[Show abstract][Hide abstract] ABSTRACT: Under inflammatory conditions or during tumor progression macrophages acquire distinct phenotypes, with factors of the microenvironment such as hypoxia and transforming growth factor β (TGFβ) shaping their functional plasticity. TGFβ is among the factors causing alternative macrophage activation, which contributes to tissue regeneration and thus, resolution of inflammation but may also provoke tumor progression. However, the signal crosstalk between TGFβ and hypoxia is ill defined.
Exposing human primary macrophages to TGFβ elicited a rapid SMAD2/SMAD3 phosphorylation. This early TGFβ-signaling remained unaffected by hypoxia. However, with prolonged exposure periods to TGFβ/hypoxia the expression of SMAD2 declined because of decreased protein stability. In parallel, hypoxia increased mRNA and protein amount of the calpain regulatory subunit, with the further notion that TGFβ/hypoxia elicited calpain activation. The dual specific proteasome/calpain inhibitor MG132 and the specific calpain inhibitor 1 rescued SMAD2 degradation, substantiating the ability of calpain to degrade SMAD2. Decreased SMAD2 expression reduced TGFβ transcriptional activity of its target genes thrombospondin 1, dystonin, and matrix metalloproteinase 2.
Hypoxia interferes with TGFβ signaling in macrophages by calpain-mediated proteolysis of the central signaling component SMAD2.
Cell and Bioscience 07/2015; 5(1):36. DOI:10.1186/s13578-015-0026-x · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.
PLoS ONE 06/2015; 10(6):e0130893. DOI:10.1371/journal.pone.0130893 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1-/- PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1-/- macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.
[Show abstract][Hide abstract] ABSTRACT: Tumor-associated lymphangiogenesis was identified as an important clinical determinant for the prognosis of HCC and significantly influences patient survival. However, in this context little is known about regulation of lymphangiogenesis by hypoxia inducible factors. In HCC mainly HIF-1α was positively correlated with lymphatic invasion and metastasis, while a defined role of HIF-2α is missing.
We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies. This constitutes an in vitro tumor model mimicking the cancer microenvironment and allows addressing the role of distinct HIF isoforms in regulating HCC lymphangiogenesis. In cocultures with a HIF-2α knockdown lymphangiogenesis was significantly increased, while the knockdown of HIF-1α showed no effect. The HIF-2α-dependent lymphangiogenic phenotype was confirmed in vivo using matrigel plug assays with supernatants of HIF-2α knockdown HepG2 cells. We identified and verified IGFBP1 as a HIF-2α target gene. The potential of HepG2 cells to induce lymphangiogenesis in two independent functional assays was significantly enhanced either by a knockdown of HIF-2α or by silencing IGFBP1. Moreover, we confirmed IGF as a potent pro-lymphatic growth factor with IGFBP1 being its negative modulator.
We propose that HIF-2α acts as an important negative regulator of hepatic lymphangiogenesis in vitro and in vivo by inducing IGFBP1 and thus, interfering with IGF-signaling. Therefore, HIF-2α may constitute a critical target in HCC therapy. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Biology of the Cell 03/2015; 107(6). DOI:10.1111/boc.201400079 · 3.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species (ROS), recently emerged to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a pro-inflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8+ T cell activation by using an antigen-driven co-culture model consistent of Nrf2-/- and Nrf2+/+ bone marrow derived macrophages (BMDMΦ) and transgenic OT-1 CD8+ T cells. OT-1 CD8+ T cells encode a T cell receptor that specifically recognizes MHCI-presented OVA(257-264) peptide, thereby causing a downstream T cell activation. Interestingly, co-culture of OVA(257-264)-pulsed Nrf2-/- BMDMΦ with transgenic OT-1 CD8+ T cells attenuated CD8+ T cell activation, proliferation, and cytotoxic function, compared to Nrf2+/+ BMDMΦ.Since the provision of low molecular thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8+ T cell activation, we quantified the amount of intracellular and extracellular GSH and Cys in both co-cultures. Indeed, GSH levels were strongly decreased in Nrf2-/- co-cultures compared to wildtype counterparts. Supplementation of thiols in Nrf2-/- co-cultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine importer xCT in Nrf2+/+ BMDMΦ mimicked the effect of Nrf2-/- BMDMΦ on CD8+ T cell function. Our findings demonstrate, that reduced levels of GCLM as well as xCT in Nrf2-/- BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8+ T cell function.
Free Radical Biology and Medicine 02/2015; 83. DOI:10.1016/j.freeradbiomed.2015.02.004 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The sphingolipid sphingosine-1-phosphate (S1P) has various functions in immune cell biology, regulating survival, proliferation, and, most prominently, migration. S1P couples to five G protein-coupled receptors (S1PR1-5) to transduce its effects on immune cell function. Expression of S1PR4 is restricted to immune cells. However, its impact on immune cell biology is largely elusive. In the current study, we intended to answer the question whether S1P might affect plasmacytoid dendritic cell (pDC) migration, which dominantly express S1PR4. pDC are highly specialized cells producing large amounts of type I interferon in response to TLR7/9 ligands after viral infection or during autoimmunity. Surprisingly, we noticed a reduced abundance of pDC, particularly CD4- pDC, in all organs of S1PR4-deficient versus wildtype mice. This effect was not due to altered migration of mature pDC, but rather due to a reduced potential of pDC progenitors, especially common DC progenitors, to differentiate into pDCs. In vitro studies suggested that reduced S1PR4-deficient pDC progenitor differentiation into mature pDC might be explained by both migration and differentiation of pDC progenitors in the bone marrow. Since S1PR4 also affected the differentiation of CD34+ human hematopoietic stem cells into pDC, interfering with S1PR4 might be useful to reduce pDC numbers during autoimmunity.
[Show abstract][Hide abstract] ABSTRACT: Sepsis still emerges as a major cause of patient death in intensive care units. Therefore, new therapeutic approaches are mandatory. Because during sepsis progression cytotoxic T lymphocytes (CTLs) can be activated in an autoimmune fashion contributing to multiorgan damage, it remains unclear whether CTLs are activated toward alloantigenic cells. This is important for patients receiving an immunosuppressive therapy to permit organ transplantation and, thus, known to be at high risk for developing sepsis. Therefore, we analyzed whether sepsis activates CTL toward alloantigenic target cells and whether this can be inhibited by PPARγ activation, known to block T helper cell responses. To mimic septic conditions, CTLs were isolated from cecal ligation and puncture-operated mice. CTL cytotoxicity was analyzed following a direct alloantigenic activation regime or following classical ex vivo splenocyte-driven activation in a cytotoxicity assay. With this readout, we found that CTL derived from septic mice enhanced cytotoxicity toward alloantigenic target cells, which was lowered by in vivo and ex vivo PPARγ activation. With CTL derived from T cell-specific PPARγ knockout mice, PPARγ activation was ineffective, pointing to a PPARγ-dependent mechanism. In vivo and ex vivo PPARγ activation reduced Fas and granzyme B expression in activated CTL.
In the sepsis CLP mouse model, CTLs are activated toward alloantigenic target cells.
Sepsis-mediated alloantigenic CTL activation is blocked in vivo by PPARγ activation.
PPARγ deletion or antagonization restored rosiglitazone-dependent inhibition of CTL cytotoxicity.
PPARγ inhibits the expression of Fas and granzyme B in CTLs.
Journal of Molecular Medicine 01/2015; 93(6). DOI:10.1007/s00109-014-1249-8 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages are central in regulating iron homeostasis, which is tightly linked to their versatile role during innate immunity. They sequester iron by phagocytosis of senescent erythrocytes and represent a major source of available iron in the body. Macrophage iron homeostasis is coupled to the functional heterogeneity and plasticity of these cells, with their extreme roles during inflammation, immune modulation, and resolution of inflammation. It is now appreciated that the macrophage polarization process dictates expression profiles of genes involved in iron metabolism. Therefore, macrophages have evolved a multitude of mechanisms to sequester, transport, store, and release iron. A new, enigmatic protein entering the iron scene and affecting the macrophage phenotype is lipocalin-2. Iron sequestration in macrophages depletes the microenvironment, thereby limiting extracellular pathogen or tumor growth, while fostering inflammation. In contrast, iron release from macrophages contributes to bystander cell proliferation, which is important for tissue regeneration and repair. This dichotomy is also reflected by the dual role of lipocalin-2 in macrophages. Unfortunately, the iron release macrophage phenotype is also a characteristic of tumor associated macrophages and stimulates tumor cell survival and growth. Iron sequestration versus its release is now appreciated to be associated with the macrophage polarization program and can be used to explain a number of biological functions attributed to distinct macrophage phenotypes. Here we discuss macrophage iron homeostasis with a special focus on lipocalin-2 related to the formation and function of tumor associated macrophages.
[Show abstract][Hide abstract] ABSTRACT: Lipoxins belong to the family of so-called pro-resolving endogenous lipid mediators which are derived from arachidonic acid and play a key role in the counter-regulation of inflammation. Arachidonic acid is also precursor of multiple pro-inflammatory lipid mediators, such as prostaglandins and leukotrienes, which are simultaneously present in biological compartments. The close structural relationship between several of these lipid mediators and the absence of blank matrix samples enormously complicates the unequivocal identification of these compounds. The determination of lipoxin A4 has been accomplished by chromatographic separation using a C18 reversed phase column and tandem mass spectrometry detection. Samples were liquid-liquid extracted with ethyl acetate before injection. Identification of the analyte was done based on three criteria: retention time, ratio of the m/z transitions and MS/MS spectrum. To avoid false positive results due to endogenous interferences, the extracted samples were re-injected into a chiral Lux Amylose-2 chromatographic column. The authors recommend the use of chiral chromatography in the determination of pro-resolving lipid mediators, together with transition area ratio and fragmentation spectra to improve selectivity for identification and quantitation purposes.