Species-specific metabolic reprogramming in human and mouse microglia during inflammatory pathway induction

Metabolic reprogramming is a hallmark of the immune cells in response to inflammatory stimuli. This metabolic process involves a switch from oxidative phosphorylation (OXPHOS) to glycolysis or alterations in other metabolic pathways. However, most of the experimental findings have been acquired in murine immune cells, and little is known about the metabolic reprogramming of human microglia. In this study, we investigate the transcriptomic, proteomic, and metabolic profiles of mouse and iPSC-derived human microglia challenged with the TLR4 agonist LPS. We demonstrate that both species display a metabolic shift and an overall increased glycolytic gene signature in response to LPS treatment. The metabolic reprogramming is characterized by the upregulation of hexokinases in mouse microglia and phosphofructokinases in human microglia. This study provides a direct comparison of metabolism between mouse and human microglia, highlighting the species-specific pathways involved in immunometabolism and the importance of considering these differences in translational research.

On the outside, each arc represents the identity of each gene list, using the following color code: Blue, Hasselman; red, Alasso; green, this study.On the inside, each arc represents a gene list, where each gene member of that list is assigned a spot on the arc.Dark orange color represents the genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list.Purple lines link the same gene that are shared by multiple gene lists.C. Circos plot built in the same way as (B).Blue lines link the genes that, although different, fall under the same ontology term (the term has to be statistically significantly enriched and with size no larger than 100).Blue lines indicate the amount of functional overlap among the input gene lists.D. Heatmap depicting the top 20 statistically enriched terms (GO/KEGG, canonical pathways, etc..) hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships.The term with the best p-value within each cluster is shown as its representative tem in the dendrogram.Heatmap cells are colored by their p-values, while cells indicate the lack of enrichment for that term in the corresponding gene list.E. Heatmap depicting the top100 statistically enriched terms in a similar fashion to (D).

Supplementary Figure 4. Cross-species comparison of transcriptomic responses against LPS over time.
A, E. Overlap of DEGs in mouse microglia (A) and human iMGLs (E) at 4 and 24 hours after LPS stimulation.B, F. Top enriched biological processes after performing GSEA with the metascape tool with the overlapped gene lists for mouse microglia (B) and human iMGLs (F).C, G. Circos plot depicting how genes from the lists of DEGs overlap for mouse microglia (C) and human iMGLs (G) for the two timepoints.On the outside, each arc represents the identity of each gene list.On the inside, each arch represents a gene list, where each gene member of that list is assigned a spot on the arc.Dark orange color represents the genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list.Purple lines link the same gene that are shared by multiple gene lists (notice a gene that appears in two gene lists will be mapped once onto each gene list, therefore, the two positions are purple linked).Blue lines link the genes, although different, fall under the same ontology term (the term has to be statistically significantly enriched and with size no larger than 100).The greater the number of purple links and the longer the dark orange arcs imply greater overlap among the input gene lists.Blue links indicate the amount of functional overlap among the input gene lists.D, H. Scatterplots of the genes that were differentially regulated by LPS 4h and 24h treatment (red dots) with an adjusted p<0.05 in mouse microglia (D) and human iMGLs (H).A linear model was fitted among the genes that were differentially regulated and the R squared, and the equation of the line are depicted within the scatterplot.I. Gene expression of the Tlr4 and TLR4 genes depicted by the normalized measure of TPMs per species.N= 4 human independent experiments and N=3 different animals.p=0.014 by unpaired two-tailed t-test.

A B
Glycolysis -hiMGLs

Supplementary Figure 2 .
Human iMGLs response to LPS A, B. Principal Component Analysis (PCA) of the transcriptomes of untreated, and LPS-treated iMGLs for 4h and 24h.In (A), samples are colored by condition.In (B), samples are colored by collection/biological replicate.C. Heatmap depicting the top 100 most significant differentially expressed genes of 4h LPS treatment compared with the control.Color key corresponds to row Z-score.D. Volcano plot depicting fold changes and -Log2 of the adjusted p value per gene comparing responses against LPS 24h and 4h.N=4 independent experiments per condition.Supplementary Figure 3.Comparison of the transcriptomes of LPS-treated iPSC-derived human microglialike cells from this study with other iPSC-derived microglia models.A. Venn diagram with the overlap of DEGs of short-term LPS-treated iPSC-derived microglia-like cells from the study of Alasoo and colleagues, Hasselman and this study.B. Circos plot depicting the overlap in the lists of DEGs.

Figure 5 .
Transcriptomic changes in the glycolytic and TCA pathways in LPS-treated human iMGLs.A, B. Hierarchically clustered heatmaps depicting gene expression changes in the glycolytic pathway (A) and in the TCA pathway (B).Color key corresponds to row Z-score.* denotes padj <0.05 LPS 4h versus Control, # denotes padj <0.05 LPS 24h versus Control and these values can be found in Supplementary Datasets.N=4 independent experiment per condition.
and proteomic characterization of TCA cycle enzymes in mouse microglia and human iMGLs.Mouse and human microglial cells were in vitro stimulated with LPS 250ng/mL and collected 4 hours after stimulation for proteomic analysis by LC-MS/MS.A,B.Normalized counts depicting transcript abundance of genes that code for Isocitrate dehydrogenase enzymes in mouse (A) (Idh1, p=1.39E^7) and human (B) cells.C,D.Label-free quantitation values from proteomic analysis depicting relative abundance of IDH1 (p=0.00039),IDH2 (p=0.025), and IDH3A and IDH3B in mouse (C) and human (D) cells.E,F.Normalized counts depicting transcript abundance of genes that code for TCA cycle enzymes in mouse (E) and human (F) cells (ACO1, p=0.00065;FH, p=0.00038,CS, p=0.047).G,H.Label-free quantitation values depicting relative abundance of TCA cycle enzymes in mouse (I) and human (J) cells.I,J.Proteomic abundances of TCA cycle enzymes measured by LC-MS/MS with internal standards.(I) Depicts plots of relative abundances on mouse microglia and (J) depicts human cells (ACO2, for first peptide p=0.03, second peptide p=0.014;CS, p=0.03;DLST, p=0.018;FH, p=0.04;IDH2, p=0.014;IDH3A, p=0.018;MDH2, first peptide p=0.022, second peptide p=0.03;SUCLA2, p=0.045;SUCLG2, p=0.031).For all panels, data are presented as mean ± S.E.M.. Every biological replicate is depicted as a dot.For mouse RNAseq N=3 different animals, for human RNAseq N=4 independent experiments, for mouse proteomics N=4 different animals and for human label free proteomics N=5 independent experiments, for human targeted proteomics N=4 independent experiments.For RNAseq data p values were determined by the Wald test and multi testing corrected in DESEQ2.For LFQ proteomic data, p values were determined by unpaired two-tailed t-test and for targeted proteomic data, p values were determine by paired one-tailed t-test.*p<0.05,**p<0.01,and ***p<0.001

Table 2 :
Key TCA cycle enzymes changes upon LPS stimulation in human microglia.Comparison between key glycolytic genes by taking Log2Fold Changes and padjusted values upon LPS stimulation in human microglial models byAlasoo et al., 2015, Hasselman et al.,  2019and the present study.

Table 3 :
Primary microglia studies and technical aspects for microglia culture.Columns specify the study, type of culture, if pups or adult mice were used, and whether FBS was used or not.