Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages

The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-alpha release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-alpha and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-alpha mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-alpha mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent.     

IL-10 inhibits lipopolysaccharide-induced CD40 gene expression through induction of suppressor of cytokine signaling-3

Costimulation between T cells and APCs is required for adaptive immune responses. CD40, an important costimulatory molecule, is expressed on a variety of cell types, including macrophages and microglia. The aberrant expression of CD40 is implicated in diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease, and inhibition of CD40 signaling has beneficial effects in a number of animal models of autoimmune diseases. In this study, we discovered that IL-10, a cytokine with anti-inflammatory properties, inhibits LPS-induced CD40 gene expression. We previously demonstrated that LPS induction of CD40 in macrophages/microglia involves both NF-kappaB activation and LPS-induced production of IFN-beta, which subsequently activates STAT-1alpha. IL-10 inhibits LPS-induced IFN-beta gene expression and subsequent STAT-1alpha activation, but does not affect NF-kappaB activation. Our results also demonstrate that IL-10 inhibits LPS-induced recruitment of STAT-1alpha, RNA polymerase II, and the coactivators CREB binding protein and p300 to the CD40 promoter, as well as inhibiting permissive histone H3 acetylation (AcH3). IL-10 and LPS synergize to induce suppressor of cytokine signaling (SOCS)-3 gene expression in macrophages and microglia. Ectopic expression of SOCS-3 attenuates LPS-induced STAT activation, and inhibits LPS-induced CD40 gene expression, comparable to that seen by IL-10. These results indicate that SOCS-3 plays an important role in the negative regulation of LPS-induced CD40 gene expression by IL-10.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages

Proteinase-activated receptor 2 (PAR(2)), a 7-transmembrane G protein-coupled receptor, contributes to inflammation either positively or negatively in different experimental systems. Previously, we reported that concurrent activation of PAR(2) and TLRs in human lung and colonic epithelial cells resulted in a synergistic increase in NF-κB-mediated gene expression, but a down-regulation of IRF-3-mediated gene expression. In this study, the effect of PAR(2) activation on LPS-induced TLR4 signaling was examined in primary murine macrophages. The PAR(2) activation of wild-type macrophages enhanced LPS-induced expression of the anti-inflammatory cytokine, IL-10, while suppressing gene expression of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12. Similar PAR(2)-mediated effects on LPS-stimulated IL-10 and IL-12 mRNA were also observed in vivo. In contrast, PAR 2-/- macrophages exhibited diminished LPS-induced IL-10 mRNA and protein expression and downstream STAT3 activation, but increased KC mRNA and protein. PAR(2) activation also enhanced both rIL-4- and LPS-induced secretion of IL-4 and IL-13, and mRNA expression of alternatively activated macrophage (AA-M) markers, e.g. arginase-1, mannose receptor, Ym-1. Thus, in the context of a potent inflammatory stimulus like LPS, PAR(2) activation acts to re-establish tissue homeostasis by dampening the production of inflammatory mediators and causing the differentiation of macrophages that may contribute to the development of a Th2 response.     

Dendritic cell-specific ICAM-3-grabbing nonintegrin expression on M2-polarized and tumor-associated macrophages is macrophage-CSF dependent and enhanced by tumor-derived IL-6 and IL-10

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) is a human pathogen-attachment C-type lectin with no obvious murine ortholog and for which ligation leads to enhanced anti-inflammatory cytokine release and altered proinflammatory cytokine production. Although induced by IL-4 in monocytes and considered as a DC marker, DC-SIGN expression on human APCs under homeostatic conditions is so far unexplained. We report in this study that M-CSF enhances DC-SIGN expression on in vitro derived anti-inflammatory macrophages and that M-CSF mediates the induction of DC-SIGN by fibroblast- and tumor cell-conditioned media. The M-CSF-inducible DC-SIGN expression along monocyte-to-macrophage differentiation is dependent on JNK and STAT3 activation, potentiated by STAT3-activating cytokines (IL-6, IL-10), and abrogated by the M1-polarizing cytokine GM-CSF. In pathological settings, DC-SIGN expression is detected in tumor tissues and on ex vivo-isolated CD14(+) CD163(+) IL-10-producing tumor-associated macrophages. Importantly, DC-SIGN Abs reduced the release of IL-10 from macrophages exposed to Lewis(x)-expressing SKBR3 tumor cells. These results indicate that DC-SIGN is expressed on both wound-healing (IL-4-dependent) and regulatory (M-CSF-dependent) alternative (M2) macrophages and that DC-SIGN expression on tumor-associated macrophages might help tumor progression by contributing to the maintenance of an immunosuppressive environment.     

Reprogramming of IL-10 activity and signaling by IFN-gamma

One important mechanism of cross-regulation by opposing cytokines is inhibition of signal transduction, including inhibition of Janus kinase-STAT signaling by suppressors of cytokine signaling. We investigated whether IFN-gamma, a major activator of macrophages, inhibited the activity of IL-10, an important deactivator. Preactivation of macrophages with IFN-gamma inhibited two key anti-inflammatory functions of IL-10, the suppression of cytokine production and of MHC class II expression. Gene expression profiling showed that IFN-gamma broadly suppressed the ability of IL-10 to induce or repress gene expression. Although IFN-gamma induced expression of suppressor of cytokine signaling proteins, IL-10 signal transduction was not suppressed and IL-10 activation of Janus kinases and Stat3 was preserved. Instead, IFN-gamma switched the balance of IL-10 STAT activation from Stat3 to Stat1, with concomitant activation of inflammatory gene expression. IL-10 activation of Stat1 required the simultaneous presence of IFN-gamma. These results demonstrate that IFN-gamma operates a switch that rapidly regulates STAT activation by IL-10 and alters macrophage responses to IL-10. Dynamic regulation of the activation of different STATs by the same cytokine provides a mechanism by which cells can integrate and balance signals delivered by opposing cytokines, and extends our understanding of cross-regulation by opposing cytokines to include reprogramming of signaling and alteration of function.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.