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. 2023 Jun 26:17:1178504.
doi: 10.3389/fncel.2023.1178504. eCollection 2023.

In silico-in vitro modeling to uncover cues involved in establishing microglia identity: TGF-β3 and laminin can drive microglia signature gene expression

Raissa Timmerman  1 , Ella Alwine Zuiderwijk-Sick  1 , Wia Baron  2 , Jeffrey John Bajramovic  1   3
Affiliations

In silico-in vitro modeling to uncover cues involved in establishing microglia identity: TGF-β3 and laminin can drive microglia signature gene expression

Raissa Timmerman et al. Front Cell Neurosci. .

Abstract

Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in CNS development, homeostasis, and disease. Good in vitro models are indispensable to study their cellular biology, and although much progress has been made, in vitro cultures of primary microglia still only partially recapitulate the transcriptome of in vivo microglia. In this study, we explored a combination of in silico and in vitro methodologies to gain insight into cues that are involved in the induction or maintenance of the ex vivo microglia reference transcriptome. First, we used the in silico tool NicheNet to investigate which (CNS-derived) cues could underlie the differences between the transcriptomes of ex vivo and in vitro microglia. Modeling on basis of gene products that were found to be upregulated in vitro, predicted that high mobility group box 2 (HMGB2)- and interleukin (IL)-1β-associated signaling pathways were driving their expression. Modeling on basis of gene products that were found to be downregulated in vitro, did not lead to predictions on the involvement of specific signaling pathways. This is consistent with the idea that in vivo microenvironmental cues that determine microglial identity are for most part of inhibitory nature. In a second approach, primary microglia were exposed to conditioned medium from different CNS cell types. Conditioned medium from spheres composed of microglia, oligodendrocytes, and radial glia, increased the mRNA expression levels of the microglia signature gene P2RY12. NicheNet analyses of ligands expressed by oligodendrocytes and radial glia predicted transforming growth factor beta 3 (TGF-β3) and LAMA2 as drivers of microglia signature gene expression. In a third approach, we exposed microglia to TGF-β3 and laminin. In vitro exposure to TGF-β3 increased the mRNA expression levels of the microglia signature gene TREM2. Microglia cultured on laminin-coated substrates were characterized by reduced mRNA expression levels of extracellular matrix-associated genes MMP3 and MMP7, and by increased mRNA expression levels of the microglia signature genes GPR34 and P2RY13. Together, our results suggest to explore inhibition of HMGB2- and IL-1β-associated pathways in in vitro microglia. In addition, exposure to TGF-β3 and cultivation on laminin-coated substrates are suggested as potential improvements to current in vitro microglia culture protocols.

Keywords: HMGB2; IL-1β; NicheNet; TGF-β3; laminin; microglia identity; microglia signature genes; oligodendrocytes.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic overview of the NicheNet workflow. (A) We selected ligands expressed on neurons, astrocytes, oligodendrocytes, and microglia, and also included an analysis with all ligands of the NicheNet database. Next, we selected receptors expressed by microglia. We used NicheNet’s ligand-receptor data sources to analyze ligand-receptor interactions. Of note, ligands of each group were analyzed separately. Subsequently, information from NicheNet’s signaling data sources were used to analyze which transcriptional regulators are activated by the ligand-receptor interactions. Lastly, NicheNet’s gene regulatory data sources were used to further analyze which target genes (the differentially expressed genes between ex vivo and in vitro primary microglia from adult rhesus macaques) are regulated by the transcriptional regulators. Of note, upregulated and downregulated genes were analyzed separately. (B) After the ligand-target links were determined we performed a ligand-target activity analysis to rank the ligands based on the presence of its target genes. The more target interactions, the higher the ranking of the ligand. (C) The 20 ligands with the highest ligand-activity scores based on the presence of their target genes were displayed in the ligand-target heatmaps. In these heatmaps the regulatory potential scores for interaction between the top 20 ligands and target genes is displayed.
FIGURE 2
FIGURE 2
NicheNet analyses of the ligands of interest (LOI) and the upregulated target genes. (A) Ligand-target activity analysis of the LOI and the upregulated target genes. (B) Ligand-target matrix denoting the regulatory potential between the LOI and the upregulated target genes. mRNA expression levels of upregulated target genes in the presence of (C) inflachromene (ICM) and (D) interleukin 1 receptor antagonist (IL-1Ra). –, mRNA expression in the absence of the inhibitor; +, mRNA expression in the presence of the inhibitor. Symbols represent different donors, n = 4–6 dependent on the inhibitor, paired t-test on log-transformed data.
FIGURE 3
FIGURE 3
Exposure to conditioned medium of mixed glia cell spheres increased the mRNA expression levels of P2RY12. Microglia culture medium was supplemented with conditioned medium from either (A) spheres composed of microglia, oligodendrocytes, and radial glia (SCM), (B) rat oligodendrocyte precursor cells (OPC CM), or (C) rat mature oligodendrocytes (mOLG CM). Subsequently, mRNA expression levels of microglia signature genes were measured using RT-PCR. –, mRNA expression in the absence of conditioned medium; +, mRNA expression in the presence of conditioned medium. Symbols represent different donors, n = 4, paired t-test on log-transformed data, *p < 0.05.
FIGURE 4
FIGURE 4
TGF-β3 and laminin exposure increased the mRNA expression of microglia signature genes. Ligand-activity and ligand-target matrix of (A) oligodendrocyte ligands and (B) radial glia ligands. PCC, Pearson correlation coefficient. (C) Gene expression (CPM) of transforming growth factor beta 3 (TGF-β3) in monocultured microglia (M) and spheres (S), n = 4, EdgeR false discovery rate (FDR) was used to display statistical differences, ****FDR < 0.001. (D) mRNA expression levels of microglia signature genes of microglia cultured in the absence (–) or presence (+) of TGF-β3. Symbols represent different donors, n = 4, paired t-test on log-transformed data, *p < 0.05. mRNA expression levels of (E) microglia signature genes and (F) matrix metalloproteinases of microglia cultured in the absence (–) or presence (+) of laminin. Symbols represent different donors, n = 5, paired t-test on log-transformed data, *p < 0.05.
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