Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures

Sousa, Carole; Golebiewska, Anna; Poovathingal, Suresh K; Kaoma, Tony; Pires-Afonso, Yolanda; Martina, Silvia; Coowar, Djalil; Azuaje, Francisco; Skupin, Alexander; Balling, Rudi; Biber, Knut; Niclou, Simone P; Michelucci, Alessandro

We thank the reviewer for this comment and we definitely agree that the analysis of the homeostatic microglia surface markers at the protein level would strengthen our results. Thus, as suggested, we performed flow cytometry analysis for TMEM119 and P2RY12 and compared their expression levels between steady state and LPS-induced microglia. In agreement with the transcriptional results, the protein levels of TMEM119 and P2RY12 were decreased under inflammatory conditions compared to steady state. We included these results in Fig. 3D . We amended the text and the figure legend accordingly. We agree with the reviewer, therefore we carefully addressed these comments point by point. (a) As suggested, we identified and highlighted the top genes that distinguish the two populations. To do this, we listed top differentially expressed genes unique to "Main LPS" or "Subset LPS" versus steady state (FDR<0.05; upregulated genes, Log2FC≥3; downregulated genes, Log2FC≤-3). We included these results in Table 1 and discussed the potential interesting genes in the text referring to the existent literature. (b) Please see our answer given to Referee #1 comment (please refer to major point 3d). (c) As advised, to address the differences that discriminate the "subset" versus the "main" LPS subpopulations from the naïve microglia in terms of function, proliferation and cytokine expression, we extracted these information from our scRNA-seq data and built a new table (Table EV5) . We added the corresponding information in the text. (d) Although we agree with the reviewer that it would be very interesting to study the dynamics of these subpopulations along the inflammatory and resolution phases, in the present study we focused on a specific time point in order to provide a first screening of microglia heterogeneity under acute inflammatory conditions. Follow-up studies, which would analyse these subpopulations at different time points, would certainly provide highly relevant information regarding further characterization of microglia heterogeneity along the acute inflammatory process. In order to respond to the reviewer's questions, in this study we conducted a pseudotime analysis. From this analysis, we found that, along the activation process, the inflammatory mediators are upregulated first and the homeostatic gene markers are downregulated subsequently. Thus, the identified subset may correspond to an intermediate state of activated microglia having their homeostatic signature less affected than the main LPS group, thus being temporally at an earlier stage of activation. We describe these results in a new results section entitled "Pseudotime analysis of LPS-activated microglia uncovers "subset LPS" as an intermediate activated state" and included the related figures in Fig. 5 .