Attila Pap

STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells.

Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells.

Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor’s activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner.

Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

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 activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors

BackgroundPeroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs).MethodsPACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 μmol/L) or GW9662 (10 μmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model.ResultsECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells.Transcriptome analysis showed an upregulation of prooxidative and proinflammatory pathways, and downregulation of several proangiogenic genes in db/db PACs. Interestingly, db/db PACs had also a decreased level of PPARγ and changed expression of PPARγ-regulated genes. Using normoglycemic PPARγ+/- mice we demonstrated that reduced expression of PPARγ does not influence neovascularization either in wound healing or in hind limb ischemia models.ConclusionsIn summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET‐MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration.

RDH10, RALDH2, and CRABP2 are required components of PPARγ-directed ATRA synthesis and signaling in human dendritic cells

All-trans retinoic acid (ATRA) has a key role in dendritic cells (DCs) and affects T cell subtype specification and gut homing. However, the identity of the permissive cell types and the required steps of conversion of vitamin A to biologically active ATRA bringing about retinoic acid receptor-regulated signaling remains elusive. Here we present that only a subset of murine and human DCs express the necessary enzymes, including RDH10, RALDH2, and transporter cellular retinoic acid binding protein (CRABP)2, to produce ATRA and efficient signaling. These permissive cell types include CD103+ DCs, granulocyte-macrophage colony-stimulating factor, and interleukin-4-treated bone marrow-derived murine DCs and human monocyte-derived DCs (mo-DCs). Importantly, in addition to RDH10 and RALDH2, CRABP2 also appears to be regulated by the fatty acid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) and colocalize in human gut-associated lymphoid tissue DCs. In our model of human mo-DCs, all three proteins (RDH10, RALDH2, and CRABP2) appeared to be required for ATRA production induced by activation of PPARγ and therefore form a linear pathway. This now functionally validated PPARγ-regulated ATRA producing and signaling axis equips the cells with the capacity to convert precursors to active retinoids in response to receptor-activating fatty acids and is potentially amenable to intervention in diseases involving or affecting mucosal immunity.

Is the mouse a good model of human PPARγ-related metabolic diseases?

With the increasing number of patients affected with metabolic diseases such as type 2 diabetes, obesity, atherosclerosis and insulin resistance, academic researchers and pharmaceutical companies are eager to better understand metabolic syndrome and develop new drugs for its treatment. Many studies have focused on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a crucial role in adipogenesis and lipid metabolism. These studies have been able to connect this transcription factor to several human metabolic diseases. Due to obvious limitations concerning experimentation in humans, animal models—mainly mouse models—have been generated to investigate the role of PPARγ in different tissues. This review focuses on the metabolic features of human and mouse PPARγ-related diseases and the utility of the mouse as a model.

Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism

Significance Metastasis formation from malignant tumors is the leading cause of cancer-related deaths. There is an increasing body of evidence indicating that immune cells in distant organs actively contribute to this process by establishing a tissue environment that is hospitable for cancer cells. In this study, we show that deletion of retinoid X receptor (RXR), a cellular sensor of vitamin A metabolites, specifically in the myeloid lineage of the immune system, leads to an enhanced metastasis rate. We also demonstrate that RXR inhibits the expression of a number of genes that encode proteins involved in the promotion of metastasis formation. Surprisingly, our results suggest that this activity of RXR is independent of the presence of its activators. Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3

Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell–derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell–derived DCs (ES-DCs) represented less mature cells compared with bone marrow–derived DCs. This finding prompted us to compare the gene expression profile of the ES cell– and adult progenitor-derived, GM-CSF–instructed, nonconventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow–derived DCs. In light of this altered gene expression, we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo–generated ES-DCs had a compromised maturation ability and immunogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells.