Mario Kuttke

Macrophage PTEN Regulates Expression and Secretion of Arginase I Modulating Innate and Adaptive Immune Responses

The activation of innate immune cells triggers numerous intracellular signaling pathways, which require tight control to mount an adequate immune response. The PI3K signaling pathway is intricately involved in innate immunity, and its activation dampens the expression and release of proinflammatory cytokines in myeloid cells. These signaling processes are strictly regulated by the PI3K antagonist, the lipid phosphatase, PTEN, a known tumor suppressor. Importantly, PTEN is responsible for the elevated production of cytokines such as IL-6 in response to TLR agonists, and deletion of PTEN results in diminished inflammatory responses. However, the mechanisms by which PI3K negatively regulates TLR signaling are only partially resolved. We observed that Arginase I expression and secretion were markedly induced by PTEN deletion, suggesting PTEN−/− macrophages were alternatively activated. This was mediated by increased expression and activation of the transcription factors C/EBPβ and STAT3. Genetic and pharmacologic experimental approaches in vitro, as well as in vivo autoimmunity models, provide convincing evidence that PI3K/PTEN-regulated extracellular Arginase I acts as a paracrine regulator of inflammation and immunity.

Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis

Significance Multiple sclerosis (MS) imposes substantial economic burdens on patients, their families, and society. Until now, there are few therapies available, but often unattractive parenteral application or severe side effects are serious issues. This study highlights the use of circular peptides as orally active T-cell-specific immunosuppressive therapeutics against the MS model experimental autoimmune encephalomyelitis, without inducing major adverse effects. Our work provides a proof of principle that nature-derived cyclic peptides serve as oral active therapeutics, utilizing their intrinsic bioactivity and stable three-dimensional structure. Cyclotides are considered a combinatorial peptide library and they can be anticipated to complement the existing collections of natural products that are used in drug discovery. Multiple sclerosis (MS) is the most common autoimmune disease affecting the central nervous system. It is characterized by auto-reactive T cells that induce demyelination and neuronal degradation. Treatment options are still limited and several MS medications need to be administered by parenteral application but are modestly effective. Oral active drugs such as fingolimod have been weighed down by safety concerns. Consequently, there is a demand for novel, especially orally active therapeutics. Nature offers an abundance of compounds for drug discovery. Recently, the circular plant peptide kalata B1 was shown to silence T-cell proliferation in vitro in an IL-2–dependent mechanism. Owing to this promising effect, we aimed to determine in vivo activity of the cyclotide [T20K]kalata B1 using the MS mouse model experimental autoimmune encephalomyelitis (EAE). Treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of EAE by oral administration. Cyclotide application substantially impeded disease progression and did not exhibit adverse effects. Inhibition of lymphocyte proliferation and the reduction of proinflammatory cytokines, in particular IL-2, distinguish the cyclotide from other marketed drugs. Considering their stable structural topology and oral activity, cyclotides are candidates as peptide therapeutics for pharmaceutical drug development for treatment of T-cell-mediated disorders.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: A novel hypothesis to explain steatohepatitis

BACKGROUND & AIMS Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Sustained PI3K Activation exacerbates BLM-induced Lung Fibrosis via activation of pro-inflammatory and pro-fibrotic pathways

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease with limited treatment options. Additionally, the lack of a complete understanding of underlying immunological mechanisms underscores the importance of discovering novel options for therapeutic intervention. Since the PI3K/PTEN pathway in myeloid cells influences their effector functions, we wanted to elucidate how sustained PI3K activity induced by cell-type specific genetic deficiency of its antagonist PTEN modulates IPF, in a murine model of bleomycin-induced pulmonary fibrosis (BIPF). We found that myeloid PTEN deficient mice (PTENMyKO), after induction of BIPF, exhibit increased TGF-β1 activation, mRNA expression of pro-collagens and lysyl oxidase as well as augmented collagen deposition compared to wild-type littermates, leading to enhanced morbidity and decreased survival. Analysis of alveolar lavage and lung cell composition revealed that PTENMyKO mice exhibit reduced numbers of macrophages and T-cells in response to bleomycin, indicating an impaired recruitment function. Interestingly, we found dysregulated macrophage polarization as well as elevated expression and release of the pro-fibrotic cytokines IL-6 and TNF-α in PTENMyKO mice during BIPF. This might point to an uncontrolled wound healing response in which the inflammatory as well as tissue repair mechanisms proceed in parallel, thereby preventing resolution and at the same time promoting extensive fibrosis.

Loss of Phosphatase and Tensin Homolog in APCs Impedes Th17-Mediated Autoimmune Encephalomyelitis

The PI3K signaling cascade in APCs has been recognized as an essential pathway to initiate, maintain, and resolve immune responses. In this study, we demonstrate that a cell type–specific loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) in myeloid cells renders APCs toward a regulatory phenotype. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell–polarizing cytokines. Furthermore, PTEN deficiency leads to upregulation of markers for alternative activation, such as Arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN−/− APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type–specific deletions of PTEN-targeting APCs were protected from experimental autoimmune encephalomyelitis, which was accompanied by a pronounced reduction of IL-17– and IL-22–producing autoreactive T cells and reduced CNS influx of classically activated monocytes/macrophages. These observations support the notion that activation of the PI3K signaling cascade promotes regulatory APC properties and suppresses pathogenic T cell polarization, thereby reducing the clinical symptoms and pathology of experimental autoimmune encephalomyelitis.

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

Loss of MAPK-activated protein kinase 2 enables potent dendritic cell-driven anti-tumour T cell response.

Maintaining dendritic cells (DC) in a state of dysfunction represents a key mechanism by which tumour cells evade recognition and elimination by the immune system. Limited knowledge about the intracellular mediators of DC dysfunction restricts success of therapies aimed at reactivating a DC-driven anti-tumour immune response. Using a cell type-specific murine knock-out model, we have identified MAPK-activated protein kinase 2 (MK2) as a major guardian of a suppressive DC phenotype in the melanoma tumour microenvironment. MK2 deletion in CD11c+ cells led to an expansion of stimulatory CD103+ DCs, mounting a potent CD8+ T cell response that resulted in elimination of highly aggressive B16-F10 tumours upon toll-like receptor (TLR) activation in the presence of tumour antigen. Moreover, tumour infiltration by suppressive myeloid cells was strongly diminished. These insights into the regulation of DC functionality reveal MK2 as a targetable pathway for DC-centred immunomodulatory cancer therapies.

Myeloid PTEN deficiency impairs tumor-immune surveillance via immune-checkpoint inhibition.

ABSTRACT Tumor–host interaction is determined by constant immune surveillance, characterized by tumor infiltration of myeloid and lymphoid cells. A malfunctioning or diverted immune response promotes tumor growth and metastasis. Recent advances had been made, by treating of certain tumor types, such as melanoma, with T-cell checkpoint inhibitors. This highlights the importance of understanding the molecular mechanisms underlying the crosstalk between tumors and their environment, in particular myeloid and lymphoid cells. Our aim was to study the contribution of the myeloid PI3K/PTEN-signaling pathway in the regulation of tumor-immune surveillance in murine models of cancer. We made use of conditional PTEN-deficient mice, which exhibit sustained activation of the PI3K-signaling axis in a variety of myeloid cell subsets such as macrophages and dendritic cells (DCs). In colitis-associated colon cancer (CAC), mice deficient in myeloid PTEN showed a markedly higher tumor burden and decreased survival. We attributed this observation to the increased presence of immune-modulatory conventional CD8α+ DCs in the spleen, whereas other relevant myeloid cell subsets were largely unaffected. Notably, we detected enhanced surface expression of PD-L1 and PD-L2 on these DCs. As a consequence, tumoricidal T-cell responses were hampered or redirected. Taken together, our findings indicated an unanticipated role for the PI3K/PTEN-signaling axis in the functional regulation of splenic antigen-presenting cells (APCs). Our data pointed at potential, indirect, tumoricidal effects of subclass-specific PI3K inhibitors, which are currently under clinical investigation for treatment of tumors, via myeloid cell activation.

Abstract A43: MAPK-activated protein kinase MK2 exerts immune regulatory functions in the microenvironment of orthotopic tumors

The MAPK signaling pathway represents a central player in inflammatory processes. As a downstream target of p38, MAPK-activated protein kinase 2 (MK2) contributes to signal transduction regulating the expression and translation of various targets, among them several cytokines such as tumor necrosis factor (TNF) α. While MK2 was shown to promote an inflammatory macrophage phenotype, we have reported its Th1-attenuating function in dendritic cells (DCs). These observations of differential regulatory functions prompted us to investigate MK2 in the context of DCs and other myeloid cells in the tumor microenvironment. Murine DCs lacking MK2 activity exhibit an enhanced potential to differentiate Th1 cells upon Toll-like receptor (TLR) ligation in vitro and in vivo. In line with this finding, we observe significantly reduced B16.F10 melanoma growth in CD11cCre-MK2 fl/fl mice upon DC activation by administration of lipopolysaccharide (LPS) together with whole tumor cell lysate as compared to wildtype littermate controls. Interestingly, CD11cCre-MK2 fl/fl mice show an overall reduced myeloid cell tumor infiltration. Looking at MK2 expression in DCs isolated from orthotopic B16.F10 tumor-bearing mice, we find elevated MK2 levels in tumor-resident as opposed to splenic DCs, which has also been confirmed in a model of intracranial glioma (GL-261). This MK2 up-regulation in glioma-infiltrating DCs is associated with an enhanced expression of IL-10. Furthermore, tumor-resident myeloid-derived suppressor cells (MDSCs) show elevated MK2 expression, further underlining its potential involvement in immunosuppressive mechanisms in the myeloid lineage. In DCs we have observed cross-regulation of MAPK signaling by MK2, which promotes ERK1/2 while attenuating p38 activation and is further implicated to enhance STAT3 phosphorylation – all together contributing to a tolerogenic DC phenotype. Therefore we are now looking to unravel mechanisms by which MK2 impacts on signaling networks involved in myeloid immunosuppression in the tumor microenvironment. Our data contrast the previously described role of MK2 in pro-inflammatory mechanisms of the p38 signaling route and suggest an additional immunosuppressive feedback function in myeloid cells, which might be exploited by tumor cells to escape immune recognition and elimination. These indications are particularly interesting with regard to glioma, since the glioma microenvironment has been shown to be rich in various cytokines promoting favourable conditions for tumor growth. Finally, MK2 represents a promising target for combination therapy, as it has been reported to mediate chemo- as well as radioresistance in different types of tumors. Nevertheless, its function in tumor-infiltrating immune cells remains to be elucidated. We therefore aim to contribute to a deeper understanding of the complex interplay between extra- and intracellular signaling molecules regulating tumor as well as immune cell phenotypes. Citation Format: Klara Soukup, Angela Halfmann, Fiona Poyer, Katharina Martin, Bernadette Blauensteiner, Bastien Huber, Mario Kuttke, Gernot Schabbauer, Lydia Zopf, Jelena Zinnanti, Alexander Michael Dohnal. MAPK-activated protein kinase MK2 exerts immune regulatory functions in the microenvironment of orthotopic tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A43.

Abstract 527: Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Bluml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Gunther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 527.