Pro-inflammatory stimuli of macrophage activation are not altered in obese T2D:
Plasma levels of IFN-γ were not different in OT2D versus controls (676±62 vs 610±20 relative fluorescent units [RFU] of IFN-γ, OT2D vs control, p=ns) (Supplementary figure 1A). Plasma levels of IL-4 and IL-13 were also not different in OT2D compared to control (294±10 vs 278±6 RFU of IL-4 and 598±23 vs 663±59 RFU of IL-13, OT2D vs control, p=ns) (Supplementary figure 1B, 1C). This data suggests that no increased Th1 or Th2-mediated immune response is present in obese OT2D subjects (basal condition).
M1 macrophage activation may be induced by Lipopolysaccharide (LPS) in obese subjects with T2D (OT2D):
Classically activated M1 macrophages constitute the first line of defense against intracellular pathogens and therefore exhibit a high level of phagocytic activity. A significant reduction of plasma lipopolysaccharide binding protein (LBP) was found in OT2D versus controls (85311±1453 vs 91747± 3048 RFU of LBP, OT2D vs control, p<0.05) suggesting LPS-mediated activation of M1 macrophages in OT2D (Figure 1A). Further suggestion of LPS-induced activation of M1 macrophages in OT2D was shown by no change of plasma toll like receptor-4 (TLR4) levels in OT2D (235±11 vs 254±13 RFU of TLR4, OT2D vs control, p=ns) (Figure 1B) that suggested elevated LPS-mediated endocytosis of TLR-4 in OT2D (25).
One of the markers that best characterizes human M1-like macrophages activation in response to IFN-γ and ILPS is CD80 (26). Plasma CD80 was increased in T2D (714±24 vs 626±21 RFU of CD80, T2D vs control, p<0.01) (Figure 1C). In addition, the basal level of plasma CD38, another LPS-induced M1 macrophage marker was elevated in OT2D (441±15 vs 408±7 RFU of CD38, OT2D vs control, p<0.05) (Figure 1D). There were no changes in plasma levels of LBP, TLR4, CD80 and CD38 level in OT2D in response to insulin-induced acute normalization of glycemia (Figure 1A–D).
Pro-inflammatory cytokines associated with LPS-induced activated M1 macrophages in OT2D
To determine the biomarkers (pro-inflammatory cytokines and chemokines released by LPS-TLR4 interaction) of activated M1 macrophages, basal levels of cytokines TNF-α, IL-6, IL-1β, IL-12 and chemokines CXCL-1, CXCL3, CXCL5, CXCL8, CXCL9, CXCL10 were measured. Basal levels of TNF-α, IL-1β, IL-6 and IL-12 were not changed in OT2D subjects (419±19 vs 423±21 RFU of TNF-α; 959±49 vs 971±54 RFU of IL-1β; 192±6 vs 208±17 RFU of IL-6 and 244±42 vs 196 vs 18 RFU of IL-12, OT2D vs control, p=ns) (Supplementary figure 2A–D). For chemokines, the basal levels of CXCL1 and CXCL5 were higher in OT2D (2487±177 vs 2060±67 RFU of CXCL1 and 543±17 vs 500±10, RFU of CXCL5, OT2D vs control, p<0.05) (Figure 2A-B). The level of CXCL3 and CXCL8 were not different in OT2D (377±35 vs 370±56 RFU of CXCL3; 543±17 vs 499±10 RFU of CXCL5 and 838±33 vs 814±67 RFU of CXCL8, OT2D vs control, p=ns) (Supplementary figure 2E-F). No difference in the basal level of plasma MFG-E8 level, associated with increased phagocytosis, in OT2D was found (1114±68 vs 1216±100 RFU of MGF-E8, OT2D vs control, p=ns) (Supplementary figure 2G). Two chemokines that have been shown to be increased in LPS-induced M1 polarized macrophages are CXCL-8 (IL-8) and RANTES (CCL5): basal IL-8 level did not differ in OT2D; however, plasma RANTES level was significantly higher (~2-fold) in OT2D (36961±5692 vs 18162±2393 RFU of RANTES, OT2D vs control, p<0.01) (Figure 2C).
Measurement of two classically activated M1 macrophage markers, CXCL9 and CXCL10, showed a significant reduction of the basal level of plasma CXCL9 (455±26 vs 722±113 RFU of CXCL9, OT2D vs control, p<0.05) and CXCL10 (1825±121 vs 2706±352 RFU of CXCL10, OT2D vs control, p<0.05) in OT2D compared to control (Figure 2D-E). Correction to normoglycemic did not alter the plasma levels of M1 macrophage activation markers in OT2D (Figure 2A-E).
M2 macrophage activation in obese individuals with type 2 diabetes (OT2D)
With the findings described above showing no change in basal level of plasma IL-4/IL-3 but likely elevated LPS level in OT2D, we hypothesized that M2 macrophages in OT2D are activated either by LPS (M2b) or TGB-𝛽 (M2c). Consistent with our hypothesis, we found a significant elevation of basal TGF-β1 level in OT2D (1123±72 vs 933±27 RFU of TGF-β1, OT2D vs control, p<0.01) (Figure 3A) [recently reported in (18)]. Basal level of plasma CD163 was higher in OT2D compared to control (2748±256 vs 2297±124 RFU of CD163, OT2D vs control, p<0.05) (Figure 3B). We further measured the plasma CD206 (soluble CD206), another potent M2 macrophage receptor, and found that there was no change in the basal level of plasma CD206 (10600±477 vs 9885±431 RFU of CD206, OT2D vs control, p=ns) (Supplementary Figure 3A).
We next explored other potent M2 macrophage receptor markers to determine if the basal concentration was also increased in the plasma of OT2D compared to control subjects. Our data indicate that there were no changes in plasma arginase and CD200R1 (888.2 ± 45.3 vs 963.1 ± 97.2 RFU of arginase and 1055.6 ± 41.4 vs 1102.8 ± 40.6 RFU of CD200R1, OT2D vs control, p=ns) (Supplementary Figure 3B, 3C) in OT2D compared to control. Interestingly, the level of CD200 was downregulated in OT2D (2241±85 vs 2426± 67 RFU of CD200, OT2D vs control, p<0.05) (Supplementary Figure 3D). Basal levels of dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN, also known as CD209) and CD36 were significantly lower in OT2D compared to control (1221±63 vs 1440±102 RFU of CD209 and 9752±614 vs 11360±627 RFU of CD36, OT2D vs control, p<0.05) (Supplementary Figure 3E, 3F).
The matrix metalloproteinases (MMPs) MMP7 and MMP9 were found to be elevated in OT2D compared to control (1242±93 vs 1005±42 RFU of MMP7 and 30193± 3746 vs 19532±1562 RFU of MMP9, OT2D vs control, p<0.05) (Figure 3C-D) [recently reported in (18)]. Correction to normoglycemic had no effect on plasma levels of M2 macrophage activation markers in OT2D (Figure 3A-D).
Adipose tissue macrophage (ATM) activation in OT2D:
Since the T2D subjects in this study were obese, we sought to determine the circulatory markers for activation of adipose tissue macrophages (ATMs) in OT2D. Human ATMs are characterized by their expression of CD163 and we showed in the previous section that the plasma CD163 level was increased in OT2D, suggesting activation of ATMs in OT2D. To determine if those ATMs are derived from circulating monocytes, we measured the monocyte chemoattractant protein-1 (MCP-1, also known as CCL2). Our data indicated that circulatory MCP-1 level did not differ in OT2D compared to control (698±49 vs 760±73 RFU of MCP-1, OT2D vs control, p=ns) (Figure 4A), suggesting reduced or no migration of monocyte-derived macrophages in adipose tissue. This observation was supported by the level of macrophage migration inhibitory factor (MIF) which was higher in OT2D (1387±223 vs 1007±29 RFU of MIF-1, OT2D vs control, p<0.05) (Figure 4B). We further measured the level of netrin-1 to determine if the ATM activation is mediated by a local modulator. Plasma netrin-1 level was higher in OT2D (558±30 vs 477±19 RFU of netrin-1, OT2D vs control, p<0.05) (Figure 4C). No changes were observed in the plasma levels of adipose tissue macrophages in response to acute normalization of glycemia in OT2D (Figure 4A-C).
Association of macrophage-related proteins with BMI
Since the T2D subjects in this study were obese, we next considered which of the macrophage-associated circulatory proteins associated with BMI. Six proteins showed an association with BMI: RANTES (p=0.002), CXCL1 (p=0.002), MIF (p=0.03), LBP (p=0.03), IL13 (p=0.03) and CD200R1 (p=0.03). Whilst the above proteins associated with BMI, suggesting that BMI may serve as a surrogate for insulin resistance or increased adipokine activity, other proteins were BMI-independent, namely CD80 (p=0.004), CXCL10 (p=0.012), TGF-β1 (p=0.02), and MMP9 (p=0.03), indicating that these BMI-independent proteins may relate specifically to T2D.