Gyöngyike Majai

PPARγ‐dependent regulation of human macrophages in phagocytosis of apoptotic cells

Macrophages acquire their capacity for efficient phagocytosis of apoptotic cells during their differentiation from monocytes. The peroxisome proliferator‐activated receptor gamma (PPARγ) is highly up‐regulated during this maturation program. We report that addition of PPARγ antagonist during differentiation of human monocytes to macrophages significantly reduced the capacity of macrophages to engulf apoptotic neutrophils, but did not influence phagocytosis of opsonized bacteria. Macrophage‐specific deletion of PPARγ in mice also resulted in decreased uptake of apoptotic cells. The antagonist acted in a dose‐dependent manner during the differentiation of human macrophages and could also reverse the previously observed augmentation of phagocytosis by glucocorticoids. Blocking activation of PPARγ led to down‐regulation of molecular elements (CD36, AXL, TG2 and PTX3) of the engulfment process. Inhibition of PPARγ‐dependent gene expression did not block the anti‐inflammatory effect of apoptotic neutrophils or synthetic glucocorticoid, but significantly decreased production of IL‐10 induced by LPS. Our results suggest that during differentiation of macrophages natural ligands of PPARγ are formed, regulating the expression of genes responsible for effective clearance of apoptotic cells and macrophage‐mediated inflammatory responses.

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages.

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K+ efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K+ efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.

Differentiation and Glucocorticoid Regulated Apopto-Phagocytic Gene Expression Patterns in Human Macrophages. Role of Mertk in Enhanced Phagocytosis

The daily clearance of physiologically dying cells is performed safely mainly by cells in the mononuclear phagocyte system. They can recognize and engulf dying cells utilizing several cooperative mechanisms. In our study we show that the expression of a broad range of apopto-phagocytic genes is strongly up-regulated during differentiation of human monocytes to macrophages with different donor variability. The glucocorticoid dexamethasone has a profound effect on this process by selectively up-regulating six genes and down-regulating several others. The key role of the up-regulated mer tyrosine kinase (Mertk) in dexamethasone induced enhancement of phagocytosis could be demonstrated in human monocyte derived macrophages by gene silencing as well as blocking antibodies, and also in a monocyte-macrophage like cell line. However, the additional role of other glucocorticoid induced elements must be also considered since the presence of autologous serum during phagocytosis could almost completely compensate for the blocked function of Mertk.

Transglutaminase 2 is expressed and active on the surface of human monocyte-derived dendritic cells and macrophages

The multifunctional enzyme, transglutaminase 2 (TG2), can be found intracellularly, in the extracellular matrix and on the cell surface. Cell surface TG2 (csTG2) could not be detected by TG2-specific antibodies or autoantibodies on immunocompetent cells. A supposedly csTG2-specific antibody, 6B9, was recently shown to actually react with CD44. Though the importance of TG2-mediated deamidation of gluten in the pathogenesis of celiac disease has been well recognized, it is not known in which intestinal cells or cell compartment the deamidation occurs. Duodenal dendritic cells (DCs) can be directly involved in gluten-reactive T-cell activation. Here we use blood monocyte-derived dendritic cells (iDC) and macrophages (MPhi) as a model for intestinal antigen-presenting cells (APCs) and show that they contain large amounts of TG2. We found that TG100, a commercial TG2-specific monoclonal antibody can recognize TG2 on the surface of these cells, that is monocyte-derived APCs express surface-associated TG2. TG2 expression was found on the surface of individual tunica propria cells in frozen small bowel tissue sections from both normal and celiac subjects. We also demonstrate that the pool of TG2 on the surface of iDCs can be catalytically active, hence it might directly be involved in the deamidation of gliadin peptides. Bacterial lipopolysaccharide (LPS) increased the level of TG2 on the surface of maturing DCs, supporting the hypothesis that an unspecific inflammatory process in the gut may expose more transglutaminase activity.

The glucocorticoid dexamethasone programs human dendritic cells for enhanced phagocytosis of apoptotic neutrophils and inflammatory response

GCs are powerful anti‐inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte‐derived human DCs develop their capacity to engulf apoptotic cells by up‐regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow‐derived DCs of ADORA3 KO mice were treated with Dex. The GC‐treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN‐γ, the inflammatory cytokine TNF‐α. Furthermore, the Dex‐treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.

PPARγ modulated inflammatory response of human dendritic cell subsets to engulfed apoptotic neutrophils.

The means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by human monocyte‐derived DCs is slow and less efficient than that of macrophages, and CD1a– DCs are more active in the engulfment of apoptotic neutrophils than CD1a+ DCs. Blocking DC‐SIGN function partially interferes with the uptake of apoptotic cells, and long‐term interaction of apoptotic neutrophils with DCs makes them prone to proinflammatory cytokine responses. Engulfment of apoptotic cells sensitizes CD1a– DCs for high IL‐8, TNF‐α, IL‐6, and CD1a+ cells for IL‐12 and IL‐10 cytokine secretion elicited by additional inflammatory stimuli, which also result in the polarization of autologous T lymphocytes to Th1 effector cells. Ligand‐induced activation of PPARγ by RSG results in enhanced phagocytosis, but the proinflammatory response and the capacity to trigger Th1 cell activation of CD1a– DCs are not enhanced. These results demonstrate that DCs are able to respond to allogeneic, apoptotic neutrophils with inflammatory cytokines and T cell responses in a subtype‐specific manner that is modulated by the anti‐inflammatory effects of PPARγ.

Altered sialylation on the cell-surface proteins of dexamethasone-treated human macrophages contributes to augmented uptake of apoptotic neutrophils.

Macrophages eliminate apoptotic granulocytes before their secondary necrosis during resolution of inflammation. A well-known glucocorticoid, the anti-inflammatory dexamethasone augments phagocytosis capacity of macrophages with a so far not fully clarified mechanism. We have found that sialylation of cell-surface proteins on human macrophages is markedly altered by dexamethasone. Compared to non-treated cells, dexamethasone-treated macrophages can bind significantly less Sambucus nigra lectin specific for sialic acids on their surfaces as a result of undersialylation of annexin-II and an HLA-II protein. Non-treated macrophages covered by S. nigra lectin had increased uptake of apoptotic cells; however, the significantly higher phagocytosis capacity of dexamethasone-treated macrophages could not be stimulated further this way. Our results suggest that dexamethasone treatment leads to decreased number of sialic acids on the surfaces of human macrophages promoting recognition and uptake of apoptotic cells.