Author Response
On behalf of the authors of the article "Elevated glycolytic metabolism of monocytes limits the generation of HIF-1α-driven migratory dendritic cells in tuberculosis", I would like to provide interim responses noting some relevant points about eLife assessment and public reviews,
eLife assessment
This useful study tests the hypothesis that Mycobacterium tuberculosis infection increases glycolysis in monocytes, which alters their capacity to migrate to lymph nodes as monocyte-derived dendritic cells. The authors conclude that infected monocytes are metabolically pre-conditioned to differentiate, with reduced expression of Hif1a and a glycolytically exhaustive phenotype, resulting in low migratory and immunologic potential. Unfortunately, the evidence for the conclusions is currently incomplete, as the use of dead mycobacteria will affect bioenergetic readouts. The study will be of interest to microbiologists and infectious disease scientists.
We would like to clarify what may be a misunderstanding. Indeed, the study did not deal with “infected monocytes” per se, but rather with the ability of monocytes purified from TB patients vs. healthy control to differentiate into DCs with different migratory capacities upon Mtb infection or stimulation. Since there is no evidence for the presence of Mtb in the patient’s blood, the metabolic effects we observed are likely a consequence of systemic pulmonary disease rather than of direct interaction of monocytes with Mtb. Although irradiated Mtb was used in most experiments, in particular because Seahorse and other technologies cannot be used in our BSL3 laboratory, we provide evidence (Figure 1) that infecting DCs with live Mtb or stimulating DCs with irradiated Mtb generates comparable glycolytic profiles (release of lactate, glucose consumption, HIF1a expression and LDHA expression). To strengthen the relevance of our data, we will characterize the metabolism of DCs infected with live Mtb using SCENITH.
Reviewer #1 (Public Review):
The manuscript by Maio and colleagues looks at the impact of the heightened glycolytic activity induced by Mtb in monocytes, and its impact on Hif1-a dependent migration of DCs.
Data concerning the biological significance of the impact of enhanced glycolysis on DC migration is strong and convincing. While Hif1-a is obviously a key factor, the evidence that it is a linear component in the cascade falls a little short as the main inhibitor used PX-478 does not have a clear, single mode of action. Additional characterization with the alternative inhibitor (Echinomycin) would make the argument more convincing.
We would like to thank the reviewer for their positive assessment of our manuscript. Although Echinomycin has been used for validating some of the representative experiments performed in our study (see supplementary figure 2E-F), we agree with the reviewer’s suggestion. Therefore, additional experiments using echinomycin will be carried out to confirm our results.
Reviewer #2 (Public Review):
The manuscript by Maio et al attempts to examine the bioenergetic mechanisms involved in the delayed migration of DC's during Mtb infection. The authors performed a series of in vitro infection experiments including bioenergetic experiments using the Agilent Seahorse XF, and glucose uptake and lactate production experiments. This is a well-written manuscript and addresses an important question in the TB field. A major weakness is the use of dead Mtb in virtually all the experiments. Unfortunately, the authors did not attempt to address this critical confounding factor. As a result, data was interpreted, and conclusions were made as if live Mtb was used. Also, previous studies (PMID: 30444490 and PMID: 31914380) have shown that live Mtb suppresses glycolysis, which contradicts findings in this study, perhaps because dead Mtb was used here. For these reasons, obtaining any pertinent conclusions from the study is not possible, which diminishes the significance of the work.
We thank the reviewer for their evaluation of our study. We agree that using live Mtb in all experiments would have been ideal. However, we do not have a Seahorse Analyzer in our BSL3 facility. Thus, we will characterize the metabolism of DCs infected with live Mtb using SCENITH during revision of our manuscript.
With regard to the differences between our results and those of previous studies showing Mtb-induced suppression of glycolysis, they could be explained by the use of different Mtb strains, different multiplicity of infection (MOI), macrophages of different origins, and different measurement timepoints, as discussed in one of these publications (PMID 30444490). For instance, in PMID 30444490, hMDMs infected at an MOI of 1 showed increased extracellular acidification and glycolytic parameters, as opposed to higher MOI or the same MOI but measured in THP1 cells. Importantly, the aforementioned articles studied macrophage and not DC metabolism. These aspects will be discussed in a revised manuscript.
