Downregulation of macrophage Irs2 by hyperinsulinemia impairs IL-4-indeuced M2a-subtype macrophage activation in obesity

M2a-subtype macrophage activation is known to be impaired in obesity, although the underlying mechanisms remain poorly understood. Herein, we demonstrate that, the IL-4/Irs2/Akt pathway is selectively impaired, along with decreased macrophage Irs2 expression, although IL-4/STAT6 pathway is maintained. Indeed, myeloid cell-specific Irs2-deficient mice show impairment of IL-4-induced M2a-subtype macrophage activation, as a result of stabilization of the FoxO1/HDAC3/NCoR1 corepressor complex, resulting in insulin resistance under the HF diet condition. Moreover, the reduction of macrophage Irs2 expression is mediated by hyperinsulinemia via the insulin receptor (IR). In myeloid cell-specific IR-deficient mice, the IL-4/Irs2 pathway is preserved in the macrophages, which results in a reduced degree of insulin resistance, because of the lack of IR-mediated downregulation of Irs2. We conclude that downregulation of Irs2 in macrophages caused by hyperinsulinemia is responsible for systemic insulin resistance via impairment of M2a-subtype macrophage activation in obesity.

In this manuscript entitled 'Inhibition of FoxO1/HDAC3/1 NCoR by IL-4/Irs2 is impaired in macrophages, causing decreased M2a-subtype activation and systemic insulin resistance in obesity' , Kubota et al. describe the role of the IL4/IRS2 pathway in the development of obesityinduced inflammation and insulin resistance. By using various approaches including myeloidspecific deletion of IRS2 and IR, the authors conclude that the inhibition of the IL-4/IRS2 axis in macrophages leads to a reduced number of M2a macrophages translating into adipose tissue inflammation and insulin resistance in obesity. It is proposed that hyperinsulinemia promotes downregulation of IRS2 in adipose tissue macrophages leading to a reduced ability of adipose tissue macrophages to differentiate towards the anti-inflammatory M2a phenotype. The manuscript reads well and contains an impressive set of experiments. The data reveal a unique future of the IRS2 molecule in macrophages controlling their polarization potential in adipose tissue. However, the IL-4/IRS2 axis has already been identified as an important regulator of M2 differentiation. The novel part in this manuscript relates to the identification of hyperinsulinemia as suppressor of IRS2 mediated by the IR in macrophages. This in turn leads to a reduction in M2a polarized macrophages due to lower efficacy of IL-4.
Comments and suggestions -As stated above, earlier papers have already established a role for the IL-4/IRS2 axis in the regulation of macrophage polarization. Several papers show similar findings related to the polarization of M2-like macrophages as reported in the current manuscript. What do the current results add, in a mechanistic way, to the existing data on the role of the IL-4/IRS2 axis in the regulation of M2a ?
-The m1 vs. m2 classification in the adipose tissue is a somewhat outdated concept. It would be very interesting if the authors could also demonstrate the importance of this pathway in metabolically activated macrophages that are known to reside in obese adipose tissue. Moreover, insulin has been shown to be one of the factors, along with glucose and palmitic acid, driving the metabolically activated macrophages in obese adipose tissue. This would be a very relevant issue for the authors to address.
-IRS2 ko in myeloid cells results in liver and adipose tissue specific insulin resistance without any differences on total BW. In adipose tissue of the IRS2 ko animals, more CLS were observed. The authors conclude, based upon data presented in figure 2, that the ratio of M1/M2 was increased in the ko animals and demonstrate that some markers of M2 macrophages were lower expressed in the ko animals. I am not really sure you can interpret these results as a reduction in M2a macrophages, since all data is reported as arbitrary units or percentage. The gene expression values and M1/M2 ration may actually reflect a strong upregulation in M1-like macrophages during HFD-feeding that may be accompanied by a less pronounced increase of M2 cells in the KO animals. In other words, I am not really convinced that the data presented in figure 2 would allow the authors to state that there is a significant reduction in M2a-like macrophages. It might also be an overwhelming influx of monocytes differentiating towards M1-like cells.
-Did the authors consider to measure IL-4 levels and eosinophils in the adipose tissue of the IRS2ko versus WT after HFD-feeding? Also, MCP-1 levels are higher in the KO animals suggestive of more influx into the adipose tissue that would account for more M1 cells not necessarily being accompanied by a reduction in M2a like cells.
-The authors go to great lengths to establish the contribution of the FOXO-NCOr1/HDAC3 corepressor complex being responsible for the suppression of the M2a-subtype (data presented in figure 3). However, do the authors have any data to support the in vivo relevance of this pathway?
For example, what are the gene expression levels of these regulators in ATMs ? How are these factors expressed/regulated in adipose tissue macrophages? In addition, in an in vitro setting, does insulin treatment affect this pathway? Presumably, prolonged insulin exposure should directly impact on this pathway. -I think the in vitro experiments using insulin together with IL-4 as presented in figure 5 are interesting. What is the concentration of insulin that was used in these experiments and how long are the cells treated ? On a more functional level, is the cytokine production of these cells also affected? Hence, do the cells produce different/lower levels of typical M2-like cytokines? -I am a bit confused about certain figure legends used by the authors. For example, in figure 2g the authors have presented BMDMs from C57/bl6 animals, yet in the figure legends it reads ' in the C57/Bl6 mice after stimulation with IL-4'. This needs to be clarified. -As explained by the authors in figure 7, hyperinsulinemia is the driving force in the development of inflammation and adipose tissue due to the dysregulation of M2a polarization. However, it has also been established that hyperinsulinemia drives expression of MCP-1 in adipose tissue leading to the recruitment of monocytes during obesity. Would it be possible to somehow lower plasma insulin levels (STZ treatment or metformin treatment for example) to reveal the potential contribution of hyperinsulinemia to the regulation of IRS2 levels in macrophages in vivo? -Overall, I feel that most of the conclusions are justified based upon the presented data. However, some of the results are over interpreted and figure 7 is an oversimplified model. Currently, no data in the paper clearly supports the role of hyperinsulinemia in lowering IRS2 in macrophages. Although the absence of the IR provides some proof, this would not explain why normal insulin levels would not affect the pathway and hyperinsulinemia would lead to downregulation of IRS2.
Reviewer #2 (Remarks to the Author): Adipose tissue macrophages play a pivotal role for adipose tissue inflammation and insulin resistance in obesity, and macrophage subtypes can impact insulin sensitivity. This manuscript shows that 1) Irs2 expression is downregulated in obese adipose tissue macrophages, 2) Myeloidspecific Irs2 KO mice exhibit reduced M2 macrophage numbers, increased adipose tissue inflammation, and increased insulin resistance upon HFD feeding, 3) Foxo1/HDAC3/NCoR1 inhibits M2-related gene expression, 4) Hyperinsulinemia represses Irs2 expression and enhances Foxo1 activity, which leads to repression of M2 marker genes. Although many studies suggest the reduced number of M2 macrophages contribute to enhanced pro-inflammatory responses in adipose tissue inflammation and insulin resistance, the mechanisms for M2 macrophage reduction has not been clearly elucidated. This study provides new insights for how M2 macrophages may be reduced in obese adipose tissue. However, there are a few shortcomings that need attention: 1. The authors propose that reduced Irs2 expression impairs M2a macrophage differentiation. Although they show the Irs2 expression level in peritoneal macrophages and adipose tissue macrophages (SiglecF-CD11b+F4/80+), the downregulation of Irs2 expression itself might be secondary to reduced M2 macrophages. Perhaps the expression levels of Irs2 in M1 and M2 macrophages in adipose tissues in NCD and HFD fed mice could be determined. If there is no difference in Irs2 expression in M2 macrophages in NCD-fed mice, it would be also helpful to explain no difference in M2 macrophage in NCD-fed myeloid-specific Irs2 KO mice.  5. figure 3 shows the regulation of M2-related genes by Foxo1/HDAC3/NCoR1 complex. To explore this complex in more depth, the promoter activity of Arg1, FIZZ1, and Ym1 in CA-Foxo1 with siHDAC3 and siNCoR1 transfected macrophages could be measured.