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The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The aim of my Ph.D. thesis was to approach the mechanisms as well as the characteristics of macrophage phenotype alterations induced by AC, and to elucidate a possible connection between tumor cell apoptosis and TAM generation. In the first part of my studies, I investigated the impact of AC on macrophage viability. I could show that macrophage survival against pro-apoptotic agents increased after the interaction with AC. Protection of macrophages against cell death required activation of phosphatidylinositol-3 kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ signaling, and correlated with Bcl-XL and Bcl-2 up-regulation as well as Ser136-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. AC released the bioactive lipid sphingosine-1-phosphate (S1P), dependent on sphingosine kinase (SphK) 2, as a survival messenger. These data indicated an active role of AC in preventing cell destruction in their neighborhood. My next aim was to elucidate the mechanism of S1P production by AC. During cell death, SphK 2 was cleaved at its N-terminus by caspase-1. Thereupon, the truncated but enzymatically active fragment of SphK 2 was released from cells. This release was coupled to phosphatidylserine exposure, a hallmark of apoptosis and a crucial signal for the phagocyte/apoptotic cell interaction. Thus, I observed a link between common signaling events during apoptosis and the extracellular production of S1P, which is known to affect immune cell attraction and polarization as well as angiogenesis in cancer. In the next part of my studies, I asked for a correlation between tumor cell apoptosis and TAM polarization. During co-culture of human macrophages with human breast cancer carcinoma cells (MCF-7), the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α; and interleukin (IL)-12-p70 production, but increased formation of IL-8 and IL-10. Alternative macrophage activation required tumor cell death, because a co-culture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. These phenotype alterations were also achieved with conditioned media from apoptotic tumor cells, which again argued for a soluble factor being involved. Knock-down of SphK2, but not SphK1, to attenuate S1P formation in MCF-7 cells, repressed the otherwise observed alternative macrophage polarization during co-culture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P was characterized by suppression of pro-inflammatory nuclear factor (NF)-κB DNA binding. These findings suggested that tumor cell apoptosis-derived S1P contributes to the macrophage polarization present in human tumors. To validate these in vitro data, I used an in vivo tumor model to clarify the relevance of SphK2 and S1P in tumor development. The growth of, as well as blood vessel infiltration into SphK2 knock-down MCF-7 (MCF-7-siSphK2) xenografts in nude mice was markedly decreased in comparison to control MCF-7 xenografts. In contrast, macrophage infiltration was similar or even more pronounced. These data provided a first hint for an in vivo role of SphK2-derived S1P in macrophage polarization associated with tumor promotion. In summary, these data indicate a new mechanism how AC themselves shape macrophage polarization, which results in the termination of inflammatory responses and macrophage survival. Furthermore, my studies present evidence that human tumors may utilize this mechanism to foster growth via increased angiogenesis.
Smac (second mitochondria-derived activator of caspase) mimetics are considered as promising anticancer therapeutics and used to induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are often abundantly expressed in cancer cells. Here, we identify interferon regulatory factor 1 (IRF1) as a novel critical regulator of Smac mimetic BV6-induced apoptosis and proinflammatory cytokine secretion with impact on the immune response. IRF1 knockdown rescues cells from BV6-induced apoptosis and attenuates BV6-stimulated upregulation of tumor necrosis factor-α (TNFα), indicating that IRF1 mediates BV6-triggered cell death, at least in part, by inducing TNFα. This notion is supported by data showing that exogenous supply of TNFα restores BV6-induced cell death in IRF-knockdown cells. Interestingly, IRF1 selectively controls the induction of nuclear factor-κB (NF-κB) target genes, as IRF1 depletion attenuates BV6-stimulated upregulation of TNFα and interleukin-8 (IL-8) but not p100 and RelB. Concomitant knockdown of IRF1 and p65 cooperate to inhibit BV6-induced cell death, implying a cooperative interaction of IRF1 and NF-κB. In addition, IRF1 silencing hampers TNFα induction by TNFα itself as an another prototypical NF-κB stimulus. Importantly, IRF1 depletion impedes BV6-stimulated secretion of additional proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IL-6 and monocyte chemoattractant protein-1, and migration of primary monocytes to BV6-treated tumor cells. In conclusion, this identification of IRF1 as a dual regulator of BV6-induced apoptosis and inflammatory cytokine secretion provides novel insights into determinants of sensitivity towards Smac mimetic and possible implications of Smac mimetic treatment on tumor microenvironment and immune response.
Second mitochondria-derived activator of caspase (Smac) mimetics are considered as promising anticancer therapeutics that are currently under investigation in early clinical trials. They induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are frequently overexpressed in cancer. We previously reported that Smac mimetics, such as BV6, additionally exert non-apoptotic functions in glioblastoma (GBM) cells by stimulating migration and invasion in a nuclear factor kappa B (NF-κB)-dependent manner. Because NF-κB target genes mediating these effects are largely unknown, we performed whole-genome expression analyses. Here, we identify chemokine (C-C motif) ligand 2 (CCL2) as the top-listed NF-κB-regulated gene being upregulated upon BV6 treatment in GBM cells. BV6-induced upregulation and secretion of CCL2 are required for migration and invasion of GBM cells because knockdown of CCL2 in GBM cells abolishes these effects. Co-culture experiments of GBM cells with non-malignant astroglial cells reveal that BV6-stimulated secretion of CCL2 by GBM cells into the supernatant triggers migration of astroglial cells toward GBM cells because CCL2 knockdown in BV6-treated GBM cells impedes BV6-stimulated migration of astroglial cells. In conclusion, we identify CCL2 as a BV6-induced NF-κB target gene that triggers migration and invasion of GBM cells and exerts paracrine effects on the GBM's microenvironment by stimulating migration of astroglial cells. These findings provide novel insights into the biological functions of Smac mimetics with important implications for the development of Smac mimetics as cancer therapeutics.
Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+), cytotoxic-T cells (CD8+), T-helper cells (CD4+), B cells (CD20+), macrophages (CD68+), mast cells (CD117+), mononuclear cells (CD11c+), plasma cells, activated-T cells (MUM1+), B cells, myeloid cells (PD1+) and neutrophilic granulocytes (myeloperoxidase+) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.
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.
The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μm, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μm RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.
Hypoxia potentiates palmitate-induced pro-inflammatory activation of primary human macrophages
(2015)
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Background: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells.
Aims: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets.
Methods: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used.
Results: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented.
Conclusions: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.
The sphingolipid sphingosine-1-phosphate (S1P) emerges as an important regulator of immunity, mainly by signaling through a family of five specific G protein-coupled receptors (S1PR1–5). While S1P signaling generally has the potential to affect not only trafficking but also differentiation, activation, and survival of a diverse range of immune cells, the specific outcome depends on the S1P receptor repertoire expressed on a given cell. Among the S1PRs, S1PR4 is specifically abundant in immune cells, suggesting a major role of the S1P/S1PR4 axis in immunity. Recent studies indeed highlight its role in activation of immune cells, differentiation, and, potentially, trafficking. In this review, we summarize the emerging data that support a major role of S1PR4 in modulating immunity in humans and mice and discuss therapeutic implications.
S1P provokes tumor lymphangiogenesis via macrophage-derived mediators such as IL-1β or lipocalin-2
(2017)
A pleiotropic signaling lipid, sphingosine-1-phosphate (S1P), has been implicated in various pathophysiological processes supporting tumor growth and metastasis. However, there are only a few descriptive studies suggesting a role of S1P in tumor lymphangiogenesis, which is critical for tumor growth and dissemination. Corroborating own data, the literature suggests that apoptotic tumor cell-derived S1P alters the phenotype of tumor-associated macrophages (TAMs) to gain protumor functions. However, mechanistically, the role of TAM-induced lymphangiogenesis has only been poorly described, mostly linked to the production of lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C) and VEGF-D, or transdifferentiation into lymphatic endothelial cells. Recent findings highlight a rather underappreciated role of S1P in tumor lymphangiogenesis, referring to the production of interleukin-1β (IL-1β) and lipocalin-2 (LCN2) by a tumor-promoting macrophage phenotype. In this review, we aim to provide to the readers with the current understanding of the molecular mechanism how apoptotic cell-derived S1P triggers TAMs to promote lymphangiogenesis.