Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Loss of adenosine A3 receptors accelerates skeletal muscle regeneration in mice following cardiotoxin-induced injury.

Skeletal muscle regeneration is a complex process orchestrated by multiple interacting steps. An increasing number of reports indicate that inflammatory responses play a central role in linking initial muscle injury responses to timely muscle regeneration following injury. The nucleoside adenosine has been known for a long time as an endogenously produced anti-inflammatory molecule that is generated in high amounts during tissue injury. It mediates its physiological effects via four types of adenosine receptors. From these, adenosine A3 receptors (A3Rs) are not expressed by the skeletal muscle but are present on the surface of various inflammatory cells. In the present paper, the effect of the loss of A3Rs was investigated on the regeneration of the tibialis anterior (TA) muscle in mice following cardiotoxin-induced injury. Here we report that regeneration of the skeletal muscle from A3R-/- mice is characterized by a stronger initial inflammatory response resulting in a larger number of transmigrating inflammatory cells to the injury site, faster clearance of cell debris, enhanced proliferation and faster differentiation of the satellite cells (the muscle stem cells), and increased fusion of the generated myoblasts. This leads to accelerated skeletal muscle tissue repair and the formation of larger myofibers. Though the infiltrating immune cells expressed A3Rs and showed an increased inflammatory profile in the injured A3R-/- muscles, bone marrow transplantation experiments revealed that the increased response of the tissue-resident cells to tissue injury is responsible for the observed phenomenon. Altogether our data indicate that A3Rs are negative regulators of injury-related regenerative inflammation and consequently also that of the muscle fiber growth in the TA muscle. Thus, inhibiting A3Rs might have a therapeutic value during skeletal muscle regeneration following injury.

Nur77 and PPARγ regulate transcription and polarization in distinct subsets of M2-like reparative macrophages during regenerative inflammation.

Macrophage polarization is a process whereby macrophages develop a specific phenotype and functional response to different pathophysiological stimuli and tissue environments. In general, two main macrophage phenotypes have been identified: inflammatory (M1) and alternatively activated (M2) macrophages characterized specifically by IL-1ß and IL-10 production, respectively. In the cardiotoxin-induced skeletal muscle injury model bone marrow-derived macrophages (BMDMs) play the central role in regulating tissue repair. Bone marrow-derived monocytes arriving at the site of injury differentiate first to M1 BMDMs that clear cell debris and trigger proliferation and differentiation of the muscle stem cells, while during the process of efferocytosis they change their phenotype to M2 to drive resolution of inflammation and tissue repair. The M2 population is formed from at least three distinct subsets: antigen presenting, resolution-related and growth factor producing macrophages, the latest ones expressing the transcription factor PPARγ. Nuclear receptor subfamily 4 group A member 1 (NR4A1; also termed Nur77) transcription factor is expressed as an early response gene, and has been shown to suppress the expression of pro-inflammatory genes during efferocytosis. Here we demonstrate that (1) Nur77 null BMDMs are characterized by elevated expression of PPARγ resulting in enhanced efferocytosis capacity; (2) Nur77 and PPARγ regulate transcription in different subsets of M2 skeletal muscle macrophages during muscle repair; (3) the loss of Nur77 prolongs M1 polarization characterized by increased and prolonged production of IL-1ß by the resolution-related macrophages normally expressing Nur77; whereas, in contrast, (4) it promotes M2 polarization detected via the increased number of IL-10 producing CD206+ macrophages generated from the PPARγ-expressing subset.

Palmitate Inhibits Mouse Macrophage Efferocytosis by Activating an mTORC1-Regulated Rho Kinase 1 Pathway: Therapeutic Implications for the Treatment of Obesity.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome-lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered.

Retinoids Promote Mouse Bone Marrow-Derived Macrophage Differentiation and Efferocytosis via Upregulating Bone Morphogenetic Protein-2 and Smad3.

Clearance of apoptotic cells by bone marrow-derived macrophages differentiated from monocytes plays a central role in the resolution of inflammation, as the conversion of pro-inflammatory M1 macrophages to M2 macrophages that mediate the resolution process occurs during efferocytosis. Thus, proper efferocytosis is a prerequisite for proper resolution of inflammation, and failure in efferocytosis is associated with the development of chronic inflammatory diseases. Previous studies from our laboratory have shown that (13R)-all-trans-13,14-dihydroretinol (DHR), the product of retinol saturase, acting from day 4 of monocyte differentiation enhances the efferocytosis capacity of the resulted macrophages. Loss of retinol saturase in mice leads to impaired efferocytosis, and to development of autoimmunity. In the present paper, we report that in differentiating monocytes DHR, retinol, and all-trans retinoic acid all act directly on retinoic acid receptors and enhance the clearance of apoptotic cells by upregulating the expression of several efferocytosis-related genes. The effect of retinoids seems to be mediated by bone morphogenetic protein (BMP)-2, and the Smad3 transcription factor. In addition, retinoids also upregulate the expression of the vitamin D receptor and that of vascular endothelial growth factor A, indicating that altogether retinoids promote the generation of a pro-reparative M2 macrophage population during monocyte differentiation.

Involvement of phosphatidylserine receptors in the skeletal muscle regeneration: therapeutic implications.

Sarcopenia is a progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life, and death. Increasing evidence indicates that diminished ability of the muscle to activate satellite cell-dependent regeneration is one of the factors that might contribute to its development. Skeletal muscle regeneration following myogenic cell death results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibres. Satellite cell differentiation is not a satellite cell-autonomous process but depends on signals provided by the surrounding cells. Infiltrating macrophages play a key role in the process partly by clearing the necrotic cell debris, partly by producing cytokines and growth factors that guide myogenesis. At the beginning of the muscle regeneration process, macrophages are pro-inflammatory, and the cytokines produced by them trigger the proliferation and differentiation of satellite cells. Following the uptake of dead cells, however, a transcriptionally regulated phenotypic change (macrophage polarization) is induced in them resulting in their transformation into healing macrophages that guide resolution of inflammation, completion of myoblast differentiation, myoblast fusion and growth, and return to homeostasis. Impaired efferocytosis results in delayed cell death clearance, delayed macrophage polarization, prolonged inflammation, and impaired muscle regeneration. Thus, proper efferocytosis by macrophages is a determining factor during muscle repair. Here we review that both efferocytosis and myogenesis are dependent on the cell surface phosphatidylserine (PS), and surprisingly, these two processes share a number of common PS receptors and signalling pathways. Based on these findings, we propose that stimulating the function of PS receptors for facilitating muscle repair following injury could be a successful approach, as it would enhance efferocytosis and myogenesis simultaneously. Because increasing evidence indicates a pathophysiological role of impaired efferocytosis in the development of chronic inflammatory conditions, as well as in impaired muscle regeneration both contributing to the development of sarcopenia, improving efferocytosis should be considered also in its management. Again applying or combining those treatments that target PS receptors would be expected to be the most effective, because they would also promote myogenesis. A potential PS receptor-triggering candidate molecule is milk fat globule-EGF-factor 8 (MFG-E8), which not only stimulates PS-dependent efferocytosis and myoblast fusion but also promotes extracellular signal-regulated kinase (ERK) and Akt activation-mediated cell proliferation and cell cycle progression in myoblasts.

Regenerating Skeletal Muscle Compensates for the Impaired Macrophage Functions Leading to Normal Muscle Repair in Retinol Saturase Null Mice.

Skeletal muscle repair is initiated by local inflammation and involves the engulfment of dead cells (efferocytosis) by infiltrating macrophages at the injury site. Macrophages orchestrate the whole repair program, and efferocytosis is a key event not only for cell clearance but also for triggering the timed polarization of the inflammatory phenotype of macrophages into the healing one. While pro-inflammatory cytokines produced by the inflammatory macrophages induce satellite cell proliferation and differentiation into myoblasts, healing macrophages initiate the resolution of inflammation, angiogenesis, and extracellular matrix formation and drive myoblast fusion and myotube growth. Therefore, improper efferocytosis results in impaired muscle repair. Retinol saturase (RetSat) initiates the formation of various dihydroretinoids, a group of vitamin A derivatives that regulate transcription by activating retinoid receptors. Previous studies from our laboratory have shown that RetSat-null macrophages produce less milk fat globule-epidermal growth factor-factor-8 (MFG-E8), lack neuropeptide Y expression, and are characterized by impaired efferocytosis. Here, we investigated skeletal muscle repair in the tibialis anterior muscle of RetSat-null mice following cardiotoxin injury. Our data presented here demonstrate that, unexpectedly, several cell types participating in skeletal muscle regeneration compensate for the impaired macrophage functions, resulting in normal muscle repair in the RetSat-null mice.

Impaired Skeletal Muscle Development and Regeneration in Transglutaminase 2 Knockout Mice.

Skeletal muscle regeneration is triggered by local inflammation and is accompanied by phagocytosis of dead cells at the injury site. Efferocytosis regulates the inflammatory program in macrophages by initiating the conversion of their inflammatory phenotype into the healing one. While pro-inflammatory cytokines induce satellite cell proliferation and differentiation into myoblasts, growth factors, such as GDF3, released by healing macrophages drive myoblast fusion and myotube growth. Therefore, improper efferocytosis may lead to impaired muscle regeneration. Transglutaminase 2 (TG2) is a versatile enzyme participating in efferocytosis. Here, we show that TG2 ablation did not alter the skeletal muscle weights or sizes but led to the generation of small size myofibers and to decreased grip force in TG2 null mice. Following cardiotoxin-induced injury, the size of regenerating fibers was smaller, and the myoblast fusion was delayed in the tibialis anterior muscle of TG2 null mice. Loss of TG2 did not affect the efferocytic capacity of muscle macrophages but delayed their conversion to Ly6C-CD206+, GDF3 expressing cells. Finally, TG2 promoted myoblast fusion in differentiating C2C12 myoblasts. These results indicate that TG2 expressed by both macrophages and myoblasts contributes to proper myoblast fusion, and its ablation leads to impaired muscle development and regeneration in mice.

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice.

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer-/- mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.

Heme Oxygenase-1 Contributes to Both the Engulfment and the Anti-Inflammatory Program of Macrophages during Efferocytosis.

Heme oxygenase-1 (HO-1) plays a vital role in the catabolism of heme and yields equimolar amounts of biliverdin, carbon monoxide, and free iron. We report that macrophages engulfing either the low amount of heme-containing apoptotic thymocytes or the high amount of heme-containing eryptotic red blood cells (eRBCs) strongly upregulate HO-1. The induction by apoptotic thymocytes is dependent on soluble signals, which do not include adenylate cyclase activators but induce the p38 mitogen-activated protein (MAP) kinase pathway, while in the case of eRBCs, it is cell uptake-dependent. Both pathways might involve the regulation of BTB and CNC homology 1 (BACH1), which is the repressor transcription regulator factor of the HO-1 gene. Long-term continuous efferocytosis of apoptotic thymocytes is not affected by the loss of HO-1, but that of eRBCs is inhibited. This latter is related to an internal signaling pathway that prevents the efferocytosis-induced increase in Rac1 activity. While the uptake of apoptotic cells suppressed the basal pro-inflammatory cytokine production in wild-type macrophages, in the absence of HO-1, engulfing macrophages produced enhanced amounts of pro-inflammatory cytokines. Our data demonstrate that HO-1 is required for both the engulfment and the anti-inflammatory response parts of the efferocytosis program.

Retinol Saturase Knock-Out Mice are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity.

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvß3/ß5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Loss of transglutaminase 2 sensitizes for diet-induced obesity-related inflammation and insulin resistance due to enhanced macrophage c-Src signaling.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes clearance of apoptotic cells (efferocytosis) acting as integrin ß3 coreceptor. Accumulating evidence indicates that defective efferocytosis contributes to the development of chronic inflammatory diseases. Obesity is characterized by the accumulation of dead adipocytes and inflammatory macrophages in the adipose tissue leading to obesity-related metabolic syndrome. Here, we report that loss of TG2 from bone marrow-derived cells sensitizes for high fat diet (HFD)-induced pathologies. We find that metabolically activated TG2 null macrophages express more phospho-Src and integrin ß3, unexpectedly clear dying adipocytes more efficiently via lysosomal exocytosis, but produce more pro-inflammatory cytokines than the wild type ones. Anti-inflammatory treatment with an LXR agonist reverts the HFD-induced phenotype in mice lacking TG2 in bone marrow-derived cells with less hepatic steatosis than in wild type mice proving enhanced lipid clearance. Thus it is interesting to speculate whether LXR agonist treatment together with enhancing lysosomal exocytosis could be a beneficial therapeutic strategy in obesity.

Macrophages engulf apoptotic and primary necrotic thymocytes through similar phosphatidylserine-dependent mechanisms.

One of the major roles of professional phagocytes is the removal of dead cells in the body. We know less about the clearance of necrotic cells than apoptotic cell phagocytosis, despite the fact that both types of dead cells need to be cleared together and necrotic cells appear often in pathological settings. In the present study, we examined phagocytosis of heat- or H2O2-killed necrotic and apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs) in vitro and found that the two cell types are engulfed at equal efficiency and compete with each other when added together to BMDMs. Phagocytosis of both apoptotic and necrotic thymocytes was decreased by (a) blocking phosphatidylserine on the surface of dying cells; (b) inhibition of Mer tyrosine kinase, Tim-4, integrin ß3 receptor signaling, or Ras-related C3 botulinum toxin substrate 1 activity; or (c) using BMDMs deficient for transglutaminase 2. Stimulation of liver X, retinoid X, retinoic acid or glucocorticoid nuclear receptors in BMDMs enhanced not only apoptotic, but also necrotic cell uptake. Electron microscopic analysis of the engulfment process revealed that the morphology of phagosomes and the phagocytic cup formed during the uptake of dying thymocytes is similar for apoptotic and necrotic cells. Our data indicate that apoptotic and necrotic cells are cleared via the same mechanisms, and removal of necrotic cells in vivo can be facilitated by molecules known to enhance the uptake of apoptotic cells.

Transglutaminase 2 in human diseases.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. In addition to being an enzyme, TG2 also serves as a G protein for several seven transmembrane receptors and acts as a co-receptor for integrin ß1 and ß3 integrins distinguishing it from other members of the transglutaminase family. TG2 is ubiquitously expressed in almost all cell types and all cell compartments, and is also present on the cell surface and gets secreted to the extracellular matrix via non-classical mechanisms. TG2 has been associated with various human diseases including inflammation, cancer, fibrosis, cardiovascular disease, neurodegenerative diseases, celiac disease in which it plays either a protective role, or contributes to the pathogenesis. Thus modulating the biological activities of TG2 in these diseases will have a therapeutic value.

Anti-inflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance.

In the human body, billions of cells die by apoptosis every day. The subsequent clearance of apoptotic cells by phagocytosis is normally efficient enough to prevent secondary necrosis and the consequent release of cell contents that would induce inflammation and trigger autoimmunity. In addition, apoptotic cells generally induce an anti-inflammatory response, thus removal of apoptotic cells is usually immunologically silent. Since the first discovery that uptake of apoptotic cells leads to transforming growth factor (TGF)-ß and interleukin (IL)-10 release by engulfing macrophages, numerous anti-inflammatory mechanisms triggered by apoptotic cells have been discovered, including release of anti-inflammatory molecules from the apoptotic cells, triggering immediate anti-inflammatory signaling pathways by apoptotic cell surface molecules via phagocyte receptors, activating phagocyte nuclear receptors following uptake and inducing the production of anti-inflammatory soluble mediators by phagocytes that may act via paracrine or autocrine mechanisms to amplify and preserve the anti-inflammatory state. Here, we summarize our present knowledge about how these anti-inflammatory mechanisms operate during the clearance of apoptotic cells.

Involvement of adenosine A3 receptors in the chemotactic navigation of macrophages towards apoptotic cells.

The first step in the clearance of apoptotic cells is chemotactic migration of macrophages towards the apoptotic cells guided by find-me signals provided by the dying cells. Upon sensing the chemotactic signals, macrophages release ATP. ATP is then degraded to ADP, AMP and adenosine to trigger purinergic receptors concentrated at the leading edge of the cell. Previous studies have shown that in addition to the chemotactic signals, this purinergic autocrine signaling is required to amplify and translate chemotactic signals into directional motility. In the present study the involvement of adenosine A3 receptors (A3R) was studied in the chemotactic migration of macrophages directed by apoptotic thymocyte-derived find-me signals. By taking video images in vitro, we demonstrate 1, by administering apyrase, which degrades ATP and ADP, that the purinergic autocrine signaling is required for maintaining both the velocity and the directionality of macrophage migration towards the apoptotic thymocytes; 2, by readding 5'-N-ethylcarboxamidoadenosine, an adenosine analogue, to apyrase treated cells that the adenosine receptor signaling alone is sufficient to act so; and 3, by studying migration of various adenosine receptor null or adenosine receptor antagonist-treated macrophages, that the individual loss of the A3R signaling leads to the loss of chemotactic navigation. Though loss of A3Rs does not affect the phagocytotic capacity of macrophages, intraperitoneally-injected apoptotic thymocytes were cleared with a delayed kinetics by A3R null macrophages in vivo due to the impaired chemotactic navigation. All together these data demonstrate the involvement of macrophage A3Rs in the proper chemotactic navigation and consequent in vivo clearance of apoptotic cells. Interestingly, loss of A3Rs did not affect the in vivo clearance of apoptotic thymocytes in the dexamethasone-treated thymus.

Adenosine produced from adenine nucleotides through an interaction between apoptotic cells and engulfing macrophages contributes to the appearance of transglutaminase 2 in dying thymocytes.

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-ß (TGF-ß) and retinoids, are produced by macrophages engulfing apoptotic cells. However, in addition to TGF-ß and retinoids, engulfing macrophages produce adenosine as well. Here, we show that in vitro adenosine, adenosine, and retinoic acid or adenosine, TGF-ß 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.

Adenosine in the Thymus.

Adenosine is an ancient extracellular signaling molecule that regulates various biological functions via activating four G-protein-coupled receptors, A1, A2A, A2B, and A3 adenosine receptors. As such, several studies have highlighted a role for adenosine signaling in affecting the T cell development in the thymus. Recent studies indicate that adenosine is produced in the context of apoptotic thymocyte clearance. This review critically discusses the involvement of adenosine and its receptors in the complex interplay that exists between the developing thymocytes and the thymic macrophages which engulf the apoptotic cells. This crosstalk contributes to the effective and immunologically silent removal of apoptotic thymocytes, as well as affects the TCR-driven T-cell selection processes.

Transmembrane TNF-alpha reverse signaling leading to TGF-beta production is selectively activated by TNF targeting molecules: Therapeutic implications.

Tumor necrosis factor (TNF)-α is a potent pro-inflammatory cytokine exerting pleiotropic effects on various cell types. It is synthesized in a precursor form called transmembrane TNF-α (mTNF-α) which, after being processed by metalloproteinases, is released in a soluble form to mediate its biological activities through Type 1 and 2 TNF receptors in TNF receptor expressing cells. In addition to acting in soluble form, TNF-α also acts in the transmembrane form both as a ligand by activating TNF receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into mTNF-α bearing cells. Since the discovery that TNF-α plays a determining role in the pathogenesis of several chronic inflammatory diseases, anti-TNF agents are increasingly being used in the treatment of a rapidly expanding number of rheumatic and systemic autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, ankyloting spondylitis, Wegener granulomatosis and sarcoidosis. There are 5 TNF antagonists currently available: etanercept, a soluble TNF receptor construct; infliximab, a chimeric monoclonal antibody; adalimumab and golimumab, fully human antibodies; and certolizumab pegol, an Fab' fragment of a humanized anti-TNF-α antibody. Though each compound can efficiently neutralize TNF-α, increasing evidence suggests that they show different efficacy in the treatment of these diseases. These observations indicate that in addition to neutralizing TNF-α, other biological effects induced by TNF-α targeting molecules dictate the success of the therapy. Recently, we found that mTNF-α reverse signaling leads to transforming growth factor (TGF)-ß production in macrophages and anti-TNF agents selectively trigger this pathway. In this review we will focus on the potential contribution of the activation of the mTNF-α signaling pathway to the success of the anti-TNF therapy.