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.

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.

Transmembrane TNF-α Reverse Signaling Inhibits Lipopolysaccharide-Induced Proinflammatory Cytokine Formation in Macrophages by Inducing TGF-ß: Therapeutic Implications.

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-ß. Based on inhibitory experiments, the production of TGF-ß1 is regulated via Jun kinases, whereas that of other TGF-ßs 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-ß 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-ß. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-ß, we show that they do not use the mTNF-α signaling pathway. Because TGF-ß possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-ß 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.

Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1.

Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

Transcriptional control of transglutaminase 2 expression in mouse apoptotic thymocytes.

Transglutaminase 2 (TGM2) is a ubiquitously expressed multifunctional protein, which participates in various biological processes including thymocyte apoptosis. As a result, the transcriptional regulation of the gene is complex and must depend on the cell type. Previous studies from our laboratory have shown that in dying thymocytes the expression of Tgm2 is induced by external signals derived from engulfing macrophages, such as retinoids, transforming growth factor (TGF)-ß and adenosine, the latter triggering the adenylate cyclase signaling pathway. The existence of TGF-ß and retinoid responsive elements in the promoter region of Tgm2 has already been reported, but the intergenic regulatory elements participating in the regulation of Tgm2 have not yet been identified. Here we used publicly available results from DNase I hypersensitivity analysis followed by deep sequencing and chromatin immunoprecipitation followed by deep sequencing against CCCTC-binding factor (CTCF), H3K4me3, H3K4me1 and H3K27ac to map a putative regulatory element set for Tgm2 in thymocytes. By measuring eRNA expressions of these putative enhancers in retinoid, rTGF-ß or dibutiryl cAMP-exposed thymocytes we determined which of them are functional. By applying ChIP-qPCR against SMAD4, retinoic acid receptor, retinoid X receptor, cAMP response element binding protein, P300 and H3K27ac under the same conditions, we identified two enhancers of Tgm2, which seem to act as integrators of the TGF-ß, retinoid and adenylate cyclase signaling pathways in dying thymocytes. Our study describes a novel strategy to identify and characterize the signal-specific functional enhancer set of a gene by integrating genome-wide datasets and measuring the production of enhancer specific RNA molecules.

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.

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.

Glucocorticoids enhance prolonged clearance of apoptotic cells by upregulating liver X receptor, peroxisome proliferator-activated receptor-δ and UCP2.

Efficient phagocytic clearance of apoptotic cells (efferocytosis) is essential to prevent the development of chronic inflammation and autoimmunity. Glucocorticoids are widely used in the therapy of chronic inflammatory diseases, and increasing evidence suggests that they act partly via enhancing efferocytosis by macrophages. Glucocorticoids were previously shown to promote both protein S- and MFG-E8-dependent efferocytosis. Since previous studies in our laboratory have demonstrated that glucocorticoids induce the expression of retinaldehyde dehydrogenases in macrophages, in the present experiments the possible involvement of retinoids in the glucocorticoid-induced efferocytosis was studied in mouse bone marrow derived macrophages. Here we show that glucocorticoids promote not only short-term, but also long-term clearance of apoptotic cells. Glucocorticoids seem to directly induce the expression of the phagocytosis-related genes MERTK, C1q, UCP2, and the transcription factor C/EBPß. C/EBPß contributes to the further induction of the phagocytosis-related genes, and is required for the induction of lipid sensing receptors LXRs, PPARδ, RARα, RXRα and RALDH1, the latter one in an LXR- and RARα-dependent manner. Glucocorticoid-induced enhancement in long-term efferocytosis was dependent on the induction of lipid sensing receptors known to be triggered by the lipid content of the engulfed cells to enhance phagocytic capacity. Retinoids did not affect the glucocorticoid-induced short term phagocytosis of apoptotic cells, but were required for the glucocorticoid-induced enhancement of efferocytosis during prolonged clearance of apoptotic cells by promoting efficient LXR and PPARδ upregulation. Our data indicate that retinoids could be considered as potential promoters of the efficacy of glucocorticoid treatment in inflammatory diseases.

Retinoids induce Nur77-dependent apoptosis in mouse thymocytes.

Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis.

Macrophages engulfing apoptotic cells produce nonclassical retinoids to enhance their phagocytic capacity.

Previous work in our laboratory has shown that transglutaminase 2 (TG2) acting as a coreceptor for integrin ß3 is required for proper phagocytosis of apoptotic cells. In the absence of TG2, systemic lupus erythematosus-like autoimmunity develops in mice, similarly to other mice characterized by a deficiency in the clearance of apoptotic cells. In this study, we demonstrate that increasing TG2 expression alone in wild-type macrophages is not sufficient to enhance engulfment. However, during engulfment, the lipid content of the apoptotic cells triggers the lipid-sensing receptor liver X receptor (LXR), which in response upregulates the expression of the phagocytic receptor Mer tyrosine kinase and the phagocytosis-related ABCA1, and that of retinaldehyde dehydrogenases leading to the synthesis of a nonclassical retinoid. Based on our retinoid analysis, this compound might be a dihydro-retinoic acid derivative. The novel retinoid then contributes to the upregulation of further phagocytic receptors including TG2 by ligating retinoic acid receptors. Inhibition of retinoid synthesis prevents the enhanced phagocytic uptake induced by LXR ligation. Our data indicate that stimulation of LXR enhances the engulfment of apoptotic cells via regulating directly and indirectly the expression of a range of phagocytosis-related molecules, and its signaling pathway involves the synthesis of a nonclassical retinoid. We propose that retinoids could be used for enhancing the phagocytic capacity of macrophages in diseases such as systemic lupus erythematosus, where impaired phagocytosis of apoptotic cells plays a role in the pathogenesis of the disease.

Thymocyte death by neglect: contribution of engulfing macrophages.

The thymus provides the microenvironment in which thymocytes develop into mature T cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize the peptide-loaded self-MHC molecules found in the thymus, and therefore lack a TCR signal, undergo a default death pathway named "death by neglect" in the thymic cortex. In the absence of this TCR signaling, it has been suggested that binding of glucocorticoids to - or the ligation of certain cell surface molecules, such as CD8, CD24, CD45, or CD99 on - these neglected thymocytes will induce them to enter the apoptotic program. Apoptotic thymocytes are cleared by the surrounding macrophages and, as a consequence, these macrophages are known to release various molecules, such as adenosine, retinoids, TGF-ß, ATP, and carbon monoxide. Interestingly, all these molecules have been described to induce or promote apoptosis in thymocytes in the absence of TCR signaling. Here, we propose that thymic macrophages, because they continually engulf apoptotic cells, might constantly provide these cell death-inducing signals, and thus contribute to the formation of a thymic milieu that ensures the effective induction of "death by neglect".

Retinoids produced by macrophages engulfing apoptotic cells contribute to the appearance of transglutaminase 2 in apoptotic 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 was strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 could be detected, when thymocytes were induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the in vivo induction of the enzyme in apoptotic thymocytes. Previous studies have shown that one of these signals is transforming growth factor-ß (TGF-ß) which is released by macrophages engulfing apoptotic cells. Besides TGF-ß, the TG2 promoter contains retinoic acid response elements as well. Here we show that in vitro retinoic acids, or TGF-ß and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes, and the apoptotic signal contributes to the TG2 induction. Inhibition of retinoic acid synthesis either by alcohol or retinaldehyde dehydrogenases significantly attenuates the in vivo induction of TG2 following apoptosis induction indicating that retinoids indeed might contribute in vivo to the apoptosis-related TG2 expression. What is more, the in vivo apoptosis induction in the thymus is accompanied by an enhanced retinoid-dependent transcriptional activity due to the enhanced retinoid synthesis by macrophages engulfing apoptotic cells. Our data reveal a new crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-induced retinoid synthesis in macrophages enhances TG2 expression in the dying thymocytes.

Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation.

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 proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) 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, as a result of possible 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.

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.

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.

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.

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.

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.

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.