HDL induces the expression of the M2 macrophage markers arginase 1 and Fizz-1 in a STAT6-dependent process

Abstract

Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro ("M1") to an anti-inflammatory ("M2") phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNγ. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6(-/-) mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6.

Figure 1. HDL promotes and enhances the expression of specific anti-inflammatory genes in primary macrophages.

BMDM were treated for 6h with HDL (50μg/mL), IL-4 (10ng/mL) or both. Gene expression of Arg-1 (A), Fizz-1 (B) and IL-4R (C) was assessed by real-time qPCR. Results are representative of four independent experiments. Asterisks indicate statistically significant differences, either compared to control, or between 2 conditions when linked by a bar (*p<0.05, **p<0.01).

Figure 2. HDL inhibits the basal and induced expression of pro-inflammatory genes in primary macrophages.

BMDM were treated for 6h with HDL (50μg/mL), IFNγ (10ng/mL) or both. Gene expression for iNOS (A), IL-6 (B) or TNFα (C) was assessed by real-time qPCR. Results are representative of three independent experiments. Asterisks indicate statistically significant differences, either compared to control, or between 2 conditions when linked by a bar (*p<0.05, **p<0.01, ***p<0.001).

Figure 3. HDL induces STAT6 phosphorylation and enhances IL-4-induced STAT6 activation, but has no effect on STAT3 phosphorylation status.

BMDM were grown as previously described and stimulated for 15min with either HDL alone (50μg/mL), IL-4 alone (10ng/mL) or both. Phosphorylation state for STAT6 (A) and STAT3 (C) was assessed by western blot. As a positive control for STAT3 activation, cells were treated with IL-10 (10ng/mL) for 30 min. Signal quantification for phospho-STAT6 (B) is the result of three independent experiments. Asterisks indicate statistically significant difference, either compared to control, or between 2 conditions when linked by a bar (*p<0.05, **p<0.01).

Figure 4. STAT6 inhibition affects macrophages alternative activation by HDL.

BMDM were isolated from STAT6^+/+ (black bars) and STAT6-/- (hatched bars) mice and were stimulated for 6h with HDL (50μg/mL) or IL-4 (10ng/mL). The induction of Arg-1 or Fizz-1 mRNA in the STAT6+/+ cells by HDL was ~3X for each and by IL-4, ~1800 or 3300, respectively; these values were set to 100%. Results are representative of three independent experiments. Asterisks indicate statistically significant differences compared to corresponding STAT6^+/+ conditions (***p<0.001).

Figure 5. Jak inhibition affects macrophages alternative activation by HDL.

BMDM were pre-treated for 2h with Jak pharmacological inhibitors (hatched bars. Ruxolitinib/Jak1/2 inhibitor: 1μM. TG101348/Jak2 inhibitor: 1μM: Kaempferol/Jak3 inhibitor: 40μM) or vehicle (black bars), and then treated for 6h with HDL (50μg/mL) or IL-4 (10ng/mL). Arg-1 (A, C, E) and Fizz-1 (B, D, F) expression are presented as the percentage of activation compared to non-inhibited conditions. The induction of Arg-1 and Fizz-1 by HDL was ~2X for each, and for IL-4 was >300 for Arg-1 and Fizz-1 and these values were set to 100%. Results are representative of three independent experiments. Asterisks indicate statistically significant differences compared to the vehicle-treated conditions (*p<0.05, **p<0.01, ***p<0.001).