Crosstalk between purinergic receptor P2Y11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages.

P2Y11 is a G protein-coupled ATP receptor that activates IL-1 receptor (IL-1R) in a cyclic AMP dependent manner. In human macrophages, P2Y11/IL-1R crosstalk with CCL20 as a prime target is controlled by phosphodiesterase 4 (PDE4), which mediates breakdown of cyclic AMP. Here, we used gene expression analysis to identify activation of CXCR4 and CXCR7 as a hallmark of P2Y11 signaling. We found that PDE4 inhibition with rolipram boosts P2Y11/IL-1R-induced upregulation of CXCR7 expression and CCL20 production in an epidermal growth factor receptor dependent manner. Using an astrocytoma cell line, naturally expressing CXCR7 but lacking CXCR4, P2Y11/IL-1R activation effectively induced and CXCR7 agonist TC14012 enhanced CCL20 production even in the absence of PDE4 inhibition. Moreover, CXCR7 depletion by RNA interference suppressed CCL20 production. In macrophages, the simultaneous activation of P2Y11 and CXCR7 by their respective agonists was sufficient to induce CCL20 production with no need of PDE4 inhibition, as CXCR7 activation increased its own and eliminated CXCR4 expression. Finally, analysis of multiple CCL chemokines in the macrophage secretome revealed that CXCR4 inactivation and CXCR7 activation selectively enhanced P2Y11/IL-1R-mediated secretion of CCL20. Altogether, our data establish CXCR7 as an integral component of the P2Y11/IL-1R-initiated signaling cascade and CXCR4-associated PDE4 as a regulatory checkpoint.

P2Y11/IL-1 receptor crosstalk controls macrophage inflammation: a novel target for anti-inflammatory strategies?

Although first cloning of the human ATP receptor P2Y11 was successful 25 years ago, the exact downstream signaling pathways of P2Y11 receptor, which can couple to Gq and Gs proteins, have remained unclear. Especially the lack of rodent models as well as the limited availability of antibodies and pharmacological tools have hampered examination of P2Y11 expression and function. Many meaningful observations related to P2Y11 have been made in primary immune cells, indicating that P2Y11 receptors are important regulators of inflammation and cell migration, also by controlling mitochondrial activity. Our recent studies have shown that P2Y11 is upregulated during macrophage development and activates signaling through IL-1 receptor, which is well known for its ability to direct inflammatory and migratory processes. This review summarizes the results of the first transcriptomic and secretomic analyses of both, ectopic and native P2Y11 receptors, and discusses how P2Y11 crosstalk with the IL-1 receptor may govern anti-inflammatory and pro-angiogenic processes in human M2 macrophages.

The P2Y11 receptor of human M2 macrophages activates canonical and IL-1 receptor signaling to translate the extracellular danger signal ATP into anti-inflammatory and pro-angiogenic responses.

The cytoprotective ATP receptor P2Y11 is upregulated during M2 macrophage differentiation and contributes to the anti-inflammatory properties of this macrophage subset. Here, we studied P2Y11-induced reprogramming of human M2 macrophages at the level of mRNA and protein expression. Upregulation of IL-1 receptor (IL-1R) and its known downstream effectors VEGF, CCL20 and SOCS3 as well as downregulation of the ATP-degrading ecto-ATPase CD39 emerged as hallmarks of P2Y11 activation. The anti-inflammatory signature of the P2Y11 transcriptome was further characterized by the downregulation of P2RX7, toll-like receptors and inflammasome components. P2Y11-induced IL-1R upregulation formed the basis for reinforced IL-1 responsiveness of activated M2 macrophages, as IL-1α and IL-1ß each enhanced P2Y11-induced secretion of VEGF and CCL20 as well as the previously reported shedding of soluble tumor necrosis factor receptor 2 (sTNFR2). Raising intracellular cyclic AMP (cAMP) in M2 macrophages through phosphodiesterase 4 inhibition enhanced P2Y11-driven responses. The cAMP-binding effector, exchange protein activated by cAMP 1 (Epac1), which is known to induce SOCS3, differentially regulated the P2Y11/IL-1R response because pharmacological Epac1 inhibition enhanced sTNFR2 and CCL20 release, but had no effect on VEGF secretion. In addition to cAMP, calcium and protein kinase C participated in P2Y11 signaling. Our study reveals how P2Y11 harnesses canonical and IL-1R signaling to promote an anti-inflammatory and pro-angiogenic switch of human M2 macrophages, which may be controlled in part by an Epac1-SOCS3 axis.

Peptides from allergenic lipocalins bind to formyl peptide receptor 3 in human dendritic cells to mediate TH2 immunity.

BACKGROUND: How TH2-mediated allergic immune responses are induced is still under investigation. OBJECTIVE: In an in vitro system we compared the effect of lipocalin allergens and nonallergenic homologues on human monocyte-derived dendritic cells (DCs) to investigate how they polarize naive CD4+ TH cells. Microarray data gained with these DCs showed a significant difference in expression of formyl peptide receptors (FPRs). Activation of FPR3 in human monocyte-derived DCs leads to inhibition of IL-12 production. Low concentrations of IL-12 during T-cell priming biases immune responses toward TH2. We hypothesize that binding of allergenic lipocalins to FPR3 might be a mechanism for induction of allergic immune responses. METHODS: We examined whether lipocalins and FPR3 colocalize within the cells by using confocal microscopy. With calcium mobilization assays of FPR3-transfected HEK 293 cells, we measured FPR3 signaling in response to allergenic and nonallergenic lipocalins. Silencing of FPR3 in DCs and pretreatment with an antagonistic peptide were used to assess the function of FPR3 in TH2 induction. RESULTS: FPR3 and lipocalins colocalize in the same vesicles in DCs. Cathepsin S-digested allergenic lipocalins, but not digestion products of nonallergenic homologues, activate FPR3 signaling. FPR3 silencing in DCs or pretreatment with an antagonistic peptide restores IL-12 and induces IL-10 expression by DCs treated with lipocalin allergens, attenuating the TH2 bias and inducing IL-10 production in cocultured TH cells. CONCLUSION: We describe a novel molecular mechanism for induction of TH2-mediated allergic immune responses.

Antibody opsonization enhances MAIT cell responsiveness to bacteria via a TNF-dependent mechanism.

Mucosal-associated invariant T (MAIT) cells are an abundant human T-cell subset with antimicrobial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for T-cell receptor-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here, we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to Escherichia coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1, but not B, cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering.

Control of Macrophage Inflammation by P2Y Purinergic Receptors.

Macrophages comprise a phenotypically and functionally diverse group of hematopoietic cells. Versatile macrophage subsets engage to ensure maintenance of tissue integrity. To perform tissue stress surveillance, macrophages express many different stress-sensing receptors, including purinergic P2X and P2Y receptors that respond to extracellular nucleotides and their sugar derivatives. Activation of G protein-coupled P2Y receptors can be both pro- and anti-inflammatory. Current examples include the observation that P2Y14 receptor promotes STAT1-mediated inflammation in pro-inflammatory M1 macrophages as well as the demonstration that P2Y11 receptor suppresses the secretion of tumor necrosis factor (TNF)-α and concomitantly promotes the release of soluble TNF receptors from anti-inflammatory M2 macrophages. Here, we review macrophage regulation by P2Y purinergic receptors, both in physiological and disease-associated inflammation. Therapeutic targeting of anti-inflammatory P2Y receptor signaling is desirable to attenuate excessive inflammation in infectious diseases such as COVID-19. Conversely, anti-inflammatory P2Y receptor signaling must be suppressed during cancer therapy to preserve its efficacy.

The human G protein-coupled ATP receptor P2Y11 is a target for anti-inflammatory strategies.

BACKGROUND AND PURPOSE: The ATP receptor P2Y11 , which couples to Gq and Gs proteins, senses cell stress and promotes cytoprotective responses. P2Y11 receptors are upregulated during differentiation of M2 macrophages. However, it is unclear whether and how P2Y11 receptors contribute to the anti-inflammatory properties of M2 macrophages. EXPERIMENTAL APPROACH: Transcriptome and secretome profiling of ectopic P2Y11 receptors was used to analyse their signalling and function. Findings were validated in human monocyte-derived M2 macrophages. The suramin analogue NF340 and P2Y11 receptor-knockout cells confirmed that agonist-mediated responses were specific to P2Y11 receptor stimulation. KEY RESULTS: Temporal transcriptome profiling of P2Y11 receptor stimulation showed a strong and tightly controlled response of IL-1 receptors, including activation of the IL-1 receptor target genes, IL6 and IL8. Secretome profiling confirmed the presence of IL-6 and IL-8 proteins and additionally identified soluble tumour necrosis factor receptor 1 and 2 (sTNFR1 and sTNFR2) as targets of P2Y11 receptor activation. Raised levels of intracellular cAMP in M2 macrophages, after inhibition of phosphodiesterases (PDE), especially PDE4, strongly increased P2Y11 receptor-induced release of sTNFR2 through ectodomain shedding mediated by TNF-α converting enzyme (TACE/ADAM17). Both IL-1α and IL-1ß synergistically enhanced P2Y11 receptor- induced IL-6 and IL-8 secretion and release of sTNFR2. During lipopolysaccharide-induced activation of TLR4, which shares the downstream signalling pathway with IL-1 receptors, P2Y11 receptors specifically prevented secretion of TNF-α. CONCLUSIONS AND IMPLICATIONS: Targeting P2Y11 receptors activates IL-1 receptor signalling to promote sTNFR2 release and suppress TLR4 signalling to prevent TNF-α secretion, thus facilitating resolution of inflammation.