Comparative Analysis of HMC3 and C20 Microglial Cell Lines Reveals Differential Myeloid Characteristics and Responses to Immune Stimuli

Abstract

Microglia are the primary resident immune cells of the central nervous system (CNS) that respond to injury and infections. Being critical to CNS homeostasis, microglia also have been shown to contribute to neurodegenerative diseases and brain cancer. Hence, microglia are regarded as a potential therapeutic target in CNS diseases, resulting in an increased demand for reliable in vitro models. Two human microglia cell lines (HMC3 and C20) are being used in multiple in vitro studies, however, the knowledge of their biological and immunological characteristics remains limited. Our aim was to identify and compare the biological changes in these immortalised immune cells under normal physiological and immunologically challenged conditions. Using high-resolution quantitative mass spectrometry, we have examined in-depth proteomic profiles of non-stimulated and LPS or IFN-γ challenged HMC3 and C20 cells. Our findings reveal that HMC3 cells responded to both treatments through upregulation of immune, metabolic, and antiviral pathways, while C20 cells showed a response associated with mitochondrial and immune activities. Additionally, the secretome analysis demonstrated that both cell lines release IL-6 in response to LPS, while IFN-γ treatment resulted in altered kynurenine pathway activity, highlighting distinct immune and metabolic adaptations.

Keywords: immune; infection: Myeloid; inflammation; microglia.

FIGURE 1

(A) Morphological features of HMC3 and C20 cells. Epifluorescence microscopy, staining with DAPI (blue) for cell nuclei and AlexaFluor 488 phalloidin (green, f‐Actin cytoskeleton). Scale bar 200 μm. The contrast of the images was enhanced for presentation purposes. Note a smaller size and reduced density of f‐Actin in perinuclear region in untreated HMC3 cells, compared with C20 cell line, which demonstrates a more even and dense distribution of f‐Actin filaments across the whole cell body. This feature of f‐Actin intracellular distribution is unaffected by LPS and IFN‐γ stimulation. However, the size and shape parameters of HMC3 and C20 cells are changing in different ways. (B–M) Effect of LPS and IFN‐γ treatment on the morphological characteristics of HMC3 and C20 cells.

FIGURE 2

Untreated HMC3 and C20 microglial cells (A) Venn diagram of the shared and unique proteins (found in at least two biological replicates) of both cell lines, (B) Heatmap of all 802 DEPs between HMC3 in comparison to C20, with examples of some DEPs highlighted. 802 DEPs (609 upregulated, 193 downregulated in HMC3), (C) Volcano plot comparing 3122 proteins commonly expressed between HMC3 and C20 cells, with the top 5 down‐ and up‐regulated proteins in HMC3 highlighted in red. The x‐axis represents the fold change of protein expression. Proteins significantly upregulated (green, FC ratio > 2) and downregulated (lilac, FC ratio > −2) in HMC3 relative to C20. The y‐axis represents the −log10 p‐value (p < 0.05). 802 DEPs (609 upregulated, 193 downregulated in HMC3), (D, E) Bar plot illustrating the results of IPA analysis, highlighting (D) the top 8 and bottom 2 pathways of HMC3 in relation to C20, and (E) the top 5 and bottom 5 pathways of C20 in relation to HMC3 with the highest and lowest z‐score impacted by the DEPs. Bars are colour‐coded to indicate the regulation status of the genes within each pathway. The total number of genes involved in each up/down regulated pathway is annotated in the middle of the respective bars. The bar height reflects the percentage of DEPs implicated in each pathway, providing a visual overview of the significant pathways affected by DEPs in human microglia cells (z‐score + 2, p < 0.05), and (F, G) Dot pots illustrating pathway enrichment analysis of (F) up‐, and (G) down‐ regulated DEPs between HMC3 and C20, representing GO terms belonging to one of the three root GO ontologies: Biological process (BP), cellular component (CC), or molecular function (MF) respectively.

FIGURE 3

(A) Venn diagram showing overlapping and unique altered proteins associated to the innate immunity response (GO:0045087), HMC3, and C20, (B) Volcano plot comparing 198 common proteins between the innate immunity response (GO:0045087), HMC3, and C20. Of which, 50 proteins were differentially expressed (10 downregulated, and 40 upregulated) in HMC3 compared to C20 (FC ratio + 2, p < 0.05), (C) Heatmap of differentially expressed common proteins between HMC3, C20 and the innate immunity response (GO:0045087). A total of 50 proteins were differentially expressed, with some examples highlighted: 10 proteins were downregulated, and 40 proteins were upregulated in HMC3 (indicating that these 10 proteins were upregulated, and the 40 proteins were downregulated in C20) (FC ratio + 2, p < 0.05).

FIGURE 4

Cell protein quantification in LPS and IFN‐γ treated groups in (A–D) HMC3 and (E–H) C20. Volcano plot representations on DEPs (FC ratio + 2, p < 0.05) between control and treatment groups. Red dots indicate upregulated proteins, green dots indicate upregulated low value imputed proteins, blue dots indicate down‐regulated proteins, and orange dots indicate down‐regulated low value imputed proteins. Dotted lines on the X‐axis are log2 FC = −1 and 1.

FIGURE 5

(A–C) Bar plot illustrating results of IPA analysis, identifying cellular pathways impacted by the differential expression of proteins (A) LPS treated HMC3, (B) IFN‐γ treated HMC3, and (C) IFN‐γ treated C20 (z‐score > 2 or < −2, −log10 p < 1.3), and (D, E) Venn Diagram displaying common and unique DEPs associated to the innate immunity response (GO:0045087), HMC3, and C20 when challenged with (D) LPS, or (E) IFN‐γ.

FIGURE 6

Regulation of the kynurenine pathway (A–F) HMC3 (G–L) C20, and (M, N) Effect of LPS and IFN‐γ treatments on the concentration of IL‐6 in the secretome of (M) HMC3 and (N) C20 cells. Effect of LPS and IFN‐γ treatments on the concentration of IL‐6 in the secretome of HMC3 and C20 cells.