Polygenic risk for alcohol use disorder affects cellular responses to ethanol exposure in a human microglial cell model.

Characterization of iPSC-derived PMPs and microglia.(A) Protocols for generation of PMPs and microglial cells from iPSC. (B) Representative images of CD235+ and CD43+ cells in PMPs (line 8864). (C and D) Quantification of CD235+ PMPs derived from all iPSC lines with different PRS (low-PRS, n = 10; high-PRS, n = 8). (E and F) Quantification of CD43+ PMPs derived from all iPSC lines with different PRS (low-PRS, n = 10; high-PRS, n = 8). (G) Representative images of IBA1+, P2Y12+, CX3CR1+, TMEM119+, and PU.1+ microglia from high-PRS line 8864 and low-PRS line 9618. BMP-4, bone morphogenetic protein 4; VEGF, vascular endothelial growth factor; EB, embryonic body; SCF, stem cell factor; IL-3, interleukin-3; M-CSF, macrophage colony stimulating factor; IL-34, interleukin-34; GM-CSF, granulocyte-macrophage colony stimulating factor; DAPI, 4′,6-diamidino-2-phenylindole.

Whole-genome transcriptome profile comparison between human microglial cells derived from high-PRS and low-PRS iPSC lines.(A) Volcano plot depicts the DEGs between the high-PRS and low-PRS microglial cells. (B to D) GO analysis depicting the significant terms (top 10 up- and down-regulated terms) associated with microglia when comparing high-PRS versus low-PRS. It includes biological processes (BPs, B), molecular functions (MFs, C), and cellular components (D). P adjusted values are displayed. (E) Heatmap illustrates gene expression patterns related to chromosome separation in both high-PRS (red, n = 4 lines) and low-PRS (blue, n = 5 lines) microglia. (F) Heatmap illustrates gene expression patterns related to antigen processing and presentation in both high-PRS (red, n = 4 lines) and low-PRS (blue, n = 5 lines) microglia.

The impact of ethanol exposure on the activation of iPSC-derived microglia.(A) Representative images of CD68+ and IBA1+ microglia derived from high-PRS (line 8528) and low-PRS lines (line 9206) following a 7-day exposure to ethanol (0, 20, and 75 mM). (B) Quantification of CD68 corrected fluorescence-integrated density normalized to each control (0 mM) replicate. Low-PRS (n = 6 lines) versus high-PRS (n = 5 lines), **P < 0.01 and ***P < 0.001. Data are presented as mean ± SEM. (C) Representative images showing the process traced with regions of interest (ROI) using the IBA1 signal in iPSC-derived microglia with high-PRS (line 8864) or low-PRS (line 8838). (D) Morphological analysis of microglia binary outlines using FracLac ImageJ. n = 177 cells in five high-PRS lines, 211 cells in six low-PRS lines for 0 mM; n = 188 cells in five high-PRS lines, 214 cells in six low-PRS lines for 20 mM; n = 164 cells/five high-PRS lines, 219 cells/six low-PRS lines for 75 mM. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are presented as medium ± quartiles.

Transcriptomic profiles in high-PRS versus low-PRS human microglia after ethanol exposure.(A and B) Volcano plots depict the DEGs between the different ethanol concentrations (75 mM versus 0 mM) in four high-PRS lines (A) and five low-PRS lines (B) of microglial cells. (C and D) GO analysis of BPs highlighting significant terms (top 10 up- and down-regulated terms) associated with ethanol exposure (75 mM versus 0 mM) in both high-PRS (C) and low-PRS (D) microglia. Adjusted P values (P adjust.) are displayed. (E) GSEA plot depicting the enrichment of the phagocytosis-associated pathway specific to high-PRS microglia following ethanol exposure. P values and P adjust. are displayed. (F) GSEA plot of the MHCII and antigen processing–associated pathway enrichment specific to high-PRS microglia following ethanol exposure. P values and P adjust. are displayed.

Differential expression of phagocytosis-related genes between high-PRS and low-PRS microglia in response to ethanol exposure.(A) A heatmap illustration of the gene expression profiles related to antigen processing and presentation in both high-PRS (red) and low-PRS (blue) microglia, with and without ethanol exposure. (B) Box blots comparisons of the differential expression of genes associated with antigen processing and presentation in microglia from both high-PRS (red) and low-PRS (blue) lines, with and without ethanol exposure. Low-PRS/high-PRS lines = 5/4. *P < 0.05 and **P < 0.01. Data are presented as means ± SEM. (C) A heatmap illustration of the gene expression profiles related to the phagocytosis pathway in both high-PRS (red) and low-PRS (blue) microglia, with and without ethanol exposure. (D) Box blots comparison of the representative differential expression of genes associated with the phagocytosis pathway in microglia from both high-PRS (red) and low-PRS (blue) lines, with and without ethanol exposure. Low-PRS/high-PRS lines = 5/4. *P < 0.05 and **P < 0.01. Data are presented as means ± SEM.

CLEC7A expression between high-PRS and low-PRS microglia after ethanol exposure.(A) Representative images of CLEC7A+ and IBA1+ microglia derived from PRS lines following ethanol exposure (0 and 75 mM). (B and C) Quantification of CLEC7A+ fluorescence-integrated density (B) and corrected fluorescence-integrated density normalized to each control (0 mM) replicate (C). n = 331 cells/eight high-PRS lines and 365 cells/10 low-PRS lines for 0 mM; n = 315 cells/eight high-PRS lines and 373 cells/10 low-PRS lines for 75 mM; ***P < 0.001. Data are presented as means ± SEM.

Enhanced phagocytosis ability in high-PRS iPSC-derived microglia in response to ethanol.(A and B) Representative live-cell imaging of iPSC-derived microglia from low-PRS (line 8092, A) and high-PRS (line 8528, B) engaged in the phagocytosis of zymosan particles at different ethanol concentrations (0, 20, and 75 mM). The upper images are overlapped with bright-field microscopy for reference. (C) Quantification of the fluorescence-integrated density of zymosan bioparticles (beads): The top panel shows the fluorescence-integrated density between high-PRS and low-PRS microglial cells, while the bottom panel shows the corrected fluorescence-integrated density normalized to each control (0 mM). Low-PRS/high-PRS lines = 10/8, **P < 0.01 and ***P < 0.001. Data are presented as means ± SEM. (D) Quantification of the proportion of microglia with zymosan particles (beads): The top panel shows the proportion of microglia with beads, while the bottom panel displays the corrected proportion, which has been normalized to each control (0 mM) replicate. Low-PRS/high-PRS lines = 10/8, **P < 0.01 and ***P < 0.001. Data are presented as means ± SEM.

High-PRS iPSC-derived microglia decreases excitatory neurotransmission in coculture.(A) Representative images of coculture consisting of MAP2+ neurons and IBA1+ microglia derived from both high-PRS and low-PRS lines, following treatment with ethanol (0 and 75 mM). (B) Quantification of the proportion of microglia and neurons in coculture with high-PRS and low-PRS microglial cells under 0 and 75 mM conditions (low-PRS = 7 lines, high-PRS = 6 lines). Data are presented as means ± SEM. (C) Representative images illustrating synaptic puncta (green, labeled by synapsin immunofluorescence) associated with dendrites (red, visualized by MAP2 immunofluorescence) in iNs without microglia, with low-PRS microglial cells or with high-PRS microglial cells. (D) Quantification of synapsin puncta densities per MAP2 area. (iN without microglia: 0 mM = 73 images, 75 mM = 73 images; iN + low-PRS microglial cells: 0 mM = 93 images, 75 mM = 85 images; iN + high-PRS microglial cells: 0 mM = 70 images, 75 mM = 75 images). (E) Representative traces of miniature excitatory postsynaptic currents (mEPSCs) in Ngn2-iNs without microglia (gray, 0 mM = 43 cells, 75 mM = 46 cells), Ngn2-iNs with low-PRS microglial cells (blue, 0 mM = 53 cells, 75 mM = 55 cells), and Ngn2-iNs with high-PRS microglial cells (red, 0 mM = 43 cells, 75 mM = 46 cells). (F) Cumulative distribution and quantification (inserts) of mEPSC frequency (top) and amplitude (bottom) in Ngn2-iNs without microglia (gray), Ngn2-iNs with low-PRS microglial cells (blue), and Ngn2-iNs with high-PRS microglial cells (red). Data are means ± SEM; statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001) was evaluated with the Kolmogorov-Smirnov test (cumulative probability plots) and t test (bar graphs).