Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2.

TLR3 drives IRF6-dependent IL-23p19 expression and p19/EBI3 heterodimer formation in keratinocytes.

Interferon regulatory factor (IRF) family members impart cell-type specificity to toll-like receptor (TLR) signalling, and we recently identified a role for IRF6 in TLR2 signalling in epithelial cells. TLR3 has a well-characterized role in wound healing in the skin, and here, we examined TLR3-dependent IRF6 functions in human keratinocytes. Primary keratinocytes responded robustly to the TLR3 agonist poly(IC) with upregulation of mRNAs for interferon-ß (IFN-ß), the interleukin-12 (IL-12) family member IL-23p19 and the chemokines IL-8 and chemokine (C-C motif) ligand 5 (CCL5). Silencing of IRF6 expression enhanced poly(IC)-inducible IFN-ß mRNA levels and inhibited poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Consistent with these data, co-transfection of IRF6 increased poly(IC)-inducible IL-23p19 promoter activity, but inhibited poly(IC)-inducible IFN-ß promoter activity in reporter assays. Surprisingly, poly(IC) did not regulate IL-12p40 expression in keratinocytes, suggesting that TLR3-inducible IL-23p19 may have an IL-23-independent function in these cells. The only other IL-12 family member that was strongly poly(IC) inducible was EBI3, which has not been shown to heterodimerize with IL-23p19. Both co-immunoprecipitation and proximity ligation assays revealed that IL-23p19 and EBI3 interact in cells. Co-expression of IL-23p19 and EBI3, as compared with IL-23p19 alone, resulted in increased levels of secreted IL-23p19, implying a functional role for this heterodimer. In summary, we report that IRF6 regulates a subset of TLR3 responses in human keratinocytes, including the production of a novel IL-12 family heterodimer (p19/EBI3). We propose that the TLR3-IRF6-p19/EBI3 axis may regulate keratinocyte and/or immune cell functions in the context of cell damage and wound healing in the skin.

Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein C5a.

Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-ß (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation.

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 Mϕ) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF Mϕ). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 Mϕ and GM-CSF Mϕ exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 Mϕ versus GM-CSF Mϕ, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 Mϕ and GM-CSF Mϕ (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 Mϕ contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways.

Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages.

Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.