The Kat in the HAT: The Histone Acetyl Transferase Kat6b (MYST4) Is Downregulated in Murine Macrophages in Response to LPS.

Epigenetic modulators, including histone methylases, demethylases, and deacetylases, have been implicated previously in the regulation of classical and alternative macrophage activation pathways. In this study, we show that the histone acetyl transferase (HAT) Kat6B (MYST4) is strongly suppressed (>80%) in macrophages by lipopolysaccharide (LPS) (M1 activation), while Kat6A, its partner in the MOZ/MORF complex, is reciprocally upregulated. This pattern of expression is not altered by LPS together with the adenosine receptor agonist NECA (M2d activation). This is despite the observation that miR-487b, a putative regulator of Kat6B expression, is mildly stimulated by LPS, but strongly suppressed by LPS/NECA. Other members of the MYST family of HATs (Kat5, Kat7, and Kat8) are unaffected by LPS treatment. Using the pLightswitch 3'UTR reporter plasmid, the miR-487b binding site in the Kat6b 3'UTR was found to play a role in the LPS-mediated suppression of Kat6B expression, but other as-yet unidentified factors are also involved. As Kat6B is a HAT that has the potential to modulate gene expression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigenetic modulator in macrophage activation pathways.

The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Rα) signaling.

Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as "alternatively activated" (M2a) macrophages. We have shown previously that adenosine A2A receptor (A(2A)R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an "M2-like" phenotype that we have termed "M2d." Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα(-/-) mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify "M2" macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Regulation of Macrophage Polarization and Wound Healing.

BACKGROUND: Macrophages (Mφs) participate in wound healing by coordinating inflammatory and angiogenic processes. Mφs respond to environmental cues by adopting either "classically" activated (M1) proinflammatory or "alternatively" activated (M2a, M2b, M2c, M2d) wound healing phenotypes. THE PROBLEM: Mφ polarization is essential for wound healing and aberrations in this process are linked to several pathologies. It is important to elucidate molecular mechanisms underlying Mφ polarization. BASIC/CLINICAL SCIENCE ADVANCES: Mφs are categorized as proinflammatory (M1) or anti-inflammatory/wound healing (M2). M1 Mφs are observed in initial tissue damage responses, are induced by exogenous pathogen-associated molecular patterns or endogenous damage-associated molecular patterns, and exhibit increased phagocytosis and pro-inflammatory cytokine production, facilitating innate immunity and wound debridement. M2 Mφs predominate later in repair, express vascular endothelial growth factor, transforming growth factor beta, and interleukin 10 (IL-10), are activated by varied stimuli, assist in the resolution of inflammation, and promote tissue formation and remodeling. Recent work has characterized a novel "M2d" phenotype resulting from adenosine-dependent "switching" of M1 Mφs that exhibits a pattern of marker expression that is distinct from canonical IL-4/IL-13-dependent M2a Mφs. Recent studies have demonstrated important roles for specific transcriptional elements in M1 and M2a Mφ polarization, notably members of the interferon regulatory factor family interferon regulatory factor 5 (IRF5) and IRF4, respectively. The role of these IRFs in M2d polarization and wound healing remains to be determined. CLINICAL CARE RELEVANCE: Knowledge of microenvironmental signals and molecular mechanisms that mediate Mφ polarization should permit their manipulation to regulate important physiological processes and resolve pathological conditions. CONCLUSION: Proper Mφ polarization is essential to effective wound healing, and distinct phenotypes, such as the angiogenic M2d Mφ, may be of critical importance to this process. The IRF5 transcription factor has been shown to play a key role in M1 Mφ activation and the Jumonji domain containing-3-IRF4 pathway has been implicated in M2 Mφ activation.

Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype.

Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A(2A) receptor (A(2A)R) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A(2A)Rs by TLR agonists, down-regulation of tumor necrosis factor alpha (TNFalpha) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)beta1 and beta2 expression in macrophages by de-stabilizing their mRNAs. The PLCbeta inhibitor U73122 down-regulates TNFalpha expression by macrophages, and in the presence of A(2A)R agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A(2A)R agonists. Selective down-regulation of PLCbeta2, but not PLCbeta1, using small-interfering RNA resulted in increased VEGF expression in response to A(2A)R agonists, but did not suppress TNFalpha expression. Macrophages from PLCbeta2(-/-) mice also expressed increased VEGF in response to A(2A)R agonists. LPS-mediated suppression of PLCbeta1 and beta2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 microg/mouse, i.p.) suppressed PLCbeta1 and beta2 expression in spleen, liver, and lung of wild-type but not MyD88(-/-) mice. These studies indicate that LPS suppresses PLCbeta1 and beta2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCbeta2 plays an important role in switching M1 macrophages into an M2-like state.

Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.

Adenosine A(2A)R and TLR agonists synergize to induce an "angiogenic switch" in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alphaI.2 is expressed ubiquitously, and HIF-1alphaI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A(2A)R agonists. HIF-1alphaI.1 is induced strongly compared with HIF-1alphaI.2 upon costimulation with LPS and A(2A)R agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted approximately 4% of HIF-1alpha transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was approximately 15%, indicating a substantial contribution of HIF-1alphaI.1 to total HIF-1alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A(2A)R-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1alphaI.1 was down-regulated by Bay 11-7085 (NF-kappaB inhibitor) and ZM241385 (A(2A)R antagonist). Although VEGF and IL-10 expression by HIF-1alphaI.1-/- macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1alpha, IL-6, IL-12p40, and IL-1beta expression was significantly greater, suggesting a role for HIF-1alphaI.1 in modulating expression of these cytokines. A(2A)R expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappaB-dependent manner. LPS-induced expression of A(2A)Rs and HIF-1alpha and A(2A)R-dependent HIF-1alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A(2A)R agonists on macrophage VEGF expression.

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors.

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.

Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter.

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2A receptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1alpha expression. HIF-1alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappaB-dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1alpha promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1alpha promoter activity, indicating that the A2AR-dependent increase in HIF-1alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1alpha protein and DNA binding levels. Deletion of putative NF-kappaB-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappaB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1alpha through the HRE. HIF-1alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

Analysis of signal transduction pathways in macrophages using expression vectors with CMV promoters: a cautionary tale.

The cytomegalovirus (CMV) major immediate-early promoter is a strong promoter used for both in vitro and in vivo expression of proteins in signal transduction and gene therapy studies. CMV activity is induced by external stimuli such as endotoxin from Gram-negative bacteria (LPS), TNF-alpha and phorbol esters. This inducibility poses problems when this promoter is used to drive the expression of either wild type or dominant negative mutated proteins as tools in signal transduction studies. This report draws attention to the problem associated with this widely used approach. The role of NF-kappaB and Hypoxia Inducible Factor-1alpha (HIF-1alpha) in the transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in macrophages was investigated using CMV-promoter-driven expression of either wild type or dominant negative proteins involved in these pathways. Difficulties encountered while interpreting the data due to the inducibility of the CMV promoter by LPS are highlighted in this report and provide a cautionary note for the evaluation of data acquired using this approach.

Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin.

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.