BV-2 cells are routinely cultured in a serum-containing medium. Given the roles that MafB plays during macrophage phagocytosis and actin organization, we characterized the effect of serum on MafB protein levels in BV-2 cell cultures. Culturing BV-2 cells in reduced (0–5%) serum for 24 h decreased MafB protein levels to less than half of those cultured in 10% serum, respectively (0.44 and 0.42–fold, respectively, vs 10% FBS; p-values <0.0001; Suppl. Fig. 1A). Ionized calcium-binding adaptor molecule 1 (Iba1), a key player in phagocytic cup formation and membrane ruffling in microglia, was decreased as well in 5% serum cultures (0.70–fold vs 10% FBS; p = 0.044), but not serum-free cultures (0.83–fold vs 10% FBS; p = 0.156), relative to 10% serum cultures (Suppl. Fig. 1A, full blots shown in Suppl. Fig. 2). Because 5% of serum cultures showed significantly lower Iba1 levels compared to 10% serum cultures, we next asked whether the phagocytosis of phosphatidylserine-coated (PS) beads would be impaired in reduced serum cultures. To this end, we cultured BV-2 cells in 10% or 5% serum for 18 h before adding 3 μm diameter PS beads for another 6 h. We immunostained these cultures with the lysosomal marker Cluster of Differentiation 68 (CD68) and Iba1 to visualize particle engulfment (Suppl. Fig. 1B). For quantification, we measured the proportion of CD68 co-localized with fluorescent PS beads to the total volume of Iba1 using pseudo-confocal microscopy. BV-2 cells cultured in 5% serum showed reduced phagocytosis of fluorescent PS beads compared to those cultured in 10% serum (engulfment index 0.21 [5% serum] vs 0.27 [10% serum]; p = 0.0025, Suppl. Fig. 1C). As a negative control, we pretreated BV-2 cells with the actin polymerization inhibitor cytochalasin D to prevent phagocytosis. Although cytochalasin D-treated BV-2 cells were able to bind fluorescent PS beads (Suppl. Fig. 1B, Suppl. Fig. 3), BV-2 engulfment of them was dramatically impaired when compared to untreated 10% serum cultures (engulfment index 0.08; p <0.001) (Suppl. Fig. 1C). The lack of complete inhibition by cytochalasin D might reflect CD68’s ability to bind phosphatidylserine coating the bead surface, which could account for the apposition of these signals (see Suppl. Fig. 3), particularly given the resolution limits of pseudo-confocal microscopy.
Pure, serum-treated primary microglia cultures are largely devoid of apoptotic cells because neighboring microglia rapidly clear them. Also, peripheral macrophages upregulate Mafb when exposed to apoptotic cells. However, it is unclear whether BV-2 cells phagocytose one another in pure cultures and, if so, whether this occurs frequently enough to elicit measurable changes in MafB at the population level. Having observed decreased MafB levels in BV-2 cells cultured in reduced serum conditions (Suppl. Fig. 1A), we next asked whether this effect could be explained by the phagocytosis of neighboring and perhaps apoptotic BV-2 cells or debris. To test this, we compared three phagocytosis inhibition strategies—reduced serum (Suppl. Fig. 1C), the actin polymerization inhibitor cytochalasin D, and the phosphatidylserine-binding protein Annexin V—for their ability to modulate MafB protein levels in BV-2 cultures. As expected, 5% serum significantly reduced MafB protein levels relative to 10% serum (0.72–fold vs 10% FBS; p = 0.042, Suppl. Fig. 1D), albeit to a lesser extent than we observed in supplementary figure 1A. This inconsequential discrepancy is likely due to the technical differences between these experiments (see Suppl. Experimental Methods) and the semi-quantitative nature of western blotting. Cytochalasin D treatment in the presence of 10% serum dramatically reduced MafB protein levels (0.36–fold vs 10% FBS; p = 0.0004; Suppl. Fig. 1D; full blots shown in Suppl. Fig. 4) versus untreated 10% serum cultures. Finally, Annexin V treatment in the presence of 10% serum mimicked the result of reduced serum by decreasing MafB relative to untreated 10% serum cultures (0.71–fold vs 10% FBS; p = 0.042; Suppl. Fig. 1D). Together, these results suggest that MafB protein levels in BV-2 cells could depend on the phagocytosis of neighboring, perhaps apoptotic BV-2 cells.
To further explore the possibility that BV-2 cells phagocytose neighboring BV-2 cells under standard cell culture conditions (see Methods), we visualized BV-2 cell apoptosis using a cell-permeable, activated caspase-3/7 dye. We observed activated caspase-3/7-positive BV-2 cells in all imaged cell culture wells. Live BV-2 cells frequently surrounded activated caspase-3/7-positive (called “apoptotic” hereafter; see Limitations for a caveat to this interpretation) BV-2 cells (Suppl. Videos 1–3). In such cases, activated caspase-3/7 puncta appeared to radiate from apoptotic cells into surrounding live BV-2 cells (Suppl. Videos 1 & 2). In one case, activated caspase-3/7 debris was engulfed by a distant BV-2 cell (Fig. 1A, Suppl. Video 1). We also observed the disappearance of engulfed, activated caspase-3/7 puncta in live BV-2 cells (Fig. 1B, Suppl. Video 1). These observations provide evidence of BV-2 cells’ phagocytosing one another in pure cultures. To determine the robustness of our initial phagocytosis inhibitor experiment (Suppl. Fig. 1D), we replicated the experiment (without the serum deprivation condition) in 3 independent experiments. These experiments generated similar results (Fig. 1C, Suppl. Fig. 5). Nevertheless, the interpretation that basal MafB protein depends on BV-2 cells’ phagocytosing one another remains speculative because it is unclear whether the engulfment of BV-2 cell debris we observed is sufficient to drive changes in MafB protein at the population level. Instead, MafB levels could depend on other factors present in the cell culture environment.
How might MafB be regulated by the phagocytosis of cells and cellular debris? This effect could be mediated by the activation of liver X receptors (LXR) by hydrolyzed cholesterol obtained from engulfed apoptotic bodies. After all, engulfed apoptotic bodies supply macrophages with a substantial cholesterol load, and MafB is known to be transcriptionally induced by LXR agonists. This putative mechanism raises the possibility that other lipid sources in the cell culture environment—namely, serum, which is an abundant source of lipids and lipoproteins—could contribute to basal MafB protein levels in BV-2 cell cultures. To test this hypothesis, we compared BV-2 cells cultured in normal growth medium with those cultured in medium containing lipid-depleted (LD) serum. BV-2 cells exhibited lower MafB protein when cultured in medium containing LD serum (0.66-fold vs normal FBS, p = 0.0475; Fig. 1D). This effect was prevented by the addition of human low-density lipoproteins (LDL), another rich source of cholesterol (p = 0.0138; Fig. 1D). We replicated this result in three independent experiments (Suppl. Fig. 6). In aggregate, our results indicate that basal MafB levels in the BV-2 microglial cell line depending on the availability of lipids in the cell culture environment—either obtained directly from serum or via phagocytosis of apoptotic BV-2 cell debris.
When transferred to a cell culture environment, microglia rapidly downregulate transcripts that typify mature, homeostatic microglia in vivo. Such transcripts—e.g., P2ry12, Tmem119, and Sparc—exhibit a remarkably short half-life of <1 h in vitro, consistent with previous estimates that show downregulation of thousands of genes within 6 h of transfer to culture. Microglia also transiently upregulate many inflammatory transcripts when transferred in vitro. However, with longer exposure (1–5 days) to serum-supplemented or defined media, microglia assume an alternative gene signature characterized by increased expression of Mafb (Fig. 1E) and phagocytosis-related genes (Suppl. Fig. 7). These include known/putative MafB target genes C1qa, Fcgr3, Cd5l, Msr1, and Rab13, as well as the lysosomal marker Cd68. We speculate that the upregulation of Mafb and putative MafB target genes by serum in this culture system may be a consequence of increased lipid content in the cell culture environment.