Felix Meissner

The tumor suppressor protein p53 orchestrates cellular responses to stress by regulating the transcription of target genes involved in processes such as cell cycle control, DNA damage repair and apoptosis. The protein kinase DYRK1B, known to promote cancer cell survival and contribute to DNA damage repair, is overexpressed in various tumor types. Here, we demonstrate that expression of DYRK1B - but not its closely related paralog DYRK1A - is upregulated by cytostatic drugs (Actinomycin D, Doxorubicin) in multiple cancer cell lines. This induction required functional p53 and was mediated by p53-dependent activation of the transcription factor RFX7. Furthermore, we show that DYRK1B physically interacts with RFX7 and counteracts its activation by p53, thereby establishing a negative feedback loop that attenuates RFX7-dependent gene expression. This inhibitory effect of DYRK1B was strictly dependent on its catalytic activity and could be blocked by using small-molecule DYRK1 inhibitors. In conclusion, our study identifies DYRK1B as an indirect p53 target that suppresses p53-mediated activation of RFX7. These findings suggest that pharmacological inhibition of DYRK1B may represent a therapeutic strategy to enhance RFX7 tumor suppressor function.

Our research presents a new animal model of transient ischemic attack (TIA) that mimics brief episodes without cell loss, but results in neuronal and behavioral deficits. We identified excessive microglial reactivity, driven by acute ATP release, as a key factor in post-TIA neurological deficits, which were ameliorated by inhibiting the P2Y12 receptor, a microglia-specific purinergic receptor in the brain parenchyma responsible for activity-dependent microglial cell-cell interactions. This finding suggests that modulation of microglial reactivity offers a promising strategy to prevent cognitive impairment in TIA patients, opening avenues for future research in this underexplored area.

Organellar perturbations are linked to NLRP3 inflammasome activation, however, it remains unclear whether unrelated agonists converge on a common upstream pathway. Here, we traced intracellular organelle and protein movements by differential ultracentrifugation combined with mass spectrometry-based proteomics. We show that NLRP3 activators uniformly disrupt the endocytic Adaptor Protein 2 (AP2) complex, whereas other subcellular rearrangements are stimulus-specific. We discovered Dynasore as a K⁺-efflux–independent NLRP3 activator that engages this signaling node irrespective of endocytosis inhibition. Pharmacological and genetic perturbation of AP2 renders cells unresponsive to extracellular cues, blunting GPCR signaling, cAMP production, and chemotaxis, thereby enforcing a ’frozen’ signaling state that propagates NLRP3 inflammasome activation. Collectively, our study reveals a common surveillance checkpoint linking impaired plasma membrane signaling to the execution of inflammation and cell death. Graphical

The innate immune system is known for its ability to recognize cytosolic DNA as evidence of infection, but detailed studies of this process have been mostly limited to mice and cell lines. To investigate inflammasome responses in human primary cells, we used engineered viruses encoding the inflammasome reporter caspase-1 ^CARD -EGFP. We show that released genomes of vaccinia virus and monkeypox virus trigger robust inflammasome assembly in human primary cells. To determine the involved inflammasome sensors, we generated nanobodies against AIM2. Three of them inhibit AIM2 inflammasome assembly by blocking the polymerization of the AIM2 Pyrin domain, most potently as bivalent nanobodies. Utilizing an engineered vaccinia virus expressing bivalent AIM2 nanobodies, we demonstrate that inflammasomes in primary human macrophages and keratinocytes are nucleated by AIM2, while CD14 ⁺ monocytes assemble NLRP3 inflammasomes. This finding resolves the discrepancy between the previously reported activation of AIM2 inflammasomes in mice and NLRP3 inflammasomes in humans, and provides the first evidence for cell-type-specific regulation of DNA-triggered inflammasome activation. The newly developed AIM2-specific nanobodies offer a precise tool to dissect and potentially target AIM2 inflammasome assembly in other disease contexts.

Cellular polarization plays a crucial role in regulating immunological processes and is often associated with reorientation of the centrosome. During immune synapse formation, centrosome repositioning in lymphocytes assists in T cell activation. While a single centrosome, consisting of two centrioles, is present in T cells, antigen-presenting cells such as dendritic cells amplify centrioles during maturation and immune activation. How centriole amplification in antigen-presenting cells affects immune synapse formation and T cell activation is unclear. In this study, we combine experimental data with mathematical and computational modelling to provide evidence that extra centrioles in dendritic cells form over-active microtubule organizing centers, which cluster during dendritic cell-T cell interactions and, unlike in T cells, localize close to the cell center. Perturbing either centrosome integrity or centriole numbers and configuration in dendritic cells results in impaired T cell activation. Collectively, our results highlight a crucial role for centriole amplification and optimal centrosome positioning in antigen-presenting cells for controlling T cell responses.

Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18 , we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.

Microglia, the innate immune cells of the brain, play a defining role in the progression of Alzheimer’s disease (AD) ¹ . The microglial response to amyloid plaques in AD can range from neuroprotective to neurotoxic ² . Here we show that the protective function of microglia is governed by the transcription factor PU.1, which becomes downregulated following microglial contact with plaques. Lowering PU.1 expression in microglia reduces the severity of amyloid disease pathology in mice and is linked to the expression of immunoregulatory lymphoid receptor proteins, particularly CD28, a surface receptor that is critical for T cell activation ^3,4 . Microglia-specific deficiency in CD28, which is expressed by a small subset of plaque-associated PU.1 ^low microglia, promotes a broad inflammatory microglial state that is associated with increased amyloid plaque load. Our findings indicate that PU.1 ^low CD28-expressing microglia may operate as suppressive microglia that mitigate the progression of AD by reducing the severity of neuroinflammation. This role of CD28 and potentially other lymphoid co-stimulatory and co-inhibitory receptor proteins in governing microglial responses in AD points to possible immunotherapy approaches for treating the disease by promoting protective microglial functions.

Background: The circulating proteome may provide insights into the pathobiology of idiopathic pulmonary fibrosis (IPF) and diagnostic or prognostic biomarkers. We applied liquid chromatography coupled to mass spectrometry to quantify the peripheral blood proteome in patients with IPF and identify proteins associated with disease severity and progression. Methods: The IPF cohort comprised 299 patients from the IPF-PRO Registry. Controls ( n = 99) without known lung disease had similar distributions of age, sex and smoking status to the IPF cohort. Proteins were measured in plasma collected at enrollment using an Evosep One coupled to an Orbitrap Exploris. Data were analyzed with Spectronaut 14 with a deep experimental spectral library and were log ₂ transformed. Linear regression was used to compare protein abundances in the IPF versus control cohorts and identify proteins associated with disease severity measures at enrollment in the IPF cohort. Cox regression analyses were used to identify proteins associated with outcomes in the IPF cohort, split 75/25 into training and test sets. The false discovery rate was controlled at 5%. Results: Overall, 761 protein groups corresponding to 736 unique genes were detected. Of these, 168 protein groups were significantly different in abundance in the IPF versus control cohorts, of which 39 were ≥ 1.3-fold different. Among the top differentially expressed proteins were surfactant protein B (SFTPB), secretoglobin family 3A member 1, intercellular adhesion molecule 1, thrombospondin 1 and platelet factor 4. In patients with IPF, greater abundance of apolipoprotein A-1 was statistically significantly associated with higher forced vital capacity % predicted at enrollment, while greater abundance of fibulin-1 was statistically significantly associated with lower diffusing capacity of the lungs for carbon monoxide % predicted. Multivariable models selected 4 proteins (SERPINA7, SFTPB, alpha 2 HS glycoprotein, kininogen 1) and 3 clinical factors that best discriminated the risk of respiratory death or lung transplant in patients with IPF, with a C-index of 0.78 in the training set and 0.72 in the test set. Conclusions: Mass spectrometry-based proteomic analysis of data from the IPF-PRO Registry confirmed proteins previously associated with the presence, severity and progression of IPF and revealed new candidate biomarkers. Trial registration: ClinicalTrials.gov; No: NCT01915511; registered August 5, 2013; URL: www.clinicaltrials.gov .