De novo synthesized polyunsaturated fatty acids operate as both host immunomodulators and nutrients for Mycobacterium tuberculosis
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Evaluation Summary:
In this study, the authors highlight a role for de novo biosynthesis of Poly-unsaturated Fatty Acids and the consequence effect of these metabolites on the production of arachidonic acid. The increased bio-availability of arachidonic acid seemingly promotes mycobacterial growth whilst inhibition of arachidonic acid formation, and its resultant downstream eicosanoid products, affect macrophage function but somewhat surprisingly do not affect growth of M. tuberculosis in macrophages or in mice. The uptake of the different classes of fatty acids in axenic culture as well as in macrophages is explored and the authors demonstrate that the Mce1 transporter is largely responsible for their uptake during in vitro growth but only plays a partial role in their uptake during growth of the pathogen in host cells. This work will be of interest to bacteriologists and those studying infectious diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb’s capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However, upon activation, macrophages produce polyunsaturated fatty acids (PUFAs), mammal-specific FAs mediating the generation of immunomodulatory eicosanoids. Here, we asked how Mtb modulates de novo synthesis of PUFAs in primary mouse macrophages and whether this benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection selectively activates the biosynthesis of ω6 PUFAs upstream of the eicosanoid precursor arachidonic acid (AA) via transcriptional activation of Fads2 . Inhibiting FADS2 in infected macrophages impaired their inflammatory and antimicrobial responses but had no effect on Mtb growth in host cells nor mice. Using a click-chemistry approach, we found that Mtb efficiently imports ω6 PUFAs via Mce1 in axenic culture, including AA. Further, Mtb preferentially internalized AA over all other FAs within infected macrophages by mechanisms partially depending on Mce1 and supporting intracellular persistence. Notably, IFNγ repressed de novo synthesis of AA by infected mouse macrophages and restricted AA import by intracellular Mtb. Together, these findings identify AA as a major FA substrate for intracellular Mtb, whose mobilization by innate immune responses is opportunistically hijacked by the pathogen and downregulated by IFNγ.
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Evaluation Summary:
In this study, the authors highlight a role for de novo biosynthesis of Poly-unsaturated Fatty Acids and the consequence effect of these metabolites on the production of arachidonic acid. The increased bio-availability of arachidonic acid seemingly promotes mycobacterial growth whilst inhibition of arachidonic acid formation, and its resultant downstream eicosanoid products, affect macrophage function but somewhat surprisingly do not affect growth of M. tuberculosis in macrophages or in mice. The uptake of the different classes of fatty acids in axenic culture as well as in macrophages is explored and the authors demonstrate that the Mce1 transporter is largely responsible for their uptake during in vitro growth but only plays a partial role in their uptake during growth of the pathogen in host cells. This work will …
Evaluation Summary:
In this study, the authors highlight a role for de novo biosynthesis of Poly-unsaturated Fatty Acids and the consequence effect of these metabolites on the production of arachidonic acid. The increased bio-availability of arachidonic acid seemingly promotes mycobacterial growth whilst inhibition of arachidonic acid formation, and its resultant downstream eicosanoid products, affect macrophage function but somewhat surprisingly do not affect growth of M. tuberculosis in macrophages or in mice. The uptake of the different classes of fatty acids in axenic culture as well as in macrophages is explored and the authors demonstrate that the Mce1 transporter is largely responsible for their uptake during in vitro growth but only plays a partial role in their uptake during growth of the pathogen in host cells. This work will be of interest to bacteriologists and those studying infectious diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors show that Mtb infection of resting bone marrow derived macrophages stimulates host fatty acid synthesis including that of certain polyunsaturated fatty acids (PUFAS). The work additionally demonstrates that this has wider relevance in that other bacterial danger signals similarly induce these changes. The effect of interferon-gamma which is known to activate macrophages and effect lipid droplet formation and fatty acid homeostasis in infected macrophages is not investigated. The authors focus on the upstream increase in PUFAS and show that inhibition of their downstream conversion to arachidonic acid and eicosanoids does not affect Mtb growth in macrophages or in mice which is perhaps a bit surprising but expected effects on eicosanoid on macrophage necrosis/ autophagy/ apoptosis are not …
Reviewer #1 (Public Review):
The authors show that Mtb infection of resting bone marrow derived macrophages stimulates host fatty acid synthesis including that of certain polyunsaturated fatty acids (PUFAS). The work additionally demonstrates that this has wider relevance in that other bacterial danger signals similarly induce these changes. The effect of interferon-gamma which is known to activate macrophages and effect lipid droplet formation and fatty acid homeostasis in infected macrophages is not investigated. The authors focus on the upstream increase in PUFAS and show that inhibition of their downstream conversion to arachidonic acid and eicosanoids does not affect Mtb growth in macrophages or in mice which is perhaps a bit surprising but expected effects on eicosanoid on macrophage necrosis/ autophagy/ apoptosis are not investigated. Moreover, whether interferon-gamma stimulation which is known to affect eicosanoid production in macrophages would affect this outcome is not explored although the mouse data may suggest that Fads2 inhibition in the context of immune activation will not affect this outcome. In vitro cultured Mtb acquires the saturated, mono-unsaturated as well as PUFAS by the Mce1 transporter with competition assays with various fatty acids suggesting different binding sites depending on fatty acid conformation but acquire these fatty acids by additional mce1-independent transporters in the host macrophage. Interestingly, it is found that during host cell infection, Mtb preferentially acquires host arachidonic acid although the effect of interferon-gamma stimulation on host fatty acid acquisition are not explored.
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Reviewer #2 (Public Review):
In the manuscript by Laval et al, the authors profile fatty acids after Mtb infection in a BMDM system. This is a timely and important piece of work, since metabolic reprogramming of macrophages has emerged as directive in responses to infection and activation but can also be modulated by Mtb to evade host defence. Much of the published work thus far has focused on glucose metabolism and although it is long-known that Mtb alters lipid levels in cells (causing a foamy macrophage-like phenotype), little work has profiled the various lipid species and the mechanisms regulating their production and fate. The authors find, consistent with other work in activated macrophages, that Saturated Fatty Acid (SFAs) and Mono-unsaturated Fatty Acids (MUFAs) are increased after infection by mycobacterial species, but …
Reviewer #2 (Public Review):
In the manuscript by Laval et al, the authors profile fatty acids after Mtb infection in a BMDM system. This is a timely and important piece of work, since metabolic reprogramming of macrophages has emerged as directive in responses to infection and activation but can also be modulated by Mtb to evade host defence. Much of the published work thus far has focused on glucose metabolism and although it is long-known that Mtb alters lipid levels in cells (causing a foamy macrophage-like phenotype), little work has profiled the various lipid species and the mechanisms regulating their production and fate. The authors find, consistent with other work in activated macrophages, that Saturated Fatty Acid (SFAs) and Mono-unsaturated Fatty Acids (MUFAs) are increased after infection by mycobacterial species, but interestingly here, also characterize an increase in certain Polyunsaturated Fatty Acids, but not one of the products of PUFA metabolism, Arachidonic Acid (AA). This is surprising since AA has a major role in host/Mtb macrophage responses particularly the production of various eicosanoids and when an earlier enzyme in the PUFA metabolism pathway is targeted, FADS2, this has a major effect on the host macrophage response, down-regulating inflammatory gene expression and eicosanoid production. Using elegant technologies, the authors go on to reveal an usual dichotomy whereby Mtb itself imports AA alongside some other PUFA & FA species. This preference for AA explains why AA itself is not up-regulated after Mtb infection, but also why targeting the FADS2 enzyme which blunts AA levels, does not affect mycobacterial survival in in-vitro or in-vivo assays. This is particularly interesting since the interaction of different host signals (TLR stimulation, IFN production) in the modulation of specific lipid species in macrophages is receiving a lot of attention lately (Knight et al, 2018, Hsieh et al 2020), yet the importance of these for host immunity versus metabolism of infecting pathogens can be difficult to discern. The authors develop a strain of Mtb in which the import of AA & other FAs is decreased. However, it is still unclear if the bioavailability of AA itself is controlling this phenotype and what effect blocking its import by Mtb has on the host macrophage response. This should be examined further alongside a more complete characterization of the role of FADS2 in both bacterial survival, macrophage metabolism and inflammatory activation.
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Reviewer #3 (Public Review):
Laval et al examine the metabolism of polyunsaturated fatty acids, in particular arachidonic acid (AA), during macrophage infection with M. tuberculosis. First they demonstrate that infection results in increased levels of linoleic acid and other fatty acid precursors of AA in macrophages. Using an inhibitor of FADS2, an enzyme that mediates one step in the pathway by which linoleic acid is converted to arachidonic acid, they demonstrate that synthesis of AA is required for production of immunomodulatory eicosanoids. In addition, treatment of macrophages with the FADS2 inhibitor results in decreased expression of numerous cytokines and effectors of innate immunity. However, treatment of infected macrophages or mice with the inhibitor has no impact on bacterial growth. In parallel experiments, the authors use …
Reviewer #3 (Public Review):
Laval et al examine the metabolism of polyunsaturated fatty acids, in particular arachidonic acid (AA), during macrophage infection with M. tuberculosis. First they demonstrate that infection results in increased levels of linoleic acid and other fatty acid precursors of AA in macrophages. Using an inhibitor of FADS2, an enzyme that mediates one step in the pathway by which linoleic acid is converted to arachidonic acid, they demonstrate that synthesis of AA is required for production of immunomodulatory eicosanoids. In addition, treatment of macrophages with the FADS2 inhibitor results in decreased expression of numerous cytokines and effectors of innate immunity. However, treatment of infected macrophages or mice with the inhibitor has no impact on bacterial growth. In parallel experiments, the authors use a click chemistry approach to label specific fatty acids and demonstrate that M. tuberculosis takes up AA and other PUFAs using the known FA transporter Mce1. Finally, they demonstrate that M. tuberculosis takes up these PUFAs during macrophage infection from the host, a phenomenon that is also partially Mce1 dependent. The experiments are for the most part technically sound and well controlled, and their specific findings are plausible. However, the findings represent only an incremental advance over what is already known about lipid metabolism during M. tuberculosis infection.
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