and increases the dependency on

glutamine and fatty acid oxidation. hMDM were infected with

Mtb, BCG and heat-killed Mtb (MOI 4) for 24 hrs prior to

conducting the Mitochondrial Fuel Flex Test on the XF96. Glc,

glucose; Gln, glutamine; FA, fatty acids

• Basal respiration, ATP-linked OCR and maximalrespiration are reduced in Mtb infected hMDMs withincreasing MOI in contrast to infection with BCG andheat-killed Mtb that do not affect respiration at lowMOIs.

• Furthermore, BCG infection increases the maximalrespiration and the spare respiratory capacity ofhMDMs above that of uninfected hMDMs.

• Unlike BCG or heat killed Mtb infection, Mtb infectiondoes not increase the rate of basal glycolysis orcompensatory glycolysis in hMDMs.

• Mtb infection decelerates flux through glycolysis andthe TCA cycle of the hMDMs, confirming the observedshift to a quiescent energy phenotype.

• ADP/ATP and AMP/ATP ratios increase in Mtb infectedhMDMs supporting the overserved reduced rates ofOXPHOS and glycolysis.

• Inhibition of glycolysis or lactate production did notredirect the bioenergetic metabolism of the macrophageto OXPHOS suggesting that Mtb has an irreversibledepressive effect on mitochondrial respiration.

• Mtb infection of hMDMs changes the substratepreference of mitochondria from glucose to glutamineand fatty acids, whereas heat-killed Mtb increases thedependency of the macrophages on glucose with noflexibility.

• Mtb infection of hMDMs slows down energy metabolismof the host cell, including both OXPHOS and glycolysis.This is probably part of Mtb’s strategy to “diminish” theinnate immune functions of the macrophage and delayapoptosis of the macrophage to sustain bacilli growth.

• Future work includes investigating the “apoptotic status”of Mtb infected hMDMs displaying the quiescent energyphenotype.

Conclusions

This work was supported by the National Institutes ofHealth grants R01 AI 111 940 and R21 127182 and theUAB Center for AIDS Research.

Acknowledgements

References1. Global TB Report, 2017, WHO.

2. Gleeson et al. (2016) J. Immun. Vol. 196, pp. 2444;

3. Stanley et al. (2016) Vol. 197, pp. 1287

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human monocyte derived macrophages SUPPORTING...

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