Cytokine-induced reprogramming of human macrophages toward Alzheimer's disease-relevant molecular and cellular phenotypes in vitro

Abstract

Myeloid cells including brain-resident (microglia) and peripheral macrophages play a crucial role in various pathological conditions, including neurodegenerative disorders like Alzheimer's disease (AD). They respond to disruption of tissue homeostasis associated with disease conditions by acquiring various transcriptional and functional states. Experimental investigation of these states is hampered by the lack of tools that enable accessible and robust reprogramming of human macrophages toward Alzheimer's disease-relevant molecular and cellular phenotypes in vitro. In this study, we investigated the ability of a cytokine mix, including interleukin-4 (IL4), colony stimulating factor 1 (CSF1/MCSF), interleukin 34 (IL34) and transforming growth factor beta (TGFβ), to induce reprogramming of cultured human THP-1 macrophages. Our results indicate this treatment led to significant transcriptomic changes, driving THP-1 macrophages towards a transcriptional state reminiscent of disease-associated microglia (DAM) and lipid-associated macrophages (LAM) collectively referred to as DLAM. Transcriptome profiling revealed gene expression changes related to oxidative phosphorylation, lysosome function, and lipid metabolism. Single-cell RNA sequencing revealed an increased proportion of DLAM clusters in cytokine mix-treated THP-1 macrophages. Functional assays demonstrated alterations in cell motility, phagocytosis, lysosomal activity, and metabolic and energetic profiles. Our findings provide insights into the cytokine-mediated reprogramming of macrophages towards disease-relevant states, highlighting their role in neurodegenerative diseases and potential for therapeutic development.

Keywords: Alzheimer’s disease; DAM; IL4; LAM; THP-1 macrophages; disease-associated microglia; efferocytosis; lipid-associated macrophages.

Figure 1.. Differential abundance and pathway enrichment analysis of gene expression changes induced by cytokine treatment in THP-1 macrophages reveales changes in Lysosome, Oxidative phosphorylation and Cell cycle

A) Schematic of THP-1 differentiation using phorbol 12-myristate 13-acetate (PMA) followed by cytokine treatment B) PCA analysis comparing THP1 macrophages (with and without cytokines) with other myeloid cell types C) KEGG-selected pathways from Gene set enrichment analysis (GSEA) positively in three main cytokines group as compared to No cytokine (control) (for all GSEA see Table S1) D) Volcano plot representing top differentially expressed genes in cytokine-treated macrophages.

Figure 2.. Cytokine treatment induces a DLAM, and non-proliferative transcriptional response in THP-1 macrophages.

A) Enrichment of AD-related gene sets in Cytokine-treated macrophages. All gene sets are listed in Table S2, NES – normalized enrichment score * FDR q-val < 0.05 ** FDR q-val < 0.01 *** FDR q-val < 0.001 **** FDR q-val < 0.0001. B) Heatmap of DLAM genes Z-scores (log2 transformed TPM) based on Table S1 C) Quantification of surface expression of DLAM markers by flow cytometry. Values plotted as gMFI. Groups were tested with paired t.test (two-tailed) * p < 0.05, ** p < 0.01, *** p < 0.001. Percentage of positive cells plotted separately in Figure S2. Different dot shapes correspond to independent macrophage differentiations. N = 3–4 independent differentiation, each differentiation has 2 well replicates.

Figure 3.. Cytokine mix treatment induces DLAM, non-proliferative macrophage transcriptional states in THP-1 macrophages

A) UMAP of downsampled scRNA-seq data comparing THP1 macrophages at baseline (left) and following cytokine treatment (right). Nine independent clusters were identified using Seurat FindClusters. B) Heatmap showing top expressed genes per cluster compared to all other clusters (log2FC>0.6, qval<0.05, pct expressed>70%). The left block shows the number of genes in each geneset and representative genes are labeled on the right. Z-scores across clusters are used for the plot. C) The proportion of cells per treatment condition within each cluster after downsampling. The percentage of cells from the total number of cells per cluster is shown on each bar. D) Dotplot showing expression of the top 10 differentially expressed genes across each cluster. The size of dots are scaled to represent the percentage of cells expressing the gene within the cluster. Average expression is normalized within each feature.

Figure 4.. Comparison of THP-1 macrophages treated with cytokine mix with iPSC-derived microglia (iMGLs) exposed to phagocytic substrates and AD brain signatures reveals induction of similar transcriptional states

A) Hypergeometric overlap results showing enrichment of myeloid gene signatures in the up-regulated differentially expressed genes from pseudobulk scRNA-seq of cytokine treatment vs controls, grouped by subtypes. B) Sankey plot showing the projection of clusters found in this study to clusters identified by Dolan et al., by exposure of iMGLs to phagocytic substrates. C) UMAP projection of THP-1 macrophages dataset, cells colored by module scores of transcriptional signatures identified in DAM Clusters 2 and 8, and Proliferative Cluster 10 by Dolan et al., 2023; Lipid processing Cluster MG4 in AD brains identified by Sun et al, 2024, and DAM cluster GPNMB_EYA2 and Prolif cluster in AD biopsies identified by Gazestani et al., 2024.

Figure 5.. Cytokine mix treatment decreases phagocytosis and lysosomal processing in THP-1 macrophages.

A) Diagram showing the steps in the efferocytosis process. B) Evaluation of the migration capacity through the scratch wound assay in THP1 macrophage control and treated with the cytokine mix. B1, relative wound density over time; B2, quantification of the area under the curve. Cytochalasin D (cytD) was used as a migration inhibitor. C) Quantification of phagocytic uptake of beads C1), myelin fragments C2), Zymosan C3), and early apoptotic cells (EAJ) C4). D) Evaluation of lysosomal activity in control and cytokine stimulated macrophages by LysoTracker (lysosomal mass, D1), LysoSensor (acidification, D2) and DQ-BSA (lysosomal proteolysis, D3-D4). Different dot shapes correspond to independent macrophage differentiations. N = 3–4 independent differentiation, each differentiation has 2–3 well replicates. Groups were tested with paired t.test (two-tailed) * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6.. Cytokine mix treatment increases glycolytic and mitochondrial metabolism in THP-1 macrophages.

A) Seahorse Mitostress Test: A1, Test profile of oxygen consumption over time. Quantification of basal (A2) and maximal (A3) respiration capacity. B) Seahorse Glycolysis Stress Test: B1, Test profile of extracellular acidification rate over time. Quantification of basal glycolysis (B2) and maximal glycolytic capacity (B3). Different dot shapes correspond to independent macrophage differentiations. N = 4 independent differentiation, each differentiation has 4–6 well replicates. Groups were tested with paired t.test (two-tailed) * p < 0.05, ** p < 0.01.

Figure 7.. Cytokine mix treatment induces changes in lipidomic profiles and lipid metabolism in THP-1 macrophages.

A) Lipidomics results from control and cytokine mix stimulated THP-1 macrophages (N=4). B) Quantification of lipid droplet content (BODIPY staining) by flow cytometry in THP-1 control and stimulated macrophages. C) Abundance of selected cholesterol ester species (raw data in Table S4). D) Quantification of intracellular APOE normalized to Actin in THP-1 macrophages treated with cytokines. Quantification (left), representative images (right). E) Quantification of secreted APOE in THP-1 macrophages. F) Quantification of ABCA1, normalized to Actin, measured by western blot in control and cytokine-treated THP-1 macrophages. Quantification (left), representative image (right) G) Quantification of cholesterol efflux, in THP-1 control and stimulated macrophages into HDL acceptor N = 3–4 independent differentiation, each differentiation has 3 well replicates. Different dot shapes correspond to independent macrophage differentiations. Groups were tested by paired t.test (two-tailed) * p < 0.05, ** p <0.01.