Engskog-Vlachos et al. explored the role of the immunometabolite itaconate in microglial activation. For this purpose, the authors used various methods like metabolomics, Seahorse, pPCR and multiplex cytokine measurements in the BV-2 cell line and in dopaminergic MN9D neuronal cells, stimulated with bacterial lipopolysaccharide (LPS) or interleukin-4. Overall, the authors describe increased microglial aerobic glycolytic activity in LPS, accompanied by increased levels of endogenous itaconate and stabilization of the NF2L2/NRF2 transcription factor. They conclude an IRG1/itaconate/NF2L2 axis that may function as a feedback mechanism, reducing oxidative stress and limiting neurotoxicity while still promoting pro-inflammatory cytokine production.

General points:
1. The novelty of this manuscript is less clear. The role of itaconate in regulating inflammatory responses in microglia (and other macrophages), including neuroprotective effects has been studied in cell culture, cortical slices and animal models (for example, PMID: 23395614, PMID: 32446944, PMID: 38649932, PMID: 40073156).
2. The use of the microglia-like BV-2 cell line and dopaminergic MN9D neuronal cells - instead of primary cells and/or more complex models - is critical.
3. The use of highly artificial culture conditions that include excessive glucose concentrations, hyperoxia (room air) and antibiotics is also critical.
4. The description of Results and M+M is partially insufficient and hard to follow.

Specific points:
5. Figure 1A. Add the unit 'hour'.
6. Figure 1 (D, E) and Figure 2. Without further labeling (categorization), these figures are unclear.
7. Figure 1 and others. The font sizes and data plots are partially much too small. Revise according to guidelines.
8. Figures 1 and 2, and Line 139. 'A plentitude of features was detected based on data acquired in positive (4614 features) and negative (4489 features) ionization mode (Figure 1C).' It is very unclear what these 'features' are. 'Features' ('metabolome profiles') need to be much better explained, categorized and exemplified.
9. Line 708. Give the glucose concentration(s) in mM.
10. The culture and recording conditions (Seahorse) are highly artificial. Provide control experiments with glucose and oxygen concentrations closer to the physiological range.
11. Line 715. What are 'silenced cells'? Explain.
12. The BV2-cell line is questionable. Provide control experiments with primary microglia and critically discuss the limitations of this cell line.
13. Results and Legends. The sample sizes are less clear. Specify the sample size for each experimental group (n/N; n for cells or coverslips/dishes, N for preparations/passages) in each legend.

Overall, I find this revised manuscript to be improved (JNC-2025-0136.R1). Some of the original points have not been sufficiently addressed, however.
./1 Point 1 (response letter), novelty of the manuscript. This issue is now adequately addressed in the response letter but should be also added to the manuscript somehow (Introduction and/or Discussion), including the references.
./2 Point 10 (response letter), glucose concentrations (25 mM vs 5 mM). This is an important control experiment that should be added to the manuscript (e.g., Supplements).
./3 Point 13 (response letter), sample sizes and sampling. 'Independent biological (technical) replicate' is still unclear and needs to be clarified in M+M and individually specified in each legend (e.g., n/N; n for cells or coverslips/dishes, N for preparations/passages/animals).
./4 Some Figures seem to be incomplete in the PDF. Revise and clarify with Editorial Office for R2.