TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content

BackgroundTNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known.MethodsPrimary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients.ResultsTRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content.ConclusionsTRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization

Astragalus Saponins, Astragaloside VII and Newly Synthesized Derivatives, Induce Dendritic Cell Maturation and T Cell Activation

Astragaloside VII (AST VII), a triterpenic saponin isolated from Astragalus species, shows promise as a vaccine adjuvant, as it supported a balanced Th1/Th2 immune response in previous in vivo studies. However, the underlying mechanisms of its adjuvant activity have not been defined. Here, we investigated the impact of AST VII and its newly synthesized semi-synthetic analogs on human whole blood cells, as well as on mouse bone marrow-derived dendritic cells (BMDCs). Cells were stimulated with AST VII and its derivatives in the presence or absence of LPS or PMA/ionomycin and the secretion of cytokines and the expression of activation markers were analyzed using ELISA and flow cytometry, respectively. AST VII and its analogs increased the production of IL-1β in PMA/ionomycin-stimulated human whole blood cells. In LPS-treated mouse BMDCs, AST VII increased the production of IL-1β and IL-12, and the expression of MHC II, CD86, and CD80. In mixed leukocyte reaction, AST VII and derivatives increased the expression of the activation marker CD44 on mouse CD4+ and CD8+ T cells. In conclusion, AST VII and its derivatives strengthen pro-inflammatory responses and support dendritic cell maturation and T cell activation in vitro. Our results provide insights into the mechanisms of the adjuvant activities of AST VII and its analogs, which will be instrumental to improve their utility as a vaccine adjuvant

Improved Detection of Cytokines Produced by Invariant NKT Cells

Abstract Invariant Natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after in vivo activation, allowing for the direct detection of a number of cytokines directly ex vivo. However, for some cytokines this approach is suboptimal. Here, we report technical variations that allow the improved detection of IL-4, IL-10, IL-13 and IL-17A ex vivo. Furthermore, we describe an alternative approach for stimulation of iNKT cells in vitro that allows a significantly improved detection of cytokines produced by iNKT cells. Together, these protocols allow the detection of iNKT cell cytokines ex vivo and in vitro with increased sensitivity

Extracellular adenosine regulates naive T cell development and peripheral maintenance

Adenosine produced as a byproduct of metabolic activity is present in all tissues and produces dose-dependent suppression of TCR signaling. Naive T cell maintenance depends on inhibition of TCR signals by environmental sensors, which are yet to be fully defined. We produced mice with a floxed adenosine A(2A) receptor (A(2A)R) gene, Adora2a, and show that either global A(2A)R deletion or cre-mediated T cell deletion elicits a decline in the number of naive but not memory T cells. A(2A)R signaling maintains naive T cells in a quiescent state by inhibiting TCR-induced activation of the phosphatidylinositide 3-kinase (PI3K)-AKT pathway, thereby reducing IL-7R alpha down-regulation and naive T cell apoptosis. Patterns of IL-7R alpha expression on T cells in chimeric mice reconstituted with Adora2a(+/+) and Adora2a(-/-) bone marrow cells suggest that decreased IL-7R alpha in naive T cells is a cell-intrinsic consequence of Adora2a deletion. In addition, A(2A)R expression increases in early thymic T cell development and contributes to progression of double-negative thymic precursors to single-positive thymocytes with increased IL-7R alpha expression. Therefore, A(2A)R signaling regulates T cell development and maintenance to sustain normal numbers of naive T cells in the periphery

Adenosine 5 '-Monophosphate-Activated Protein Kinase Promotes Macrophage Polarization to an Anti-Inflammatory Functional Phenotype

Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytolkines (i.e., IL-10 and TGF beta) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPK alpha expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-alpha, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPK alpha 1 in macrophages enhanced TNF-alpha and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPK alpha 1 resulted in decreased LPS-induced TNF-alpha and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced I kappa B-alpha degradation and positively regulated Akt activation, accompanied by inhibition or glycogen synthase kinase beta and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype. The Journal of Immunology, 2008, 181: 8633-8641

The Role of TRAIL/DRs in the Modulation of Immune Cells and Responses

Expression of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by immune cells can lead to the induction of apoptosis in tumor cells. However, it becomes increasingly clear that the interaction of TRAIL and its death receptors (DRs) can also directly impact immune cells and influence immune responses. Here, we review what is known about the role of TRAIL/DRs in immune cells and immune responses in general and in the tumor microenvironment in particular