Gunther Hartmann’s research while affiliated with University of Bonn and other places

The nocebo effect, driven by negative expectations rather than pharmacological mechanisms, contributes significantly to medication non-adherence, particularly in lipid-lowering therapy. Up to 50% of reported statin-related adverse effects may result from nocebo responses, leading to unnecessary discontinuation and increased cardiovascular risk. Blinded provocation tests may offer a solution for the differentiation of true drug intolerance from nocebo-driven symptoms. Although this methodology is well-established in experimental studies, it has not been transferred to routine clinical practice so far. We present a 65-year-old female with hypercholesterolemia and cardiovascular risk factors who experienced recurrent, dose-dependent left-sided lower abdominal pain with different lipid-lowering drugs. These symptoms prompted repeated and ultimately continuous treatment discontinuations, each followed by resolution of complaints. Despite extensive evaluations, no organic cause was found. To assess the role of nocebo effects, a six-week single-blinded, placebo-controlled crossover provocation test with a commercially available placebo preparation and atorvastatin placed in neutral pill containers was conducted. Upon initiation of the provocation phase, the patient experienced similar intermittent symptoms under both treatments. The pain ratings on a numeric rating scale did not significantly differ during placebo (mean: 2.75) and atorvastatin administration (mean: 3.26), suggesting that these symptoms were not pharmacologically induced. Following information of the patient, atorvastatin therapy could be continued. During continued intake over several weeks, symptoms further diminished, reinforcing the therapeutic value of addressing nocebo effects. This case report provides for the first time the structured and detailed step-by-step description of a pragmatic approach for a prospective blinded, placebo-controlled provocation testing that can directly be implemented in routine clinical practice. This method enables the distinction of true drug intolerance from nocebo effects, thereby enabling necessary therapies and highlighting its diagnostic and therapeutic potential.

Photochemical control of oligonucleotides bears great potential for the spatio‐temporal control of therapeutic targets, such as immune sensing receptors. Retinoic acid‐inducible gene I (RIG‐I) is a cytoplasmic receptor of the innate immune system that triggers antiviral responses upon detection of viral RNA. RIG‐I can be specifically activated by short double‐stranded (ds) RNA with a blunt 5′ end bearing a triphosphate, mimicking nascent viral transcripts. Tumor cells are specifically sensitive to RIG‐I‐induced cell death. Here we developed a potent oligonucleotide ligand for spatiotemporally controlled activation of RIG‐I by light exposure. Through structural considerations and functional studies we identified a combination of two nucleoside positions in a RIG‐I oligonucleotide ligand for which the substitution of both respective 2′‐hydroxy groups of the ribose by photolabile protecting groups (2′‐photocages) resulted in a complete loss of RIG‐I ligand activity, whereas photocaging the individual positions was not sufficient to turn off RIG‐I. Light exposure fully restored RIG‐I activation by the photocaged RIG‐I ligand, enabling light‐controlled RIG‐I‐mediated cell death of human cancer cells which had internalized the photocaged RIG‐I ligand prior to light exposure. This novel photoactivatable RIG‐I oligonucleotide ligand may be applicable for precise light‐controlled induction of tumor cell death in superficial cancer such as melanoma.

The epidermal growth factor receptor (EGFR) signaling pathway is essential for cellular processes such as proliferation, survival, and migration. Dysregulation of EGFR signaling is frequently observed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis. This study aims to isolate and characterize extracellular vesicles (EVs) released by mutant EGFR lung cancer cell line PC9 and compare them with wild-type EGFR lung cancer cell line A549, while also evaluating the effect of gefitinib treatment on EV secretion and cargo composition. The two lung cancer cell lines were cultured with 2% EV-free serum, and EVs were subsequently isolated by differential ultra centrifugation. EVs were characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) for quantification size and shape determination. Western blot analysis confirmed the enrichment and purity of isolated EVs. Results showed that EGFR mutation significantly increased EV release and altered their size, compared to EVs released by wild-type EGFR cells. In addition to classical EV markers such as CD81, Flotillin- 1, and TSG101, Western blot analysis also detected phosphorylated EGFR (p-EGFR) selectively packaged into EVs from PC9 cells. Gefitinib treatment significantly reduced EV secretion in PC9 cells and led to a marked decrease in p-EGFR incorporation into EVs, indicating that EV biogenesis and compostion are modulated by active EGFR signaling. In conclusion, this study highlights the significant influence of EGFR activation on EV secretion and cargo composition while demonstrating that EGFR inhibition via gefitinib alters EV-mediated signaling in lung cancer cells. These findings provide insights into tumor behavior, EV-mediated oncogenic communication, and the potential use of EVs as biomarkers and therapeutic targets in NSCLC.

Kurzzusammenfassung Die photochemische Kontrolle von Oligonukleotiden hält großes Potenzial für die spatiotemporale Kontrolle therapeutischer Zielstrukturen wie etwa Immunrezeptoren. Retinsäure‐induzierbares‐Gen‐I (RIG‐I) ist ein zytosolischer Rezeptor des angeborenen Immunsystems, der bei Erkennung viraler RNA antivirale Immunantworten auslöst. RIG‐I kann gezielt durch kurze doppelsträngige (ds) RNA mit einem glatten, Triphosphat (ppp)‐tragenden 5′‐Ende aktiviert werden, die naszierende virale Transkripte imitiert. Tumorzellen sind besonders empfindlich gegenüber RIG‐I‐induziertem Zelltod. In dieser Arbeit entwickelten wir einen potenten Oligonukleotid‐Liganden zur spatiotemporal kontrollierten RIG‐I‐Aktivierung durch Licht. Mit Hilfe struktureller Überlegungen und funktioneller Studien identifizierten wir eine Kombination von zwei Nukleosidpositionen in einem RIG‐I‐Oligonukleotid‐Liganden, an denen der Austausch der jeweiligen 2′‐Hydroxygruppen der Ribose durch photolabile Schutzgruppen (2′‐Photocages) zu einem vollständigen Verlust der RIG‐I‐Ligandenaktivität führte. Der Austausch an nur einer einzelnen Position reichte hingegen nicht aus, um die RIG‐I‐Aktivierung vollständig zu verhindern. Lichtbestrahlung stellte die RIG‐I‐Aktivierung des photogeschützten RIG‐I‐Liganden vollständig wieder her und ermöglichte einen lichtkontrollierten, RIG‐I‐vermittelten Zelltod in humanen Krebszellen, die zuvor den lichtaktivierbaren RIG‐I‐Liganden aufgenommen hatten. Dieser neuartige lichtaktivierbare RIG‐I‐Oligonukleotidligand könnte zur präzisen lichtkontrollierten Induktion des Tumorzelltods bei oberflächlichen Krebserkrankungen, wie Melanomen anwendbar sein.

Viral infections pose a significant global burden. Host susceptibility to pathogens is determined by many factors including genetic variation that can lead to immunodeficient or dysregulated antiviral immune responses. Pax5 heterozygosity ( Pax5 −/+ ), resulting in reduced PAX5 levels in mice, mimics germline or somatic PAX5 dysregulation contributing to diseases such as childhood B-cell precursor acute lymphoblastic leukemia (B-ALL). In contrast to the well-characterized roles of PAX5 during early B-cell development, little is known about how Pax5 heterozygosity impacts antiviral responses. We infected Pax5 −/+ mice with the noncytopathic Lymphocytic Choriomeningitis Virus (LCMV) and found that infection with the chronic Docile strain resulted in decreased survival of Pax5 −/+ mice. While early adaptive CD8 ⁺ T-cell (CTL) immunity was robust in Pax5 −/+ mice, LCMV-specific neutralizing antibody production was compromised leading to impaired long-term viral clearance and a pro-inflammatory milieu in the bone marrow (BM). Here we show that survival outcomes were improved upon prophylactic treatment with the β-glucan immune trainer through induction of heterologous protection against chronic infection. β-Glucan enhanced viral clearance, CTL immunity, neutralizing antibody production and reduced monocyte immunosuppression in multiple LCMV-resident host organs. New insight from this study will help design effective prophylactic treatment strategies against chronic viral infections, particularly in genetically predisposed susceptible hosts.

Cytotoxic CD8 T lymphocytes (CTL) are crucial in antiviral immune responses. However, their recruitment to infection sites renders them at risk of viral infection that could modulate their effector activity. CTL express RIG-I that detects cytosolic viral RNA and subsequently induce antiviral gene expression. Here, we investigated how influenza A virus (IAV) infection influence TCR-dependent effector responses. IAV infection of CTL stimulated RIG-I, and activated downstream pathways including TBK1 and NF-κB, resulting in type-I interferon secretion. Transfection of CTL cells with a pure RIG-I ligand, tri-phosphorylated double stranded RNA(3p-dsRNA), not only stimulated these pathways but protected CTL from IAV infection and enhanced CTL proliferation in vitro. Analogous with a positive effect on CD8 effector function, both IAV infection and RIG-I ligands enhanced CTL degranulation and effector cytokine secretion. Conversely, activation of CTL via CD3/CD28-crosslinking increased their susceptibility to IAV infection. Altogether, RIG-I stimulation either by IAV infection or 3p-dsRNA transfection promoted cell intrinsic antiviral pathways and enhanced CD8 effector functions. These findings suggest that RIG-I agonists could enhance and prolong CTL effector function in immunotherapy.

The cytosolic nucleic acid sensors RIG-I and cGAS induce type-I interferon (IFN)-mediated immune responses to RNA and DNA viruses, respectively. So far no connection between the two cytosolic pathways upstream of IKK-like kinase activation has been investigated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a positive regulator of IRF3 phosphorylation and type-I IFN induction downstream of both cGAS and RIG-I. Combining interactome analysis with genome editing, we further uncover the RNA-binding protein ELAV-like protein 1 (ELAVL1; also known as human antigen R, HuR) as an hnRNPM interactor. Depletion of hnRNPM or ELAVL1 impairs type-I IFN induction by herpes simplex virus 1 or Sendai virus. In addition, we show that hnRNPM and ELAVL1 interact with TANK-binding kinase 1, IκB kinase ε, IκB kinase β, and NF-κB p65. Our confocal microscopy experiments demonstrate cytosolic and perinuclear interactions between hnRNPM, ELAVL1, and TBK1. Furthermore, pharmacological inhibition of ELAVL1 strongly reduces cytokine release from type-I interferonopathy patient fibroblasts. The RNA-binding proteins hnRNPM and ELAVL1 are the first non-redundant regulators to bridge the cGAS/STING and RIG-I/MAVS pathways. Overall, our study characterizes the hnRNPM-ELAVL1 complex as a novel system promoting antiviral defense, pointing to a potential therapeutic target to reduce auto-inflammation in patients with type-I interferonopathies.

The epidermal growth factor receptor signaling pathway is essential for cellular processes such as proliferation, survival, and migration. Dysregulation of EGFR signaling is frequently observed in non-small cell lung cancer and is associated with a poor prognosis. Aim: This study aims to isolate and characterize extracellular vesicles released by mutant-EGFR lung cancer cell line PC9 and compare them with wild type EGFR lung cancer cell line A549. Methodology: The two lung cancer cell lines were cultured with 2% EV-free serum, and EVs were subsequently isolated by differential (ultra)centrifugation. EVs were analyzed by nanoparticle tracking analysis for quantification and size determination. The presence and characterization of the isolated EVs was conducted with immunoblotting. Dysregulation of hsa-miR-128-3p in both cell lines and EVs was assessed by qRT-PCR. Results showed that EGFR mutation significantly increased EV release and altered their size, compared to EVs released by wild type EGFR cell line. In addition to classical EV marker such as CD9, CD81, Flotillin-1 and TSG101, Western blot analysis also detected phosphorylated EGFR. Furthermore, EGFR mutation led to a significant downregulation of hsa-miR-128-3p in EVs. In conclusion, this study highlights the significant influence of EGFR activation on EV release and cargo, providing insights into tumor behavior and potential biomarkers for lung cancer.

Key words: Trained immunity, Pax5-/+ mice, B cell precursor acute lymphoblastic leukemia (B-ALL), lymphocytic choriomeningitis virus (LCMV), bone marrow microenvironment (BME), chronic infection Background: Pax5 heterozygosity (Pax5-/+), resulting in reduced PAX5 levels in mice, mimics germline or somatic PAX5 dysregulation, contributing to diseases including B-ALL. In contrast to the well-characterized roles of PAX5 during early B cell development, little is known about how Pax5 heterozygosity impacts anti-viral responses. Viral infections pose a significant global burden. Host susceptibility to chronic pathogens is determined by many factors including genetic variation leading to immunodeficient or dysregulated anti-viral immune responses. Aims: We aimed to characterize the effects of a chronic viral infection in the BME of the Pax5-/+ mouse model using a chronic strain of the lymphocytic choriomeningitis virus (LCMV). LCMV is a non-cytopathic virus that has been extensively utilized to investigate virus-induced immunopathology, effector responses and immune tolerance. Methods: Pax5-/+ mice backcrossed to the C57BL/6J background were infected with LCMV-Docile (1*106 PFU). Using flow cytometry, multiplex ELISA, plaque and antibody neutralizing assays, innate and late adaptive immune responses in infected Pax5-/+ and WT (Pax5+/+) were assessed followed by β-glucan pre-administration as a prophylactic prevention. Results: We found that infection with the LCMV chronic Docile strain resulted in decreased survival of Pax5-/+ mice (n of 12-21, P<0.05). Early (day 15) viral titers were not different between Pax5-/+ and WT mice in multiple organs and early adaptive CD8+ T cell (CTL) immunity was also robust in Pax5-/+ mice. However, LCMV-specific neutralizing antibody production was severely compromised in Pax5-/+ mice following chronic infection leading to impaired long-term viral clearance and a pro-inflammatory milieu in the BM or serum with CXCL9, CXCL10, CCL4 and CCL3 being elevated at day 120 post infection in Pax5-/+ mice. In addition, increased CTL exhaustion as well as reduced plasma cells were observed in the BM of Pax5-/+ mice (n of 3-5, P<0.05). Importantly, survival outcomes were improved upon prophylactic treatment with the β-glucan immune trainer through induction of heterologous protection against chronic infection (n of 6-10, P<0.05). β-glucan enhanced viral clearance, CTL immunity and reduced PD-L1 expression on monocytes in multiple LCMV-resident host organs. Conclusion: Our study showed that Pax5-/+ mice succumbed to LCMV chronic Docile strain due to impaired LCMV-specific neutralizing antibody production and long-term viral clearance, as well as increased inflammation. Prophylactic treatment with β-glucan improved survival by enhancing viral clearance, CTL immunity, and reducing monocyte immunosuppression. New insight from this study will help design effective prophylactic strategies against chronic viral infections particularly in genetically-predisposed susceptible hosts.