Novel Mechanisms to Modulate Microglial Functional Phenotypes in Health and Disease

Microglia are resident innate immune cells that maintain homeostasis and sense a range of pathophysiological impairments within the central nervous system (CNS). Based on their involvement in brain disease progression, microglia-targeted therapy has emerged as a potential approach for reducing the burden of CNS disease. Microglia activation requires the activation of different signal pathways via neurotransmitters, neuropeptides and/ or other extracellular molecules, which each can be targeted. The neuropeptide VGF (non-acronym) is secreted by neurons and hydrolysed into biologically active peptides. One of these peptides is TLQP21, which binds to the complement receptors C1qbp and C3aR1. Although both receptors are expressed in microglia, the effect of TLQP21 on microglial cells has not been elucidated. The goal of the first study in the present dissertation was to determine whether TLQP21 might be a useful target in modulating microglial function. I demonstrate that exogenous application of TLQP21 stimulates microglial outward rectifying potassium (K+) currents, intracellular calcium (Ca2+) release, phagocytosis, and migration in C3aR-dependent manner. Interestingly, C3aR1 receptors were only expressed in microglia in vitro, but not in situ; suggesting that their expression might vary between different microglial activation states. Furthermore, in vitro and in situ activation of C1qbp leads to TLQP21 interference with metabotropic purinergic signalling (P2Y) in microglia, thereby, decreasing P2Y12-mediated activation of K+ conductance, microglia migration and laser lesion-induced processes outgrowth, as well as P2Y6-mediated phagocytic activity. Next, I examined other possible P2Y regulators in microglia. Neurofibromin is a known downstream effector of tyrosine kinase receptors and G protein-coupled receptors that indirectly regulates cyclic AMP (cAMP) levels through purinergic receptors. Mutations on this protein results in Neurofibromatosis type 1 disease (NF1). In the second study, a mouse model of NF1 with heterozygous neurofibromin knockout (Nf1 +/-) was employed. Interestingly, only male Nf1 +/- microglia showed impaired ATP-induced P2Y-mediated membrane currents and P2Y-dependent laser lesion-induced process movement in situ. Moreover, I found that the P2Y-control of microglial phagocytosis was only affected in male Nf1 +/- mice. In contrast, basal phagocytic activity was reduced in both male and female Nf1 +/- mice. Studying the neurofibromin downstream signaling intermediate, cAMP, revealed that male Nf1 +/- mice exhibited defects in cAMP regulation. Pharmacological blockade of phosphodiesterase III enzyme rescued these defects. The extracellular matrix (ECM) contains other factors that likely regulate the activity of microglia. In my third project, I studied Tenascin C (TNC), an ECM glycoprotein that activates Toll-like receptor 4 (Tlr4) expressed in microglia using TNC KO and Tlr4 KO mice. I found that TNC regulates microglial phagocytic activity in situ at an early postnatal age (P4) partially via Tlr4 activation. Furthermore, TNC regulates pro-inflammatory cytokine/chemokine production, chemotaxis and phagocytosis in Tlr4-dependent manner in vitro. Interestingly, the effect of TNC on microglia was linked to the expression of histone-deacetylase 1 (HDAC1) in microglia, which itself was induced by TNC. The use of MS-275 (HDAC1 inhibitor) attenuated TNC-induced microglia proinflammatory cytokines. In summary, the present dissertation demonstrates that TLQP21, NF1, and TNC are critical modulators of microglial function, suggesting that they might serve as promising targets to correct microglial dysfunction in the setting of CNS diseaseAls residente angeborene Immunzellen des ZNS sind Mikroglia wichtig für die Gewebehomöostase und erkennen jede Art von pathologischer Dysfunktion. Die zielgerichtete Mikroglia-Therapie hat sich zu einem viel versprechenden Ansatz entwickelt. Für die Aktivierung der Mikroglia über extrazelluläre Moleküle wie Pathogene, Neurotransmitter oder Neuropeptide sind verschiedene Signalwege erforderlich. Das Neuropeptid VGF wird von Neuronen sezerniert und im Golgi-Apparat zu biologisch aktiven Peptiden hydrolysiert. TLQP21 ist ein von VGF abgeleitetes Peptid, das mit metabolischen und neurologischen Störungen assoziiert ist, und die Komplementrezeptoren C1qbp und C3aR bindet, die von Mikrogliazellen exprimiert werden. Die Wirkung von TLQP21 auf Mikroglia ist jedoch noch unbekannt. In der vorgestellten Dissertation demonstriere ich, dass die exogene Anwendung von TLQP21 auswärts rektifizierende Kalium(K+)-Ströme, intrazelluläre Calcium(Ca2+)-Freisetzung, Phagozytose und Migration in Abhängigkeit von C3aR stimuliert. C3aRs werden von Mikroglia nur in vitro was darauf hindeutet, dass ihre Expression zwischen verschiedenen mikroglialen Zuständen variieren. Durch die Aktivierung von C1qbp in vitro und in situ interferierte TLQP21 mit den metabotropen purinergen Signalen (P2Y) in Mikroglia und verringerte die P2Y12-vermittelte Aktivierung der K+-Leitfähigkeit, die Migration, die durch Laserläsion Prozessauswüchse sowie die P2Y6-vermittelte phagozytische Aktivität induzierten. Um mögliche Regulatoren von P2Y in Mikroglia in einem Krankheitskontext zu untersuchen, verwendete ich in einem anderen Projekt ein transgenes Mausmodell für Neurofibromatose Typ 1 (Nf1+/-). Interessanterweise zeigten männliche Nf1+/- -Mikroglia eine Verringerung der ATP-induzierten P2Y-vermittelten Membranströme und P2Y-abhängige Laserläsion-induzierte Akkumulation mikroglialer Prozesse in situ, die weiblichen jedoch nicht. Darüber hinaus war die P2Y-Kontrolle der mikroglialen Phagozytose nur bei männlichen Nf1+/- Mäusen betroffen. Allerdings war die basale Phagozytoseaktivität sowohl bei männlichen als auch bei weiblichen Nf1+/- Mäusen reduziert. Durch Untersuchung des nachgeschalteten Botenstoffs, des zyklischen AMPs (cAMP), es wurde herausgefunden, dass männliche Nf1+/- Mäuse einen Defekt in der cAMP-Regulation aufweisen. Eine pharmakologische Blockade der Phosphodiesterase korrigierte die männlichen Nf1+/- Mikroglia-cAMP-Defekte. Als nächsten Schritt wandte ich mich der Frage zu, wie die extrazelluläre Matrix (ECM) Mikroglia beeinflussen könnte. TNC ist ein ECM-Glykoprotein, das Toll-like-Rezeptor 4 (TLR4) aktiviert. Es wurden TNC KO und TLR4 KO Mausmodelle verwendet, aus denen entweder primäre Mikrogliakulturen oder akute Hirnschnitte generiert wurden, um die Unterscheide der mikroglialen Aktivitäten zu studieren. TNC reguliert die phagozytäre Aktivität der Mikroglia in situ in einem frühen postnatalen Alter (P4) teilweise über die TLR4-Aktivierung. Die proinflammatorische Zytokin-/Chemokin-Produktion, Chemotaxis und in-vitro-Phagozytose wird hingegen in TLR4-abhängiger Weise reguliert. Darüber hinaus ist die Wirkung von TNC auf Mikroglia mit der Expression der Histon-Deacetylase 1 (HDAC1) in Mikroglia verbunden. Durch Verwendung des HDAC1-Inhibitors MS-275 konnte die TNC-induzierte Freisetzung von pro-inflammatorischen Zytokinen in Mikrogliazellen reduziert werden. Zusammengenommen zeige ich hier, dass TLQP21, NF1 und TNC Modulatoren der mikroglialen Funktionen sind und daher vielversprechende Ziele zur Korrektur mikroglialer Funktionsstörungen in Pathologien darstellen können

Site-Specific Considerations on Engineered T Cells for Malignant Gliomas

Immunotherapy has revolutionized cancer treatment. Despite the recent advances in immunotherapeutic approaches for several tumor entities, limited response has been observed in malignant gliomas, including glioblastoma (GBM). Conversely, one of the emerging immunotherapeutic modalities is chimeric antigen receptors (CAR) T cell therapy, which demonstrated promising clinical responses in other solid tumors. Current pre-clinical and interventional clinical studies suggest improved efficacy when CAR-T cells are delivered locoregionally, rather than intravenously. In this review, we summarize possible CAR-T cell administration routes including locoregional therapy, systemic administration with and without focused ultrasound, direct intra-arterial drug delivery and nanoparticle-enhanced delivery in glioma. Moreover, we discuss published as well as ongoing and planned clinical trials involving CAR-T cell therapy in malignant glioma. With increasing neoadjuvant and/or adjuvant combinatorial immunotherapeutic concepts and modalities with specific modes of action for malignant glioma, selection of administration routes becomes increasingly important

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain

Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy

Background The metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions.Methods The expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models.Results AhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice.Conclusions These findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers