Mitochondrial metabolic remodeling drives innate immune activation in Drosophila hemocytes

Reviewer #1 (Public review):

Summary:

The metabolic profiles of immune cells under steady-state or immune-activated conditions remain poorly characterized. The authors find that embryonically derived hemocytes in Drosophila larvae predominantly utilize mitochondrial respiration to generate energy and exhibit minimal glycolysis rates under unchallenged conditions. Hemocytes developmentally elevate ATP production rates. Mitochondrial respiration drives metabolic activation in larval hemocytes. More specifically, lamellocytes exhibit unique metabolic activities, including enhanced trehalose catabolism and mitochondrial remodeling, required for their encapsulation response.

Strengths:

The study shows the metabolism that is most likely to operate in different immune cells in Drosophila during development and also during infection. This is related to mitochondrial organization and proliferation and/or differentiation state.

Weaknesses:

Even though there is a rigorous analysis of mitochondrial activity using the Sea Horse analyzer, the analysis of diverse mitochondrial activities in the different immune cell types across development and in infection could be carried out using microscopy. ROS, mitochondrial membrane potential, NADH/+ and FADH/+ levels in vivo are likely to give a more specific readout of change in cellular activities. The activities of mitochondrial fusion and fission need to be collectively tested to understand their role in development and also in infection. The relevance of the change in mitochondrial activity for development or immunity remains to be tested.

Reviewer #2 (Public review):

Summary:

This study presents an analysis of the metabolism of Drosophila larval immune cells during development and activation. The authors compared the utilization of glycolysis and oxidative phosphorylation for energy metabolism. Although this topic has been widely discussed and well-studied in immune cell research, particularly in mammals, it has received little attention in insects. The authors demonstrated that quiescent and activated larval Drosophila immune cells predominantly use mitochondrial oxidative phosphorylation to produce energy. This finding is significant for the emerging field of insect immunometabolism research and is interesting in comparison to mammalian immunity, where immune cell activation is often associated with a shift toward greater reliance on glycolysis.

Strengths:

Using the Agilent Seahorse system, the authors developed and fine-tuned a method to measure the energy metabolism of Drosophila immune cells, obtaining high-quality, robust data. Through genetic manipulations targeting immune cells specifically, they analyzed metabolic changes in cells with different activations, going beyond developmental changes. They convincingly demonstrated ATP production, primarily in the mitochondria of immune cells, at various developmental stages and in various activated states. The results presented mostly support the conclusions drawn. This methodology and its results are valuable for further studies of insect immunometabolism. In a broader context, they are also valuable for comparing the metabolism of immune cells across different animal groups.

Weaknesses:

The genetic manipulations used were suitable for obtaining immune cells of various types and activation states, such as proliferation, differentiation, and immune activation. However, this method has limitations: the mixture of different cell types was always analyzed, and the specific type of interest was often a minority cell population. Had the other cells remained in their initial control state, the observed change in metabolism could have been primarily attributed to the desired cell type. However, the remaining cells that did not transform into the desired type were also usually influenced or activated in some way, making it difficult to determine to which group the observed change should be attributed. For example, consider the induction of lamellocyte differentiation using Hml>Hop[tum]. There are approximately 1,000 lamellocytes per larva, but according to Supplementary Figure 4, there are still about 5,000 Hml+ cells, and even these cells have activated Jak/Stat signaling. Therefore, it can be assumed that they are also activated. After a real infection, the proportion of lamellocytes is greater, but the remaining plasmatocytes are also activated. The authors should mention these limitations more clearly. However, as the authors correctly note, solving this problem will require single-cell approaches, which current technologies still limit. I see this as a problem when interpreting the proliferation effect. The crucial question is what percentage of the analyzed cells induced by Hml>Ras[V12] were actually in the division stage. Not all hemocytes are Hml+, so not all are induced. Of those that are induced, how many are in the division stage at the time of analysis? Meanwhile, those that were not dividing at that moment also had activated Ras, which triggers many processes besides division. Information on what percentage of the analyzed cells were dividing is missing. This information is important because the finding that dividing Drosophila immune cells primarily use mitochondria and oxidative phosphorylation to produce ATP contrasts with the debated significance of the Warburg effect in dividing mammalian cells. This finding would be significant, but unfortunately, it is not robustly supported by the presented data.

Reviewer #3 (Public review):

Summary :

This study investigates the metabolic profiles of hemocytes across multiple stage/conditions and suggests that hemocytes act as regulators of metabolism rather than merely receivers of metabolic cues. The authors show that hemocytes rely primarily on mitochondrial respiration, which is further enhanced during proliferation in development or upon genetic manipulation of plasmatocytes, but not crystal cells.

Metabolic respiration is also activated in lamellocytes, and this activation correlates with changes in mitochondrial morphology. The authors further attempt to identify mechanisms underlying this activation, proposing that mitochondrial fission may contribute to the ability of lamellocytes to encapsulate wasp eggs.

Strengths:

This work provides detailed and valuable insights into the metabolic phenotypes of hemocyte populations at different developmental stages and under both physiological and pathological conditions. The authors perform a longitudinal assessment of hemocyte metabolism and compare metabolic states across contexts.

Importantly, they provide evidence that hemocytes regulate metabolism to perform essential immunological functions, such as wasp egg encapsulation. This reinforces the view that hemocytes are key regulators and communicators that adapt their metabolic programs according to developmental and environmental demands.

Weaknesses:

The results presented are insightful, although several controls and validations could strengthen the conclusions. It would be preferable to also include responder transgenes alone as a control for leakiness, and the scRNA-seq findings would benefit from in vivo validation.

Some conclusions appear inconsistent or insufficiently supported. For instance, although mitochondrial respiration in plasmatocytes peaks at 96 h AEL, this increase is not accompanied by detectable mitochondrial rearrangement, which remains constant between 96 h AEL and 120 h AEL.

In general, the authors should temper some statements or provide further data.