The current study presents data indicating that M2-polarised macrophages are more sensitive to the lipotoxic effects of oxLDL than other forms of monocyte/macrophage. The effects of oxLDL on disruption of ER Ca2+ homeostasis, activation of the UPR transcription factor XBP-1, upregulation of the UPR target genes BiP and CHOP, and on apoptosis and cell viability, were determined in six cell types: hMΦ, hM2MΦ, THP1-Mon, THP1-M2Mon, dTHP1-MΦ, and dTHP1-M2MΦ. With regard to these parameters, a consistent pattern was observed: THP1-Mon were the least sensitive to oxLDL, while hM2MΦ and dTHP1-M2MΦ were the most sensitive, with hMΦ, dTHP1-MΦ and THP1-M2Mon being intermediate in their sensitivity. Importantly, the M2-polarised macrophages (hM2MΦ and dTHP1-M2MΦ) were the only cell types to undergo significantly increased apoptosis and decreased cell viability when exposed to oxLDL levels similar to those seen in the circulation of overweight individuals (ie. ~1 μg/ml - as seen in individuals with a body mass index of 25-30 kg/m2).
The source of oxLDL-induced lipotoxicity in this instance is likely to be cholesterol, a component of oxLDL particles. It has been known for many years that intracellular accumulation of cholesterol results when macrophages take in more lipid than can be excreted; lysosomal cholesterol esterases are responsible for the generation of free cholesterol from lipoprotein-derived cholesteryl esters, and a proportion of the resulting free cholesterol is trafficked to intracellular membranes such as the ER membrane . The ER membrane is a narrow, fluid structure containing < 10% cholesterol ; elevation of its cholesterol content results in a broader, less fluid structure which restricts the ability of ER integral membrane proteins to undergo conformational movements and so catalyse their respective enzymatic reactions [22, 23]. This results in disrupted ER function , and the resulting ER stress has been linked via the UPR/CHOP pathway to the triggering of apoptosis [19, 20]. Due to the presence of an ERSE in its 3'UTR (see Results section), CHOP mRNA can be induced by prolonged and/or severe ER stresses to act as a transcription factor that controls several genes associated with apoptosis, including Bcl-2, GADD34, TRB3 and several caspases, including caspases 3 and 7 . (NB. It should be noted that the autophagic pathway, which can lead to non-apoptotic cell death by engulfing, degrading and recycling of cell organelles and proteins , is also associated with ER stress ; because in the present study, lipotoxic cell death associated with UPRs was assessed solely by determination of CHOP expression and caspase 3/7 activity, the possibility cannot be ruled out that other forms of cell death may also play a role in obesity-related macrophage phenotypic shifts. Nevertheless, in line with several previous papers [19–22], the current study has focused on CHOP-mediated apoptosis as the predominant mode of lipotoxic cell death in macrophages.)
As stated above, several mechanisms that may contribute to the obesity-linked shift to predominantly M1 cells been identified [1, 11]. However, additional mechanism(s) may also underpin the M2-to-M1 shifts seen within pre-existing macrophage populations during the development of obesity , and as described above, lipotoxicity has recently been proposed as a potential mechanism by which such shifts may occur . In the light of our demonstration that M2 macrophages exhibit higher sensitivity to oxLDL-induced lipotoxicity than non-M2-polarised macrophages, we propose that a higher degree of oxLDL-induced lipotoxic cell death within M2 macrophages may contribute to the decrease in numbers of M2 cells. Over time, a relative increase in proportion of non-M2 cells and hence a predominantly M1 macrophage population would ensue, leading to development of a more pro-inflammatory milieu in the tissues of obese individuals.
Having demonstrated heightened sensitivity to oxLDL-induced lipotoxicity in M2-polarised macrophages, we next considered the possible sources of this heightened sensitivity (ie. we addressed the question: "At what point in the ER stress/lipotoxicity pathway do M2 cells become more sensitive?"). There were no significant differences in oxLDL's impact on [Ca2+]cyt in the different cell types (Figure 2), and dTHP1-M2MΦ did not react to a greater extent than dTHP1-MΦ in terms of XBP-1 activation (Figure 3). However, significant increases in BiP expression (cf. THP1-Mon) were only seen in dTHP1-M2MΦ after treatment with 1 μg/ml oxLDL, with only non-significant increases being seen in other cell types (Figure 4a). Moreover, as shown in Figure 4b, statistically significant increases in CHOP expression were seen only in dTHP1-M2MΦ after treatment with 40 μg/ml oxLDL (a similar pattern was seen in the case of 1 μg/ml oxLDL v. CHOP, albeit without attaining statistical significance). Finally, with regard to apoptosis and cell viability, the response of dTHP1-M2MΦ to oxLDL was significantly greater than the comparable responses of other THP-1 derived cell-types (Figure 5). As stated above, with regard to primary cells, the same general pattern was observed: hM2MΦ exhibited significantly more apoptosis and less cell viability than hMΦ when exposed to oxLDL levels similar to those seen in sedentary individuals (i.e. 1 μg/ml ; Figures 1c and 1d). Thus, it appears that a factor relevant to the UPR (perhaps involving differences in the ability of the UPR to restore normal cell physiology and so avoid the triggering of apoptosis) may be the source of M2-polarised cells' heightened lipotoxic sensitivity. Further elucidation of the source of this differential lipotoxicity may increase our understanding of the causal connections between obesity and inflammatory diseases such as T2D; however, definitive identification of this source is unfortunately beyond the scope of this preliminary study.
As stated above, macrophages are involved in lipid accumulation in a wide variety of tissues, including adipose tissue . Intriguingly, quantitation of lipid-loading in different macrophage subtypes during the progression of obesity in ob/ob mice fed a high-fat diet revealed that, while macrophage lipid-loading in general increased, very few lipid-loaded M2 macrophages could be identified, with lipid-loading and adoption of a foam cell-like appearance being characteristic only of M1 macrophages . One may speculate that this could be explained by an inability of M2 macrophages to tolerate large quantities of intracellular lipid, and thus increased M2 cell death in a high-lipid environment. Such an explanation could also account for the high levels of apoptosis and M1-predominant nature of the macrophage foam cell populations resident in the unstable regions surrounding the lipid cores of atherosclerotic plaques [8, 21]. Thus, the relevance of differential macrophage lipotoxicity is likely to extend beyond adipose tissue macrophages. For example, the M2-to-M1 shift seen in Kupffer cells during the progression of liver steatosis has recently led Mandal et al to suggest that "changes in the M1/M2 phenotypic balance can impact on diverse disease conditions" , a statement which lends increased importance to the need to understand the mechanism(s) by which macrophage polarisation can be affected by phenomena such as lipotoxicity.
Clearly, there are several limitations to the current study (eg. the exclusively in vitro nature of the experiments; the lack of direct characterisation of differences in cholesterol→ER trafficking in each cell type). In particular, although the current study has specifically investigated the lipotoxic effects of oxLDL (and demonstrated that non-oxidised LDL is not associated with the same effects), it should also be recognised that in vivo, macrophages will be exposed to many other forms of lipid. Previous studies investigating different lipids have demonstrated the need to both activate the UPR/CHOP pathway and engage a scavenger receptor if apoptosis is to be triggered , suggesting that only lipoproteins that are ligands for scavenger receptors can trigger lipotoxic cell death. However, more recent studies have employed a lipidomic approach and found that a variety of lipid species underwent obesity-associated increases in levels within adipose tissue macrophages, and were associated with shifts towards the M1 phenotype; amongst these lipids were free cholesterol and saturated fatty acids . Interestingly, the relatively rigid inflexible structures of both these lipids are compatible with disruption of ER membrane functions, and thus the triggering of the UPR. Meanwhile, lipids with more flexible structures that would not be expected to disrupt ER membrane properties (eg. polyunsaturated fatty acids, plasmalogens) were not associated with increases in the M1 subtype . Thus, a capacity for triggering ER stress/UPR-linked apoptosis - and possibly a consequent heightened degree of damage to M2-polarised cells, which appear to be more sensitive to lipotoxicity - may underpin the detrimental effects of a variety of harmful lipids (including, but not limited to, oxLDL). Further studies are required to establish whether this is the case.