This study uncovers two unexpected sources of Th2 cytokines, adipocytes and hepatocytes. This finding provides a molecular mechanism through which resident macrophage activation is modulated. Our results further show that PPARδ is induced by Th2 cytokines to control the transcriptional program of alternative activation in the macrophage. The subsequent phenotypic switch to the M2 phenotype plays an important role in dampening inflammation within WAT and the liver, thereby providing protection against insults from inflammatory stimulants. The physiological relevance of this pathway is demonstrated in myeloid specific PPARδ−/− mice, which exhibit adipocyte dysfunction and insulin resistance as well as hepatosteatosis.
Several lines of evidence support the notion that tissue-derived Th2 cytokines are biological active and metabolically relevant. First, CM collected from 3T3 adipocytes, which mainly contains IL-13, increased STAT6 activity and the expression of M2 markers. This effect is inhibited by an IL-13 neutralizing antibody. In addition, IL-13 and IL-4 protein could be detected in lysates from primary adipocytes and hepatocytes as well as from WAT and liver, although IL-4 levels appeared to be several fold lower. The production of Th2 cytokines by adipocytes and hepatocytes could be considered a regulatory mechanism to prevent uncontrolled inflammation (), as macrophages in WAT and liver are prone to activation by inflammatory mediators, including free fatty acids. The activated macrophages, in turn, release cytokines, such as TNFα, IL-1β and IL-6, which cause metabolic deregulation. In the macrophage, the signaling of Th2 cytokines is transduced through STAT6 and PPARδ. While some fatty acid species trigger inflammation, others may serve as endogenous ligands to activate PPARδ to turn on alternative activation.
In support of the proposed model, high fat fed myeloid-specific PPARδ−/− mice showed increased M1 and decreased M2 markers in WAT and the liver. This results in lipolysis and insulin resistance in adipocytes. These defects are likely mediated by elevated TNFα, as it has been shown to interfere with glucose metabolism by down-regulation of GLUT4, to inhibit insulin sensitivity through JNK activation (Hirosumi et al., 2002
; Lumeng et al., 2007b
; Ruan et al., 2002
) and to cause lipolysis by suppression of perilipin expression (Laurencikiene et al., 2007
). In the liver, the pro-inflammatory status increases lipogenesis. The induction of ACC2 further suppresses fatty acid catabolism. The combined effect leads to increased TG accumulation. Although insulin signaling in the liver showed no difference at the time of examination, the deregulated fat metabolism will likely worsen hepatic insulin resistance in Mac-PPARδ−/− mice at a later stage. We did not observe changes in inflammatory or metabolic genes in muscle (Figure S3
and data not shown). This was not unexpected, as we were unable to detect an appreciable amount of IL-13 or IL-4 by ELISA in muscle (data not shown). The metabolic phenotype of Mac-PPARδ−/− mice was less profound on a chow diet. These mice showed mild glucose intolerance and insulin resistance compared to WT controls determined by the GTT and ITT (Figure S4A and B
). There was no significant difference in the expression of M1 and M2 markers in WAT, liver or muscle (data not shown). However, the presence of adipocytes surrounded by macrophages and hepatic lipid accumulation was observed in Mac-PPARδ−/− mice (Figure S4C and D
). These results suggest that in the unchallenged, lean state, other signaling pathways are capable of maintaining basal M2 gene expression. Nevertheless, it appears that even a minor shift in the resident macrophage polarization is sufficient to induce metabolic dysregulation.
One of the potential signaling pathways known to modulate M2 activation is through PPARγ, which when deleted in myeloid cells, causes insulin resistance in multiple tissues (Hevener et al., 2007
; Odegaard et al., 2007
). However, our data suggest that PPARγ could not compensate for the loss of PPARδ. In fact, PPARδ/γ−/−macrophages show a similar defect in Mgl1 expression to that of PPARδ−/− macrophages. A similar result was seen in Mgl2 expression (data not shown). It is possible that PPARγ plays a predominant role in circulating monocytes, as it has been shown that PPARγ activation induces monocyte differentiation into M2 macrophages but does not affect resting macrophages (Bouhlel et al., 2007
). Alternatively, PPARδ may act as a competent factor for Th2 cytokine-induced M2 gene expression. Evidence supporting this idea comes from the observation that the ability of IL-13 to up-regulate Mgl1 promoter activity is highly dependent on PPARδ (). In addition, STAT6 activation and IL-4R induction by Th2 cytokines is diminished in PPARδ−/− macrophages (), which remains intact in PPARγ−/− macrophages (Odegaard et al., 2007
). PPARδ may do so by controlling the expression of PGC-1β ( and S1A
), which has been shown to co-activate STAT6 (Vats et al., 2006
) and may also be required for the full transcriptional activity of PPARγ. However, we could not rule out the possibility that PPARδ and PPARγ controls different subsets of M2 genes.
Adipocytes and hepatocytes are unique in the way that they are capable of producing both Th1 and Th2 cytokines. The balance in the production of these two counter forces therefore determines the polarization of macrophages residing in WAT and liver. The current study identifies PPARδ as a potential drug target to modulate tissue macrophage activation and insulin sensitivity. Understanding how Th1 and Th2 cytokine production is controlled in adipocytes and hepatocytes in future studies may also reveal additional therapeutic pathways to combat obesity-related metabolic diseases.