10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Chair

Prof. Dr.

Mechanisms of transcript buffering in inflammation

Immunopharmacology / inflammation / anti-infectives

AP02

Introduction: The inflammatory response, a complex biological event, hinges on the precise regulation of gene expression. We follow the hypothesis that transcript buffering, which maintains mRNA stability and protein levels amidst transcriptional fluctuations, is a hitherto unrecognized part of this regulation. This research unravels the mechanisms of transcript buffering in inflammation, specifically spotlighting XRN1 - a crucial 5'-3' exoribonuclease integral to mRNA degradation and turnover - and the implications of its depletion.Methods: Delineating these mechanisms required a combination of molecular techniques, including chase reactions of mRNA and protein decay, cytosol-nuclear fractionation, RT-PCR, western blot, and LC-MS/MS. These techniques provided insights into mRNA stability, protein levels, and cellular distribution of key transcription factors.Results: Preliminary results underscore the critical role of XRN1 in the gene-specific response of IL-1-mediated induction of inflammatory mediators. Furthermore, XRN1 depletion increased mRNA stability of four prototypical IL-1-NFkB target genes, despite inflammatory mediator protein levels remaining unaltered. Unexpectedly, the study illustrates mRNAs and proteins showing an augmented half-life of cell cycle regulatory and inflammatory proteins in the absence of XRN1. An intricate proteomic analysis identified several proteins that shuttle differentially between cytoplasm and nucleus in XRN1-deficient cells. These factors represent candidates for cytoplasmic-nuclear backward signaling during transcript buffering. Some of these proteins additionally interact with XRN1, according to our unpublished BioID data.Conclusion/Outlook: Our data show that XRN1 plays an important role in the coordination of biosynthesis versus decay both at the levels of mRNA and protein. XRN1 loss leads to changes in cytosolic-nuclear backward signaling because we identified several proteins with different distributions between the cytoplasm and the nucleus upon loss of XRN1. Future research will focus on individual depletion of recognized proteins to discern factors that regulate buffering based on XRN1-knockdown recovery of reduced de novo transcription of IL-1 target genes. Scrutiny at the synthesis, steady-state, and decay levels will lend further clarity to this intricate biological process.

Applied Pharmacology

Poster Self-Study

"Mitigating sepsis and septic shock via novel agents - the future or an idealistic dream? A state-of-the-art review"

Introduction: Since the 1960s, there has been a massive effort to find immunomodulating drugs to treat sepsis. Yet, 60 years later, the only approved causative treatments for sepsis are anti-infectives. The need for new causal medications remains critical due to immense mortality rates.Objectives: The aim of this state-of-the-art review is to elucidate current research on novel agents for mitigating sepsis and septic shock. Our focus was to develop a comprehensive understanding of past research, to identify knowledge and research gaps and to show recent advancements. Further, we want to evaluate the eligibility of those new agents and to identify promising therapeutic approaches.Materials and Methods: We performed a state-of-the-art review from the following medical databases: PubMed, Google Scholar, Wolters Kluwer and Cochrane Library. Keywords for the literature search were: sepsis, pharmacotherapy, NOS, PAR1, NETs. Our focus lies especially on studies from the last decade to ensure inclusion of recent developments.Results: Our literature search yielded multiple promising approaches targeting various arrays of pathways. Manipulating nitric oxide synthase is a complex approach considering the ambivalent role of NO. Recent research emphasizes importance of timing, site of organ failure and isoform-specific targeting: while inducing eNOS may maintain vascular integrity and microvasculature, iNOS and nNOS inhibition could mitigate inflammation. Strongly intertwined are new understandings in the ADMA and peroxynitrite - PraP1 pathways which may shed new light on approaches in sepsis treatments. New insights into PAR1 modulation, GSDMD inhibition and NET targeting reveal promising therapeutic options for sepsis regarding endothelial damage, pyroptosis and potentially even organ failure.Discussion: In the last three decades immense research was conducted and while our understanding of the pathophysiology of sepsis improved substantially, therapeutics lag behind. Promising agents were tested pre-clinically, however, many were discarded due to poor clinical results or often due to limited understanding and technology. Further extensive research is needed, with a more individualized approach tailored to the progression of sepsis, the unique patient response, or the site of organ failure. This should involve greater pharmacological precision (novel, selective agents) or draw on emerging insights into underlying pathomechanisms.

Bone cement with antibacterial substances against pathogens involved in periprosthetic joint infections

Introduction: Surgical replacement of destructive joints with prostheses is a common procedure in both human and veterinary medicine. Bacterial infections at the implant site lead to septic loosening, which often causes implant failure. To combat these infections, antibiotic-laden bone cement is commercially available and generally used to fasten joint implants. However, increasing occurrence of multi-resistant bacteria and failure to manage periprosthetic joint infections make it necessary to identify new antibacterial substances for this purpose. N-acetylcysteine (NAC) and methylglyoxal (MGO) have been chosen as candidate substances due to their reported antibacterial activity. The aim of this project is to evaluate the suitability of the candidate substances in conjunction with bone cement (BC) in a clinical context.Material and Methods: Determination of minimal inhibitory concentrations (MICs) was performed with three clinical isolates of Staphylococcus pseudintermedius in broth microdilutions. Cytotoxic concentrations of the substances were measured with viability and proliferation assays after treatment of human osteosarcoma (HOS) cells. Biocompatibility of bone cement supplemented with NAC and MGO was tested by treatment of HOS cells with cell culture medium incubated with BC platelets and by seeding HOS cells on substance-containing bone cement platelets. Enzyme-linked immunosorbent assays (ELISA) were used to measure the release of proinflammatory cytokine Interleukin-6 (IL-6). Activation of MAP kinase p38 was analysed by western blotting.Results: The half-maximal inhibitory concentration (IC50) of NAC for viability of human osteosarcoma cells (HOS) was 4.3 mg/mL and the IC50 of MGO was 0.4 mg/mL. For three clinical isolates of Staphylococcus pseudintermedius, MICs were 2.5 mg/mL for NAC and 0.15 mg/mL for MGO. Supernatants of BC platelets supplemented with 50 mg and 100 mg NAC reduced cell viability and proliferation compared to supernatants of BC without additive. After 24 hours of HOS treatment with the BC supernatants, no release of IL-6 could be detected. Incubation with 3 and 4 mg/ml NAC lead to increased phosphorylation of p38 compared to control. Supernatants of BC supplemented with 25 mg MGO reduced proliferation by 20 % compared to supernatants of BC without additive.Conclusion: Taken together, this study aims to determine effective concentrations of new antibacterial substances in bone cement, while ensuring a high cell compatibility.

Locoregional immune responses in murine chronic colitis

Inflammatory bowel diseases (IBD), such as Crohn"s disease and ulcerative colitis, are characterized by relapsing intestinal inflammation. The underlying mechanisms are poorly understood, but a dysregulation of the immune response is considered a major cause. The bone marrow (BM) niche containing hematopoietic and mesenchymal stem cells (HSC, MSC) provides a specialized environment essential for hematopoiesis. In adults, the hematopoietically active red bone marrow is primarily located in short and flat bones, as well as in the epiphyses of long tubular bones. Locoregional differences of BM responses may vary depending on proximity to inflammatory areas. Thus, we hypothesize here that the inflammation in IBD may alter BM niche composition and immune cell production at specific sites of the skeleton in a locoregional manner.Male, 6-8-week old C57BL/6J wild-type mice underwent one to three cycles of dextran sodium sulfate (DSS, 2%) treatment followed by 2-week washouts, to mimic relapsing inflammatory phases. Immune cells in the blood, along with HSCs and mature immune cells isolated from the BM of femur, tibia, sternum, thoracic, and abdominal vertebrae, were analyzed by flow cytometry.After colitis validation by assessing weight loss, colon weight/length ratio, and histological scoring, subsequent analyses showed increased circulating immune cell counts in colitis mice compared to respective control mice. Especially after the third DSS cycle, notable changes were observed with a strong rise in circulating neutrophils and monocytes. In the BM of DSS-treated mice, increased immune cell counts were mainly detected in the thoracic vertebrae, femur, and tibia, while only minor changes appeared in the sternum. During the inflammatory cycles, the distribution of immune cells in the BM shifted towards a myeloid skew, with an increase in neutrophils and reduction in lymphocytes. Additionally, HSC counts were particularly elevated in the sternum, tibia, and femur. Time course analyses showed that the vertebrae responded earlier than the femur: elevated immune cell counts and alterations in immune cell and HSPC distribution were already evident after the second cycle in the thoracic and abdominal vertebrae, whereas the femur displayed a marked increase in HSPCs only during the third DSS cycle.Our findings indicate that repetitive cycles of experimental colitis result in site-specific changes in immune cells and HSPCs, suggesting pathways for targeted therapies.

Prof.

Identifcation of protein interaction networks of the cytokine-inducible transcription factor p65 NF-kB / RELA in living cells

The proinflammatory cytokine interleukin(IL)-1 plays a pivotal role in the transcriptional induction of p65 / RELA target genes. In contrast to the wealth of information on DNA binding of p65 / RELA, we currently lack a global understanding of p65 / RELA protein–protein interactions (PPIs) as they occur in living cells and their functional contributions to IL-1-NF-kB-driven transcriptional networks. Here we used biotin-proximity labeling, a relatively new approach by which a bait protein fused to an engineered bacterial biotin ligase is employed to biotinylate all proteins within a radius of 1-10 nm to the fusion protein. After cell lysis, biotinylated proteins are captured on streptavidin affinity matrices and subsequently identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). MiniTurbo (mTb) is a recently improved small and highly efficient biotin ligase that modifies intracellular proteins within minutes in the presence of exogenously added biotin. We used p65-mTb fusion proteins to determine the protein interactomes of p65 / RELA in intact cells. This strategy revealed more than 350 RELA interactors from untreated and IL-1α-stimulated cells, including 172 transcription factors (47% of all interactors) and over 50 epigenetic regulators belonging to different classes of chromatin remodelling complexes. A comparison with the interactomes of two point mutants of p65 revealed that the interactions primarily require intact dimerization rather than DNA binding properties. A targeted RNAi screen for 38 high confidence interactors and subsequent functional transcriptome and bioinformatics studies identify gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3 / TFEB or S100A8 / A9. In summary, the data reveal hundreds of p65 / RELA interactors in a single cell type, demonstrating the enormous extent of intermolecular connectivity of a single mammalian TF. Taken together with the exemplary functional study of selected TF partners, the results have far-reaching implications for interpreting p65 / RELA-driven processes in homeostatic and diseased states.

Microscopy based analysis of global and specific RNA biosynthesis in response to Interleukin-1

Interleukin 1 (IL-1), a major regulator of the innate immune response, triggers the transcription of about 200 pro-inflammatory genes, such as IL8 and IL6, in a wide variety of cell types1. Both initiation and termination of this rapid response are tightly controlled in order to avoid harm to surrounding tissues and to preserve metabolic capacities.IL-1-specific gene sets are crucially involved in a wide range of inflammatory conditions. However, it is less clear whether the IL-1-mediated inflammatory program is involved in basic gene-regulatory or biosynthetic processes.In this project, we aim to measure the global transcription rate - by means of metabolic labeling – in conjunction with individual transcripts – by means of fluorescence in situ hybridization (FISH) - in order to correlate the global and the specific response of individual cells.The analysis of the microscopy images involves cellpose, an AI based trainable algorithm to segment unlabeled single cells, followed by measurements of fluorescence intensities and counts of mRNA spots.First, we found that IL-1 stimulation for 1 hour transiently reduces the overall transcriptional activity. To generalize this finding, we confirmed this effect in six human cell lines, including diploid cells like RPE-1 cells.Next, we investigated the IL-1 effect on bulk protein synthesis, using a similar metabolic labeling approach. Interestingly, our data showed no difference in translation rate between IL-1 stimulated and unstimulated cells in the 1-4 hour stimulation period.However, after a 6 hours of IL-1 stimulation period, A549 lung cancer cells and diploid immortalized retinal pigment epithelial (RPE-1) cells showed elevated transcription and translation rates compared to untreated controls, which were both associated with an increased cell size.To investigate if single cells that highly express pro-inflammatory genes (IL8) are also suppressing global transcription rates, we combined the metabolic labelling approach with single-molecule fluorescence in situ-hybridization. Unexpectedly, our data does not support a strong correlation between these two processes.In the future, a more comprehensive characterization of the IL-1 pathway could lead to the identification of more specific targets to manipulate a pathological cytokine-driven inflammatory response.Weber, A., P. Wasiliew, and M. Kracht, Interleukin-1 (IL-1) pathway. Sci Signal, 2010.

Targeting of CDK12/13 in cancer cells reveals additional roles in the inflammatory IL-1-NF-κB pathway

Cyclin-dependent kinase 12 (CDK12) is frequently mutated in over 30 different types of cancer. Together with its highly homologous counterpart, CDK13, these two kinases play essential roles in phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II to regulate transcriptional elongation. Beyond this, CDK12/13 are involved in translational regulation, RNA processing, and DNA damage repair, rendering them promising targets for pharmacological intervention in cancer therapy. Additionally, our unpublished data indicate that the pro-inflammatory cytokine IL-1 promotes phosphorylation of CDK12/13, suggesting a potential interaction between these CDKs and the IL-1-NF-κB signalling pathway.In this study, we tested the specific CDK12/13 inhibitors THZ531 (covalent) and SR-4835 (non-covalent) in the context of IL-1-NF-κB-mediated inflammatory responses. RT-qPCR, Western blotting and ELISA experiments were performed to analyse multiple levels of regulation. Moreover, although CDK12 and CDK13 are highly similar in sequence and function, they also exhibit unique roles. To explore these differences, we engineered the CDK12 or CDK13 genomic loci of HCT116 cells by a CRISPR/Cas-based tagging method to integrate an auxin-inducible degron domain for the rapid and reversible degradation of either kinase. The resulting cell lines were functionally characterized in the absence or presence of IL-1α.Inhibition of CDK12/13 by the ATP-competitive inhibitors THZ531 and SR-4835 led to consistent differential effects on IL-1-NF-κB-mediated gene expression and protein secretion in HCT116 cells. As both inhibitors affected IL-1 target genes in a similar manner, the observed effects are likely on-target. Specifically, inhibition of CDK12/13 led to suppression of BIRC3 and induction of CCL20, IL8 and CXCL2 gene expression. Furthermore, a reduction in phosphorylation and slower re-synthesis of IκBα was observed, as well as an induced protein secretion of IL-8 and CXCL2. Notably, these effects were absent in the CDK12/13 degron cells, possibly due to one kinase compensating for the loss of the other.Overall, our study confirms a link between CDKs and the NF-κB signalling system, suggesting non-canonical roles for CDK12 and CDK13 in inflammation that reach beyond regulating transcriptional elongation. The distinct roles of CDK12 relative to CDK13 will be further investigated using the auxin-inducible degron system in future experiments.

Pharmacological and toxicological assessment of antiviral RNAi inhalation ex vivo and in vivo

Viral respiratory tract infections (VRI) impose an immense global disease burden, often without specific approved antiviral treatment options available. RNA interference (RNAi)-based antiviral treatment is a promising new therapy approach, but pulmonary RNA delivery and clinical translation remains challenging. Here we report the investigation of an LNP formulation, that is suitable for inhaled administration and assessed compound safety and efficacy in a series of in vitro, ex vivo, and in vivo experiments.12 different LNP compositions were screened for their stability after nebulization with a Aerogen Solo mesh nebulizer by dynamic light scattering (DLS). Efficient delivery of functional antiviral siRNA was further analyzed using LLCMK2, Calu3, and human precision-cut lung slice (hPCLS) infection models by analyzing viral load via focus forming assays (FFA) and qRT-PCR. Drug safety was assessed by measuring LDH release, WST-1 metabolic activity and cytokine release. The lead candidate was tested in vivo in a cotton rat infection model of HPIV3. Cotton rats were treated twice by inhalation of nebulized LNP-formulated siRNA after infection.From the 12 tested LNP compositions, the most stable LNP formulation yielded particles of 104 nm before and 128 nm after nebulization, on average, and there was no reduction in the efficacy of the transfection following nebulization. The siRNA candidate, which was formulated with in this LNP, and which is targeting the HPIV3 L gene, reached IC50 values from 0.19 to 53.89 nM, depending on the test system, and did not show signs of cytotoxicity in this concentration range, as assessed by LDH release or metabolic activity: However, a slight increase in LDH release and in pro-inflammatory cytokines, like IL-8 or IL-1β, was induced by higher concentrations. LNP-formulated fluorescently labelled siRNA was primarily found in the lung and not in blood or any other organs after initial in vivo data from the cotton rats. A double administration in the in vivo cotton rat model reduced the viral load, but also led to increased recruitment of immune cells.We were able to show that our preclinical test systems are predictive for the pharmacological and toxicological evaluation of LNP formulated siRNAs. We also could tackle the hurdles of efficacy and delivery of antiviral RNAi using an LNP-formulated siRNA against HPIV3 infection, but were only able to achieve a small therapeutic window.

Prof., Dr.

Polarization of THP-1 monocytes to M1/M2 macrophages increases drug transporter mRNA expression levels and enhances P-gp efflux activity

Introduction:Pro-inflammatory M1 and anti-inflammatory M2 macrophages play major roles for the pathogenesis of tuberculosis and its response to drug treatment. However, data on the expression of drug transporters in monocytes-derived macrophages is largely lacking. Accordingly, this study investigated the effects of differentiation and polarization of THP-1 monocytes to M1 and M2 macrophages regarding the mRNA expression of 84 drug transporter genes and the efflux activity of P-glycoprotein (P-gp).Methods:THP-1 monocytes were differentiated to macrophages (M0) using 200 nM phorbol-12-myristate-13-acetate (PMA) for 3 days. After resting for 5 days, cells were subsequently polarized either to the M1 subtype (50 ng/mL lipopolysaccharides; 20 ng/mL interferon gamma) or the M2 subtype (interleukin-4, interleukin-13; 20 ng/mL each). Polarization was confirmed by mRNA expression of specific expression markers (e.g. interleukin 1-beta, tumor necrosis factor alpha). Transcriptomics of drug transporter genes was evaluated using the RT2 Profiler™ PCR Array from Qiagen©. P-gp protein expression and efflux activity were evaluated by western blotting and fluorescence-activated cell sorting using rhodamine 123 as a fluorescent P-gp substrate.Results:Monocyte-to-M0 differentiation upregulated 18 genes. Subsequent polarization to M1 macrophages further enhanced the expression of 48 genes, including ABCB1 (166-fold), TAP1 (30-fold), and SLCO1B1 (3-fold)), compared to monocytes. M2 polarization only upregulated 20 genes (e.g. ABCG2 (37-fold), SLCO2B1 (448-fold), SLC7A8 (104-fold)). However, on a functional level, the M2 subtype showed a significantly higher P-gp activity (1.9 ± 0.56-fold change vs. monocytes) than the M1 subtype (1.3 ± 0.46-fold change), suggesting that uptake of P-gp substrate drugs (e.g., rifampicin) into M2 macrophages might be compromised compared to M1 macrophages.Conclusion:The mRNA expression of many drug transporters is considerably increased during differentiation and polarization of monocytes to M1/M2 macrophages. Most importantly, the resulting high P-gp activity in M2 macrophages could affect tuberculosis therapy by decreasing the intracellular concentration of antibiotics such as rifampicin and explain the known poor drug responsiveness of M2-dominated tuberculosis granuloma.

Poster Walk 2
Immunopharmacology / inflammation / anti-infectives

Termin

Chair

Programm

Poster Walk 2Immunopharmacology / inflammation / anti-infectives

Termin

Chair

Programm

Poster Walk 2
Immunopharmacology / inflammation / anti-infectives