Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: (1) attraction of phagocytes via soluble `find me' signals, (2) recognition and phagocytosis via cell surface presenting `eat me' signals, and (3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of pro-inflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the present study, using wild type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, possibly via activation of activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase / protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.

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TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α–bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti–TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-β. Based on inhibitory experiments, the production of TGF-β1 is regulated via Jun kinases, whereas that of other TGF-βs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-β by Abs prevented the mTNF-α–mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-β. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-β, we show that they do not use the mTNF-α signaling pathway. Because TGF-β possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-β synthesis by those TNF-α–targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn’s disease, Wegener’s granulomatosis, or sarcoidosis. Additionally, none of the TNF-α–targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.

IntroductionTransglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied.MethodsImmunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGF-β1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model.ResultsTG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-β1, IL-1β and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-β1, but increased that of IL-1β and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1β and TNF-α, but significantly enhanced the TGF-β1 production. Neutralizing TGF-β antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-β, but in an increased IL-1β and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1β production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-β suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model.ConclusionsThese findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-β1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0592-7) contains supplementary material, which is available to authorized users.

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin β(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFβ activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFβ has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFβ production. Instead, in the absence of TG2 integrin β(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

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Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types, including T cells. Though the expression of the enzyme is strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 can be detected, when thymocytes are induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the proper in vivo induction of the enzyme. Previous studies from our laboratory have demonstrated that two of these signals, transforming growth factor-β (TGF-β) and retinoids, are produced by macrophages engulfing apoptotic cells. However, in addition to TGF-β and retinoids, engulfing macrophages produce adenosine as well. Here, we show that in vitro adenosine, adenosine, and retinoic acid or adenosine, TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes. The effect of adenosine is mediated via adenosine A2A receptors (A2ARs) and the A2AR-triggered adenylate cyclase signaling pathway. In accordance, loss of A2ARs in A2AR null mice significantly attenuates the in vivo induction of TG2 following apoptosis induction in the thymus indicating that adenosine indeed contributes in vivo to the apoptosis-related appearance of the enzyme. We also demonstrate that adenosine is produced extracellularly during engulfment of apoptotic thymocytes, partly from adenine nucleotides released via thymocyte pannexin-1 channels. Our data reveal a novel crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-related adenosine production contributes to the appearance of TG2 in the dying thymocytes.

BackgroundTumor necrosis factor (TNF)-a, a potent pro-inflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-a that is expressed on the surface TNF-a producing cells. TNF-a was shown to act both as a ligand by binding to TNF-a receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-a bearing cells. Anti-TNF-a targeting molecules which neutralize TNF-a with the same efficiency show disease specific effectiveness.ObjectivesThe aim of the present study was to characterize mTNF-a signaling and to test whether known anti-TNF-a targeting molecules trigger mTNF-a signaling in human macrophages.MethodsTNF-a expression and localization was studied in mouse bone marrow derived macrophages by RT-QPCR or FACS analysis and confocal microscopy. mTNF-a signaling was triggered by coated anti-TNF-a antibodies and tested by a Phospho-MAPK array. The cytokine induced was determined by RT-QPCR analysis and ELISA. The effect of mTNF-a signaling on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine formation was tested with a cytokine array.ResultsNon-activated macrophages express basal levels of mTNF-a and respond to anti-TNF-a antibodies by triggering the mitogen-activated protein kinase kinase 4 signaling pathway leading transforming growth factor (TGF)-b production. Exposure to LPS further induced the expression of mTNF-a, and mTNF-a signaling suppressed the LPS-induced pro-inflammatory response via TGF-b. Since apoptotic cells downregulate their TNF receptors, they do not trigger mTNF-a for TGF-b production. Etanercept, a soluble TNF-a receptor, does not, while golimumab and infliximab, which are anti-TNF-a antibodies, do trigger TGF-b production in human macrophages.ConclusionsInduction of TGF-b production by mTNF-a or by receptors triggered by apoptotic cells could be considered in the therapy of inflammatory diseases and may, in part, also account for differences in the actions of various TNF-a inhibitors.Disclosure of InterestNone declared