SAM transmethylation pathway and adenosine recycling to ATP are essential for systemic regulation and immune response

Reviewer #1 (Public review):

Summary:

In this article, Nedbalova et al. investigate the biochemical pathway that acts in circulating immune cells to generate adenosine, a systemic signal that directs nutrients toward the immune response, and S-adenosylmethionine (SAM), a methyl donor for lipid, DNA, RNA, and protein synthetic reactions. They find that SAM is largely generated through the uptake of extracellular methionine, but that recycling of adenosine to form ATP contributes a small but important quantity of SAM in immune cells during the immune response. The authors propose that adenosine serves as a sensor of cell activity and nutrient supply, with adenosine secretion dominating in response to increased cellular activity. Their findings of impaired immune action but rescued larval developmental delay when the enzyme Ahcy is knocked down in hemocytes are interpreted as due to effects on methylation processes in hemocytes and reduced production of adenosine to regulate systemic metabolism and development, respectively. Overall this is a strong paper that uses sophisticated metabolic techniques to map the biochemical regulation of an important systemic mediator, highlighting the importance of maintaining appropriate metabolite levels in driving immune cell biology.

Strengths:

The authors deploy metabolic tracing - no easy feat in Drosophila hemocytes - to assess flux into pools of the SAM cycle. This is complemented by mass spectrometry analysis of total levels of SAM cycle metabolites to provide a clear picture of this metabolic pathway in resting and activated immune cells.

The experiments show that the recycling of adenosine to ATP, and ultimately SAM, contributes meaningfully to the ability of immune cells to control infection with wasp eggs.

This is a well-written paper, with very nice figures showing metabolic pathways under investigation. In particular, the italicized annotations, for example, "must be kept low", in Figure 1 illustrate a key point in metabolism - that cells must control levels of various intermediates to keep metabolic pathways moving in a beneficial direction.

Experiments are conducted and controlled well, reagents are tested, and findings are robust and support most of the authors' claims.

Weaknesses:

The authors posit that adenosine acts as a sensor of cellular activity, with increased release indicating active cellular metabolism and insufficient nutrient supply. It is unclear how generalizable they think this may be across different cell types or organs.

The authors extrapolate the findings in Figure 3 of decreased extracellular adenosine in ex vivo cultures of hemocytes with knockdown of Ahcy (panel B) to the in vivo findings of a rescue of larval developmental delay in wasp egg-infected larvae with hemocyte-specific Ahcy RNAi (panel C). This conclusion (discussed in lines 545-547) should be somewhat tempered, as a number of additional metabolic abnormalities characterize Ahcy-knockdown hemocytes, and the in vivo situation may not mimic the ex vivo situation. If adenosine (or inosine) measurements were possible in hemolymph, this would help bolster this idea. However, adenosine at least has a very short half-life.

Reviewer #2 (Public review):

Summary:

In this work, the authors wish to explore the metabolic support mechanisms enabling lamellocyte encapsulation, a critical antiparasitic immune response of insects. They show that S-adenosylmethionine metabolism is specifically important in this process through a combination of measurements of metabolite levels and genetic manipulations of this metabolic process.

Strengths:

The metabolite measurements and the functional analyses are generally very strong and clearly show that the metabolic process under study is important in lamellocyte immune function.

Weaknesses:

The gene expression data are a potential weakness. Not enough is explained about how the RNAseq experiments in Figures 2 and 4 were done, and the representation of the data is unclear. The paper would also be strengthened by the inclusion of some measure of encapsulation effectiveness: the authors show that manipulation of the S-adenosylmethionine pathway in lamellocytes affects the ability of the host to survive infection, but they do not show direct effects on the ability of the host to encapsulate wasp eggs.

Reviewer #3 (Public review):

Summary:

The authors of this study provide evidence that Drosophila immune cells show upregulated SAM transmethylation pathway and adenosine recycling upon wasp infection. Blocking this pathway compromises the lamellocyte formation, developmental delay, and host survival, suggesting its physiological relevance.

Strengths:

Snapshot quantification of the metabolite pool does not provide evidence that the metabolic pathway is active or not. The authors use an ex vivo isotope labelling to precisely monitor the SAM and adenosine metabolism. During infection, the methionine metabolism and adenosine recycling are upregulated, which is necessary to support the immune reaction. By combining the genetic experiment, they successfully show that the pathway is activated in immune cells.

Weaknesses:

The authors knocked down Ahcy to prove the importance of SAM methylation pathway. However, Ahcy-RNAi produces a massive accumulation of SAH, in addition to blocking adenosine production. To further validate the phenotypic causality, it is necessary to manipulate other enzymes in the pathway, such as Sam-S, Cbs, SamDC, etc. The authors do not demonstrate how infection stimulates the metabolic pathway given the gene expression of metabolic enzymes is not upregulated by infection stimulus.

Author response:

We would like to thank the editors and reviewers for reviewing our work, for finding it valuable supported by convincing data, which we greatly appreciate, but also for identifying the weaknesses of the manuscript. We plan to address these weaknesses in the revised version, briefly as follows:

(1) In the Discussion, we will elaborate more on a possible generalization of our results, while being aware of the limited space in this experimental paper and therefore intend to address this in more detail and comprehensively in a subsequent perspective article.

(2) In the Discussion, we will more clearly address the limitations of our work, in particular the difference between the measurement of extracellular adenosine production ex vivo and the actual production in vivo, where the measurement is indeed very challenging, and also the limitations of manipulating the SAM pathway only at the Ahcy level.

(3) We will describe in detail and complement the supplementary RNAseq data. The RNAseq data have already been described in detail in our previous paper (doi.org/10.1371/journal.pbio.3002299), but we agree with the reviewers that we should describe the necessary details again here.

(4) We will fill in the missing data on encapsulation efficiency; we agree that it was unfortunate to omit them.

(5) We will supplement the data with methyltransferase expressions and better describe the changes in expression of some SAM pathway genes, which, especially with methyltransferase expressions, also support stimulation of this pathway by changes in expression. Although the goal of this work was to test by 13C-labeling whether SAM pathway activity is upregulated, not to analyze how the activity is regulated, we certainly agree that an explanation of possible regulation, especially in the context of the enzyme expressions we show, should be included in our work.