OP1: Sex hormones modulate how early-life inflammatory responses prime long-term protective DNA methylation and transcriptional responses

Karosham Diren Reddy ^1,2,3, Abdulkadir Kalender ^1,4, Lars Lunding ^1,4, Xiaolin Liu ^1,2, Henriette Tugiremariya ^1,2, Susanne Krauss-Etschmann ^5,6,7, Michael Wegmann ^1,4 & Markus Weckmann ^1,2,3

¹Airway Research Centre North, Member of the German Centre for Lung Research (DZL), Germany,²Department of Paediatric Pneumology & Allergology, University Clinics Schleswig-Holstein (UKSH), Lübeck, Germany,³Division of Epigenetics of Chronic Lung Diseases, Priority Area Chronic Lung Diseases, Research Centre Borstel - Leibniz Lung Centre, Borstel, Germany,⁴Division of Lung Immunology, Priority Area Chronic Lung Diseases, Research Centre Borstel - Leibniz Lung Centre, Borstel, Germany,⁵Early Origins of Chronic lung Disease, Priority Area Chronic Lung Diseases, Research Centre Borstel - Leibniz Lung Centre, Borstel, Germany,⁶DZL-Laboratory Experimental Microbiome Research, Research Centre Borstel - Leibniz Lung Centre, Borstel, Germany,⁷Institute of Experimental Asthma Research, Christian Albrecht University of Kiel

Sex hormones represent a critical early-life exposure shaping immune development and function. Estrogens promote pro-inflammatory and allergic responses, whilst androgens are immunosuppressive. These hormones govern networks contributing to sex-specific responses to inflammatory stressors, including viral infections. We investigated whether poly(I:C)-induced inflammation reprograms of sex-hormone signalling and alters DNA methylation (DNAm) and gene expression in a murine allergic asthma model.

BALB/6c mice were analysed at two stages: after initial poly(I:C) exposure (acute-model;n=8M/8F) and after completion of the poly(I:C)/HDM protocol (full-model;n=9M/7F). Allergic asthma induction was assessed by airway resistance following methacholine challenge and bronchoalveolar lavage (BAL) immune cell counts. Lung tissue was analysed by RNA-sequencing and DNAm profiling using the MouseEPIC array (n=4M/4F), processed in Rv4.6.1. Differentially expressed genes (DEGs;FDR<0.05,|logFC|>1.5) and differentially methylated sites (DMS;FDR<0.05,|logFC|>0.6) were identified, hormone pathway enrichment assessed using fgsea.

Acute poly(I:C) induced significant increases in neutrophils and lymphocytes in males only (p<0.001). poly(I:C) protected both sexes from an allergic-asthma, recognised by reduced eosinophils and airway resistance (p<0.01). HDM increased BAL lymphocytes in females, an effect reversed by poly(I:C) (p<0.05). Sex-stratified analysis identified 196 DEGs and 355 DMS, with only males indicating decreased androgen signalling after acute-poly(I:C), with this pattern maintained in the HDM-model (FDR<0.001). Integration of DNAm and gene expression demonstrated male-specific reprogramming of androgenic (p<0.05) and estrogenic (p<0.01) pathways, with no corresponding relationship in females.

Early-life poly(I:C) exposure induces male-specific molecular remodelling of the estrogenic and androgenic response pathways, suggesting differentially programming through epigenetic and transcriptional mechanisms that may contribute to sexually divergent pathways in allergic development.

OP2: TRIM28 S473 phosphorylation as a host-directed therapeutic target: Resolving lung hyperinflammation without compromising antiviral response during highly-pathogenic respiratory virus infection

S. Kumar ¹, A. Faist ², A. Mecate-Zambrano ³, K. Schughart ³, S. Ludwig ³, L. Brunotte ¹

¹Immunology of Respiratory Tract Infections, Universitätsklinikum Gießen and Marburg, Gießen, Germany,²Institute for Anesthesiology, Surgical Care and Pain Therapy, Universitätsklinikum Münster, Münster, Germany,³Institute of Virology, Universitätsklinikum Münster, Münster, Germany

Background: Severe lung disease caused by highly pathogenic respiratory viruses such as H5N1 and SARS-CoV-2 is driven by excessive inflammation. We identified the transcriptional corepressor TRIM28, phosphorylated at serine 473 (S473) during infection, as a molecular switch amplifying cytokine expression, and targeted it from cells to mice.

Methods: Upstream signaling was mapped with PKR, p38, and MSK1 inhibitors; the immune gene network was defined using knockout cells and transcriptomics. Druggability was evaluated in HPAIV- or SARS-CoV-2-infected lung epithelial cells, patient-derived lung explants, and primary alveolar type II organoids, and in H5N1-infected mice treated orally with PH-797804.

Results: During HPAIV infection, cytosolic viral RNA sensing by PKR, independently of RIG-I and MyD88, activated a p38/MSK1 cascade phosphorylating TRIM28 S473, derepressing cytokine expression. Blockade at each tier (PKR: 2-AP, C16; p38: SB202190; MSK1: SB747651) abolished S473 phosphorylation and lowered IFN-beta, IL-6, and IL-8 without impairing viral replication. PKR knockout cells and TRIM28 S473 phosphomutants provided genetic support. In SARS-CoV-2-infected explants and organoids, clinically evaluated p38 inhibitors (PH-797804, VX-702) markedly reduced IL-6, CXCL8, CXCL10, and TNF-alpha while interferon induction, viral control, and barrier integrity were preserved, and synergized with remdesivir and molnupiravir. Transcriptomics defined the extensive gene network reprogrammed by TRIM28. In vivo, oral PH-797804 delayed symptom onset and improved survival after lethal H5N1 challenge.

Conclusions: Targeting TRIM28 S473 uncouples protective immunity from inflammatory lung damage, offering an actionable host-directed therapy; defining p38 activation kinetics and timing-optimized, biomarker-guided regimens will guide translation.

OP3: Protective effects of intravenous immunoglobulins against Streptococcus pneumoniae in a murine model

Arina Ozhiganova ¹, Thomas Kohler ¹, Franziska Voß ¹ and Sven Hammerschmidt ¹

¹Department of Molecular Genetics and Infection Biology, Greifswald Universtiy, Greifswald, Germany

Introduction: Streptococcus pneumoniae remains a leading cause of community-acquired pneumonia despite widespread vaccination and antibiotic use. Intravenous immunoglobulins (IVIG) contain high levels of IgG directed against pneumococcal surface proteins including the pore-forming toxin pneumolysin (Wiebe et al., 2022), suggesting that IVIG may provide therapeutic benefits in severe pneumococcal pneumonia.

Aim: to investigate of protective effects of IVIG in a murine model of acute pneumococcal pneumonia.

Methods: C57BL/6N mice were pretreated with either vehicle control or IVIG. After 24 hours, animals were intranasally infected with S. pneumoniae strain TIGR4. Sampling was conducted at 12, 24, and 36 h post-infection (h p.i.). Bacterial burden was assessed by colony-forming unit counts, leukocyte composition was analysed using flow cytometry, IVIG concentration was measured by ELISA and total protein concentration in bronchoalveolar lavage fluid(BALF) was measured using the BCA assay.

Results: Plasma IVIG concentrations remained comparable across time points. At 24 h p.i., IVIG significantly reduced bacterial burden in BALF and systemic circulation animals (p < 0.05) compared with vehicle-treated infected animals. BALF protein concentrations were also lower following IVIG treatment. Blood leukocyte composition was comparable to that of healthy controls, while leukocyte recruitment to the lungs was unaffected by IVIG.

Conclusion: In this murine model, IVIG decreased pneumococcal dissemination to the lungs and bloodstream and limited pulmonary barrier damage. The transient nature of the protective effect of IVIG requires further investigation.

[1] Wiebe F, Handtke S, Wesche J, et al. Polyvalent Immunoglobulin Preparations Inhibit Pneumolysin-Induced Platelet Destruction. Thromb Haemost. 2022;122(7):1147-1158.

OP4: Early immunophenotypic heterogeneity is associated with treatment response in pulmonary tuberculosis

Johanna Eggeling ^1,2,3,4,5*, Martina Sester ^6,7, Christoph Lange ^1,2,3,8, Jan Heyckendorf ^9,10, Barbara Kalsdorf ^1,2,3, Anna M. Mandalakas ^1,2,8, Andrew R. DiNardo ⁸, David Lewinsohn ¹¹, Dagmar Schaub ^1,2,3, Tina Schmidt ⁶, Eva Tolosa ^12,13, Maja Reimann ^1,2,3†, and Patricia M. Sánchez Carballo ^1,2,3†* for the DZIF-TB cohort study group^#

¹Research Center Borstel, Leibniz Lung Center, Division of Clinical Infectious Diseases, Borstel, Germany,²German Center for Infection Research (DZIF), TTU TB, Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany,³Lübeck University, Respiratory Medicine & International Health, Lübeck, Germany,⁴Schleswig-Holstein University Hospital, Pediatric Oncology and Rheumatology, Kiel, Germany,⁵Schleswig-Holstein University Hospital, Neonatology, Pediatric Pneumology and Neuropediatrics, Kiel, Germany,⁶Saarland University, Department of Transplant and Infection Immunology, Homburg, Germany,⁷Saarland University, Center for Gender-specific Biology and Medicine (CGBM), Homburg, Germany.,⁸Baylor College of Medicine and Texas Children´s Hospital, Global Tuberculosis Program, Division of Global Health, Houston, Texas, USA,⁹Schleswig-Holstein University Hospital, Internal Medicine I, Leibniz Lung Clinic, Kiel, Germany,¹⁰Airway Research Center North (ARCN), German Center for Lung Research (DZL), Kiel, Germany,¹¹Oregon Health and Science University, Pulmonary, Allergy and Critical Care Medicine, Portland, OR, United States of America,¹²University Medical Center Hamburg-Eppendorf, Institute of Immunology, Hamburg, Germany,¹³German Center for Child and Adolescent Health (DZKJ), Hamburg, Germany,†These authors contributed equally.^#Additional members of the DZIF-TB cohort study group: Avsar K, DiNardo A, Glattki G, Goldmann T, Günther G, Hoelscher M, Ibraim E, Kaufmann SHE, Kontsevaya I, Maurer FP, Müller M, Nitschkowski D, Olaru ID, Popa C, Rachow A, Rolling T, Rybniker J, Salzer HJF, Schuhmann M, Spinu V, Suárez I, Terhalle E, Unnewehr M, van Leth F, Weiner 3rd J

Background: Tuberculosis (TB) is characterized by a complex inflammatory response in which excessive inflammation drives tissue pathology, whereas insufficient immune activation may impair pathogen clearance. Our goal to achieve immunophenotypic stratification of patients may therefore inform host-directed therapeutic strategies.

Methods: In this study, longitudinal whole-blood immunophenotyping was performed in adult pulmonary TB patients. For this purpose, flow cytometry analysis of whole blood cell surface markers, multiplex immunoassay of cytokines in cell plasma and microarray analysis of whole blood transcriptomics were performed on a clinically well characterized cohort.

Results: Temporal changes in T-cell and antigen-presenting-cell populations stratified patients and controls into distinct cell-based clusters. Early during treatment, one cluster exhibited a dysregulated activation-suppression phenotype characterized by increased frequencies of PD-L1⁺ myeloid cells, terminally differentiated T-cells, elevated inflammatory cytokines, and altered transcriptomic profiles consistent with simultaneous immune activation and regulatory exhaustion. Early-treatment cluster membership was associated with a high sputum mycobacterial burden, and persistence of the dysregulated phenotype after 14 days of therapy was linked to a longer time to sputum culture conversion.

Conclusions: These findings reveal substantial immunophenotypic heterogeneity associated with TB treatment response and support the development of personalized host-directed therapy approaches.

OP5: Structural and Functional Plasticity of the Staphylococcus aureus Virulence-Associated Amyloid Peptide PSMα1

Sambhasan Banerjee ¹, Valeriia Skoryk ^2,3, Bader Rayan ⁴, Eilon Barnea ⁴, Enrico Caragliano ^1,5,6, Aleksandr Golubev ¹, Nikolay Vinogradov ³, David Flores ^7,8, Shindhuja Joel ¹, Mariana Pigozzi Cali ¹, Emil Gustavsson ¹, Jens Bosse ^1,5,6, Markus Zweckstetter ^7,8,9, Thomas Gutsmann ¹⁰, Oxana Klementieva ^2,3, Meytal Landau ^1,4,11

¹CSSB, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany,²Medical Microspectroscopy Research Group, Lund University, Lund, Sweden,³MAX IV Laboratory, Lund University, Lund, Sweden,⁴Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel,⁵Institute of Virology, Hannover Medical School, Hannover, Germany,⁶Cluster of Excellence RESIST, Hannover Medical School, Hannover, Germany,⁷Department for NMR-based Structural Biology, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany,⁸German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany,⁹Department of Neurology, University Medical Center Göttingen, Göttingen, Germany,¹⁰CSSB, Hamburg, and Research Centre Borstel, Leibniz Lung Center, Borstel, Germany,¹¹Center for Experimental Medicine, Universitätsklinikum Hamburg-Eppendorf (UKE), Hamburg, Germany

Phenol-soluble modulin α1 (PSMα1) is a cytolytic peptide secreted by Staphylococcus aureus that contributes to host-cell damage and biofilm stability, yet how its assembly landscape relates to function remains incompletely understood. We combined live-cell imaging, cytotoxicity assays, NMR, cryo-EM, AFM, X-ray fibre diffraction and vibrational spectroscopy to define PSMα1 structural and functional plasticity. PSMα1 accumulated at the plasma membrane before detectable permeabilization. Epigallocatechin gallate (EGCG) markedly reduced membrane-association and cytotoxicity despite only weak, distributed interactions with soluble PSMα1. Cryo-EM revealed two canonical cross-β fibril polymorphs with distinct protofilament arrangements but a conserved hydrophobic core and interface. pH redirected PSMα1 into different supramolecular states: extreme acidic and alkaline conditions favoured cross-β amyloid fibrils, whereas pH 3.9-7.9 stabilized heterogeneous α-helical nanotubes with cross-α-like packing. At neutral pH, nanotubes remained stable after heating to 90 °C, while under acidic conditions they appeared as transient intermediates preceding cross-β fibrils. Thus, PSMα1 is an environmentally responsive assembly system rather than a structurally fixed amyloid, linking physicochemical conditions to supramolecular architecture, membrane interaction and cytotoxic activity.

OP6: Environmentally persistent free radicals enhance respiratory syncytial virus replication in respiratory epithelium

Ayaho Yamamoto ¹, Peter D Sly ¹, Lavrent Khachatryan ², Stephania A Cormier ³, Paul D Robinson ¹

¹Child Health Research Centre, The University of Queensland, South Brisbane, Queensland, Australia,²Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana, United States,³Department of Biological Sciences, and Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana, United States

Introduction: Epidemiological evidence linking exposure to poor air quality with respiratory infections is substantial. Exposure to combustion-related air pollution, particularly that associated with traffic and bushfires, is associated with an increased risk of severe respiratory infections, including respiratory syncytial virus (RSV), reduced lung function, wheezing illnesses, incident asthma, and asthma exacerbations. Environmentally persistent free radicals (EPFRs) are oxidant components of air pollution and are present in substantial numbers on particulate matter (PM). EPFRs have half-lives of days to years resulting in prolonged biological effects. However, the precise mechanism linking pollution exposure to adverse respiratory outcomes remains unclear.

Aims: To determine whether EPFR exposure increases the severity of RSV infection of the nasal epithelium and the utility of antioxidants.

Methods: Primary human nasal epithelial cells from healthy adults were grown and differentiated at an air-liquid interface and pretreated with 10 µM astaxanthin. The well-differentiated epithelia were exposed to EPFRs for 3 days using a VITROCELL® Cloud 12 system, followed by RSV infection (MOI: 0.1). Outcomes included epithelial integrity, cytokine secretion, mRNA and protein expression.

Results: EPFR exposure prior to infection increased RSV replication by 1.6-fold, whereas astaxanthin prevented this increase. EPFR exposure reduced tight junction ZO-1 expression with a significantly greater decline in combination of RSV infection. EPFR exposure and RSV infection also decreased the antioxidant enzymes, CAT, SOD1 and TRX, while astaxanthin prevented these reductions.

Discussion: Our results demonstrate that EPFR exposure increases the severity of RSV infection by disrupting tight junction integrity and oxidative stress defence in respiratory epithelium.

Poster Presentations

P1: Poster Hidden in the pipes: high prevalence of non-tuberculous mycobacteria in household water in Northern Germany

Fateme Ghoroghi ¹, Alexander Mischnik ², Stefan Niemann ³, Margo Diricks ⁴

^1,3,4MolMyk, Research Centre Borstel, Germany,^2,3National Reference Center for Mycobacteria (NRZ), Research Centre Borstel, Germany

Infections with non-tuberculous mycobacteria (NTM) are an increasing public health concern, particularly among individuals with pre-existing chronic lung disease. Despite their ubiquity in the environment, the routes of NTM persistence and transmission through water systems remain poorly understood. We investigated NTM prevalence in household water samples collected by master’s students and teachers participating in an Infection Biology course in Schleswig-Holstein, Germany. A total of 15 samples (7 tap and 8 shower water) were cultured at 30°C and 37°C using MGIT, NTM Elite agar, and 7H10 media, and analysed by qPCR. Participants also completed a questionnaire on household characteristics. NTM were detected in 7/15 samples (47%) from 6/14 households. Identified species included Mycobacterium chimaera, M. chelonae, M. gordonae, M. gilvum, and a potential novel species. Culture-based detection was more sensitive than qPCR, while no clear association was found with building age or boiler temperature. To investigate potential sources, one positive household and neighboring households with the same construction date were resampled. The same NTM species were detected, showing closely related genetic profiles. However, subsequent sampling of the upstream water-source distributor was negative. These findings suggest that NTM may become established within downstream sections of the water distribution system. In particular, underground pipes and service (lateral) lines connecting individual properties to the main water network may represent important niches for NTM biofilm formation, persistence, and local dissemination. Overall, household water systems may represent relevant reservoirs for NTM exposure, highlighting the potential role of distribution infrastructure in NTM persistence and transmission.

P2: Dissecting immunoproteasome function in lung regeneration

Kai Guo ^1,2, Silke Meiners ^1,2

¹Division of Immunology and Cell Biology, Research Center Borstel/Leibniz Lung Center, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany,²Institute of Experimental Medicine, Christian-Albrechts-University Kiel, Kiel, Germany

Immunoproteasomes (IP) are specialized proteasomes that play critical roles in multiple immune responses. Their three proteolytic subunits (PSMB8-10) are expressed at low levels in non-immune cells, such as alveolar epithelial cells, but are induced upon virus infection or stimulation with interferons (IFNs) thereby contributing to altered cellular protein degradation. As prolonged signalling of IFNs impairs proliferation of AT2 cells, we hypothesized that this effect is mediated by the induction of the immunoproteasome bestowing a role in lung regeneration.

Single cell RNA sequencing data analysis revealed that the IP is dynamically regulated upon AT2 to AT1 differentiation in mouse and human organoids with a transient increase in the AT0 stage. We confirmed this transient upregulation of the IP in a feeder-free mouse alveolar organoid system. Treatment of WT organoids with IFNg strongly stimulated IP expression and impaired organoid formation. Organoid formation, however, was similar in IFNg treated organoids derived from AT2 cells that had been isolated from IP triple KO mice. These data suggest that the IP is not mediating the anti-regenerative effects of IFNg. In contrast, we observed improved AT1 differentiation in IP deficient AT2 cells at baseline as revealed by increased AT1-marker gene expression. Gene expression analysis showed that IP deleted AT2 cells have slightly higher expression of the stress responsive genes ATF4, Ddit3 and Nrf2 compared to WT cells suggesting that the loss of IP induces a mild cellular stress response, which has previously been shown to be required for AT1 differentiation. We are currently investigating the signals leading to the transient induction of the IP in AT0 cells to better understand the role of the IP in lung regeneration.

P3: Eco-friendly Manufacturing of Al₂O₃ Nanoparticles by Urtica dioica: Structural Insights and Biomedical Applications

Dilan Jassim Khalil ¹, Nishtiman Younis Mosa ², Jasim M. Abdo ³

¹Scientific Research Center, university of Duhok, Duhok, Iraq,²Anesthesia Department, university of Duhok, Duhok, Iraq,³Biology Department, university of Zakho, Zakho, Iraq

Stinging nettle (Urtica dioica) aqueous extract was employed as a natural reducing and stabilizing agent for the environmentally friendly synthesis of aluminum oxide (Al₂O₃) nanoparticles. The biosynthesized nanoparticles were characterized using UV–visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). UV–visible analysis confirmed nanoparticle formation through a characteristic absorption peak at approximately 230 nm. FTIR analysis revealed Al–O bonds and phytochemical functional groups involved in the reduction and stabilization processes. XRD patterns confirmed the crystalline nature of the nanoparticles, with an average crystallite size of approximately 20 nm. Diffraction peaks at 2θ values of 37°, 45°, 60°, and 67° were assigned to the (311), (400), (511), and (440) planes of Al₂O₃, respectively. SEM analysis demonstrated nanoscale morphology, with particle size distribution influenced by precursor molarity. The biological activity of the synthesized nanoparticles was evaluated against Staphylococcus aureus, Escherichia coli, and Candida albicans. The nanoparticles exhibited strong antibacterial activity, with inhibition zones and minimum inhibitory concentration (MIC) values ranging from 100 to 250 µg/mL, while minimum bactericidal concentration (MBC) results confirmed bactericidal effects. No antifungal activity was observed against C. albicans. Hemolysis assays indicated good hemocompatibility and no significant harmful effects at the tested concentrations. The DPPH assay showed strong dose-dependent antioxidant activity for the plant extract, whereas Al₂O₃ nanoparticles exhibited comparatively weaker activity. Overall, this study demonstrates a sustainable phytochemical-assisted approach for producing crystalline Al₂O₃ nanoparticles with promising antibacterial activity, antioxidant potential, and acceptable blood compatibility for biomedical applications.

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P4: Quantitative Assessment of Proteasome and Immunoproteasome Activity in Human THP-1 Cells Using Activity-Based Probes

Niels Röckendorf ¹, Silke Meiners ¹

¹Immunology and Cellbiology, Research Center Borstel – Leibniz Lung Center, Borstel, Germany

The 20S proteasome is a central regulator of protein homeostasis and antigen processing in immune cells. In response to inflammatory stimuli such as IFN-γ, the constitutive proteasome is replaced by the immunoproteasome that contains catalytic subunits β1i, β2i, and β5i enhancing the generation of MHC class I–presented peptides. However, assessing the functional activity of these protein complexes remains challenging due to methodological limitations in particular under treatment with proteasome inhibiting agents. Therefore, we validated existing quantitative, and functional assays to monitor both constitutive and immunoproteasome activity in human monocytic THP-1 cells using new and established activity-based probes (ABPs). THP1 cells were lysed and incubated with covalently binding (immuno)proteasome inhibitors directed against specific active β-subunits. Subsequently, cells were treated with a set of subunit-specific ABPs targeting the same catalytically active threonine residue in the respective subunits as the inhibitors. Remaining relative activity of proteasome and immunoproteasome subunits under inhibitor treatment was quantified by an in-gel fluorescence assay. This way subunit specificity of various proteasome inhibitors at different concentrations was quantitatively analysed. In conclusion, activity-based probes enable direct, quantitative, and functional profiling of both proteasome and immunoproteasome activity in human THP-1 cells. This approach provides a powerful tool to evaluate site- and pan-specific proteasome inhibitors as immunomodulatory drugs, and investigate the role of proteasome dynamics in inflammatory and autoimmune disease.

P5: NTHi-induced exacerbation in a COPD mouse model with comorbid obesity

Sheikh Anika Rahman ¹, Barbara Hammer ¹, Bruno Steimbruech ², Xijie Zhang ¹, Matthias Merker ³, Margo Diricks ⁴, Jan Rupp ^5,6, Susanne Krauss-Etschmann ^1,2,7

¹Early Life Origins of CLD, Priority Area Asthma and Allergies, Research Center Borstel, Leibniz Lung Center, Borstel; Member of the German Center for Lung Research (DZL), Germany,²DZL Laboratory for Experimental Microbiome Research, Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,³Evolution of the Resistome, Research Center Borstel, Borstel, Germany,⁴Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany,⁵Infectious Disease -Clinic and Institute of Medical Microbiology, University Hospital Schleswig-Holstein, Lübeck, Germany,⁶Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems, Lübeck, Germany,⁷Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany

Background: COPD is frequently linked to obesity, a comorbidity associated with higher airway disease severity. Infection-induced exacerbations drive disease progression, with non-typeable Haemophilus influenzae (NTHi) being the most common pathogen. However, markers that indicate an imminent exacerbation are currently lacking. We therefore investigated these mechanisms using a mouse model of smoke-induced COPD with comorbid obesity and NTHi infection.

Methods: Female C57BL/6J wildtype mice were fed either a high-fat diet (HFD) or control diet (CD) ad libitum; in parallel, exposed to cigarette smoke (CS) or room air (RA) for 6 months, followed by infection with 1×10⁶ CFU of NTHi isolate (strain H-189).

Results: Longitudinal plethysmography revealed reduced breathing frequency and airflow limitation in CS mice. Invasive lung function showed increased hyperresponsiveness in non-infected HFD mice, while CS+HFD promoted emphysema. Consistent with these functional findings, lung histological assessment demonstrated emphysema-like structural changes. Following NTHi infection, histological inflammatory scoring revealed increased pulmonary inflammation in CS-NTHi mice, independent of HFD. At the cellular level, bronchoalveolar lavage (BAL) cytology also revealed significantly higher neutrophil levels in CS-NTHi mice at 48h post-infection (pif), which was not observed in RA-NTHi controls.

Conclusion: Smoking and obesity act additively on the COPD phenotype with a faster progression of lung function decline and emphysema-like changes. However, the altered inflammatory response to NTHi appeared to be associated with CS exposure and independent of HFD. Ongoing RNASeq analyses aim to identify early markers of recurrent exacerbation events for early intervention and prevention of irreversible lung function loss in these patients.

P6: Bioactive Green-Synthesized ZnO Nanoparticles: Antimicrobial, Antioxidant, and Cytotoxicity Profiling 

Nishtiman Younis Mosa ¹, Dilan Jassim Khalil ², Jasim M. Abdo ³

¹Anesthesia Department, university of Duhok, Duhok, Iraq,²Scientific Research Center, university of Duhok, Duhok, Iraq,³Biology Department, university of Zakho, Zakho, Iraq

The scalable and eco-friendly synthesis of nanoparticles with potent antibacterial and antioxidant properties, while ensuring biocompatibility and minimizing cytotoxicity, is crucial for their safe and effective application in biomedical fields. In this study, zinc oxide nanoparticles (ZnO NPs) were successfully synthesized under mild and environmentally friendly conditions using an aqueous extract of ribwort plantain (Plantago lanceolata) leaves. The synthesized nanoparticles were designated as ZR. Their structural and morphological properties were characterized using conventional spectroscopic and microscopic techniques. The average particle size of the synthesized ZR nanoparticles was approximately 25 ± 5 nm.

The synthesized ZR NPs exhibited significant antimicrobial activity against the tested microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans. ZR demonstrated higher antimicrobial activity against these microorganisms than the standard antibiotics azithromycin and amoxicillin. Notably, ZR exhibited greater antimicrobial efficacy against the fungal strain C. albicans than against the tested bacterial strains.

The minimum bactericidal concentration (MBC) values were 100, 25, and 100 µg/mL for C. albicans, E. coli, and S. aureus, respectively. The minimum inhibitory concentration (MIC) values were reported as 12.5, 10.5, and 12.5 µg/mL, respectively. These findings indicate that ZR possesses considerable antimicrobial activity against the tested microorganisms.

The hemolysis assay revealed that ZR caused minimal damage to red blood cells, with a hemolysis rate of only 3%, indicating a low hemolytic effect and favorable blood compatibility. Microscopic examination of blood smears before and after treatment with ZR showed no detectable morphological changes in red blood cells. In addition, the biocompatibility assay, with sodium dodecyl sulfate (SDS) and normal saline used as controls, demonstrated that ZR did not cause significant toxicity at concentrations up to 100 µg/mL. The observed lysis rate was only 1.9%, further supporting the favorable biocompatibility of the synthesized nanoparticles.

Regarding antioxidant activity, ZR exhibited only a slight antioxidant effect. Therefore, its biological activity appears to be more pronounced against the tested microbial strains than in the antioxidant assay.

Overall, the findings demonstrate that the biogenic synthesis of ZnO nanoparticles using ribwort plantain leaf extract provides an environmentally friendly and mild approach for producing nanoparticles with promising antimicrobial activity and favorable biocompatibility. The low hemolytic and lysis rates, together with the absence of observable morphological alterations in red blood cells, suggest that ZR has good biocompatibility under the tested conditions. These characteristics make ZR promising candidates for future biological and biomedical applications, particularly in antimicrobial applications.

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P7: Molecular Mechanisms Underlying the Uptake and Intracellular Fate of Methanogenic Archaea in Human Innate Immune Cells

Shoshant Sah ¹, Holger Heine ¹

¹Research Group Innate Immunity, Research Center Borstel, Leibniz Lung Center, Borstel, Germany

The human microbiome comprises not only bacteria but also archaea, whose interactions with the host immune system remain poorly understood. Unlike bacteria, archaea lack several classical bacterial pathogen-associated molecular patterns, including lipopolysaccharide and peptidoglycan, raising questions about the mechanisms underlying their recognition and uptake by innate immune cells. My PhD project investigates the host-cell receptors and signaling pathways involved in the interaction of human innate immune cells with the methanogenic archaea Methanosphaera stadtmanae and Methanobrevibacter smithii. A βestradiol-induced transdifferentiation model based on CEBRA cells has been established to investigate these interactions, and confocal microscopy has been used to examine the intracellular localization of fluorescently labelled M. stadtmanae. To identify host-cell receptors involved in archaeal uptake, the project combines targeted approaches, including siRNA-mediated gene silencing and receptor-blocking antibodies, with the development of a lentiviral CRISPR/Cas9-based screening strategy. Candidate receptors are being investigated through gene expression analysis, protein-expression assessment, and functional uptake assays in cell lines and primary human monocytes. By elucidating the molecular mechanisms governing archaeal recognition and internalization, this work aims to advance our understanding of innate immune responses to methanogenic archaea and their potential roles in immune homeostasis and inflammatory processes.

P8: Label-free identification and sorting of Mycobacterium tuberculosis-infected human monocyte-derived macrophages using autofluorescence and imaging-derived parameters

Panetas, Ioannis ¹, Ferrer-Font, Laura ², Aboutara, Nadine ^3,4, Prosser, Gareth ⁵, Hein, Manuel ⁶, Hein, Martina ⁶, Scholzen, Thomas ⁶, Reiling, Norbert ^4,5, Schwudke, Dominik ^3,4,7, Behrends, Jochen ⁶

¹FlowJo Informatics, Waters Biosciences, Ashland, OR, USA,²Scientific Applications, Waters Biosciences, Milpitas, CA, USA,³Division of Bioanalytical Chemistry, Research Center Borstel, Germany,⁴DZIF, Hamburg-Lübeck-Borstel-Riems, Germany,⁵Microbial Interface Biology, Borstel, Germany,⁶Core Facility Flow Cytometry, Borstel, Germany,⁷DZL, ARCN, Germany

Background: Conventional identification of Mycobacterium tuberculosis (Mtb)-infected human monocyte-derived macrophages (MDMs) by flow cytometry typically relies on fluorescent probes or genetically modified bacteria, potentially limiting downstream applications. We investigated whether intrinsic autofluorescence and infection-induced morphological changes could enable label-free identification and sorting of infected cells.

Methods: MDMs from three healthy donors were infected for 72 h with wild-type Mtb (H37Ra) or a Scarlet-I3-expressing strain as positive control. Cells were analyzed on the BD FACSDiscover™ S8 Cell Sorter combining spectral fluorescence with single-cell imaging. Concatenated data were visualized in FlowJo™ using t-SNE, followed by Phenograph clustering based on image-derived parameters, excluding Scarlet-I3-associated channels. BD CellView™ Lens visualized representative clusters. HyperFinder identified discriminative imaging features and generated a simplified gating strategy for sorting infected populations. Sorted cells were validated by confocal microscopy.

Results: Infected and uninfected MDMs were discriminated label-free using intrinsic autofluorescence and imaging-derived morphological features. Clusters identified in Scarlet-I3-infected macrophages, without using Scarlet-I3 fluorescence, closely mirrored wild-type infected populations and were distinct from uninfected cells. HyperFinder translated these signatures into a simplified 2D gating strategy enabling reproducible sorting of both infected populations. Confocal microscopy confirmed Mtb presence and showed increasing bacterial load with enrichment of infected-cell cluster signatures.

Conclusion: Spectral flow cytometry integrated with real-time imaging enables label-free identification and sorting of Mtb-infected macrophages using computational and simplified gating strategies. This workflow supports downstream transcriptomic and proteomic applications, expanding the utility of cytometric sorting in infectious disease research.

P9: Immunophenotyping of gut-colonised mice to study the gut-lung axis in lung health

Kodwo Appoh Odum ^1,7, Draginja Kovacevic ^1,7, Jochen Behrends ², Barbara Hammer ^3,7, Silvia Bolsega ⁴, Marijana Basic ⁴, Holger Heine ^1,5,7 and Susanne Krauss-Etschmann ^1,3,6,7

¹DZL Laboratory for Experimental Microbiome Research, Research Center Borstel-Leibniz Lung Center, Borstel, Germany; ²Core Facility Fluorescence Cytometry, Research Center Borstel, Borstel, Germany,³Research group Early Life Origins of Chronic Lung Disease, Research Center Borstel-Leibniz Lung Center, Germany; ⁴Institute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany,⁵Research group Innate Immunity, Research Center Borstel-Leibniz Lung Center, Germany; ⁶Institute of Experimental Medicine, Christian-Albrechts-Universität of Kiel, Kiel, Germany,⁷Member of the German Center for Lung Research (DZL), Airway Research Center North (ARCN), Borstel, Germany.

Development of chronic respiratory diseases such as asthma is influenced by multiple factors, including the microbiome and its circulating metabolites. With increasing evidence linking pathobionts such as Haemophilus influenza to the development of childhood asthma, efforts have been intensified to understand the underlying reasons. The gut microbiota can influence distant organs, including the lungs, potentially affecting lung health. However, experimental models to study microbiota-related effects on lung health via the gut-lung axis are largely lacking. Gnotobiotic mice are a valuable tool to study microbe-microbe and microbe-host interactions in vivo.

We immunophenotyped gnotobiotic germ-free (GF) and Oligo-mouse microbiota (OMM12), and compared them to conventionally colonized specified pathogen free (SPF) mice at different ages, to study microbiota-related influences on the developing immune system, with focus on asthma-related cells. Flow cytometric analyses revealed a strong influence of the gut microbiota on lung cell types such as eosinophils, ILCs and CD8+ T cells, all implicated in asthma development, whiles other cell-types were significantly influenced by both the microbiota and age of mice. Using hydrolysis probe-based qPCR, we observed that the lungs of the gnotobiotic GF and OMM12-colonised mice are completely sterile. This finding provides an important basis for future investigations whether Haemophilus influenzae plays a causal or consequential role in asthma development.

Next, we will investigate which microbiota-derived metabolites may play a role in influencing the immune cell development by analysing serum samples. Additionally, lung tissues will be sequenced to further confirm its sterility.

P10: Age dependent sex differences in DNA methylation Age in asthma

Henriette Tugiremariya ^1,2, Sonja Buchholz ^1,2, Antonia Bohnhorst ^1,2,3, Shauni Belana Biedermann ^1,2,3, Wasifa Nurieva ^1,2, Bianca Schaub ^{4, 5,6}, Matthias Volkmar Kopp ^{1,2, 9} , Erika von Mutius ^4,10, Nicole Maison ^4,5,10, Thomas Bahmer ^1,11, Klaus F. Rabe ^1,11,12, Carsten B. Schmidt-Weber ^4,13, Constanze A. Jackwerth ^4,13, Tobias Trojan ¹⁴, Miguel A. Alejandre Alcazar ^14,15,16, Ruth Grychtol ^1,7,8, Ayat Ismail ¹⁹, Inken Wohlers ^1,19,20, G. Hansen ^21,22,23, A. M. Dittrich ^21,22,23, Folke Brinkmann ^2,3 Markus Weckmann ^1,2,3,24 , Karosham D. Reddy ^1,2,3 & ALLIANCE Study Group

¹Airway Research Centre North, Member of the German Centre for Lung Research (DZL), Germany,²Department of Paediatric Pneumology & Allergology, University Children’s Hospital Schleswig-Holstein (UKSH), Lübeck, Germany,³Epigenetics of Chronic Lung Diseases, Priority Area Chronic Lung Diseases, Research Centre Borstel - Leibniz Lung Centre, Borstel, Germany Comprehensive Pneumology Centre, Munich (CPC-M), Germany; German Centre for Lung Research (DZL),⁴Dr. von Hauner Children’s Hospital, University Hospital, Ludwig Maximillian’s University (LMU), Munich, Germany,⁵DZKJ, Member of German Center for Child and Adolescent Health (DZKJ), Dr von Hauner Children’s Hospital, LMU, Munich, Germany,⁶Translational Paediatric pneumology, Department of Paediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany,⁷Biomedical Research in End stage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre of Lung Research (DZL), Germany,⁸Department of Paediatric Respiratory Medicine, Inselspital, University Children's Hospital of Bern, University of Bern, Bern, Switzerland,⁹Institute for Asthma and Allergy Prevention (IAP), Helmholtz Centre Munich, German Research Centre for Environmental Health (GmbH), Neuherberg, Germany,¹⁰University Hospital Schleswig-Holstein Campus Kiel, Department for Internal Medicine I, Kiel, Germany,¹¹Department of Pneumology, LungenClinic Grosshansdorf, Grosshansdorf, Germany,¹²Centre of Allergy and Environment (ZAUM), Technische Universität and Helmholtz Centre Munich, Munich, Germany,¹³University of Cologne, Faculty of Medicine and University Hospital Cologne, Translational Experimental Pediatrics, Department of Pediatric and Adolescent Medicine, associated member of the German Centre for Lung Disease (DZL), Gießen, Germany,¹⁴Excellence Cluster on Stress Responses in Aging-associated Diseases (CECAD) and Centre for Molecular Medicine Cologne (CMMC), University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.,¹⁵Institute for Lung Health (ILH), Cardiopulmonary Institute (CPI), University of Gießen and Marburg Lung Centre (UGMLC), Member of the German Centre for Lung Research (DZL), Gießen, Germany,¹⁶Division of Clinical and Molecular Allergology, PA Chronic Lung Diseases, Research Center Borstel, Borstel, Germany,¹⁷Interdisciplinary Allergy Outpatient Clinic, Department of Pneumology, University of Lübeck, Lübeck, Germany,¹⁸Biomolecular Data Science in Pneumology, Research Center Borstel, Leibniz Lung Center, Borstel,¹⁹University of Lübeck, Lübeck, Germany.,²⁰Department of Paediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany,²¹Biomedical Research in End stage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre of Lung Research (DZL), Hannover, Germany,²²Cluster of Excellence RESIST (EXC 2155), German Research Foundation (DFG), Hannover Medical School, Hannover, Germany,²³Cluster of Excellence PMI (EXC 2167), German Research Foundation (DFG), Leibniz Research Center Borstel, Borstel, Germany

Asthma exhibits age associated sex differences, being more severe in boys during childhood and in females after puberty. Underlying mechanisms remain incompletely understood. Interleukin (IL)-6 is a key factor driving accelerated biological aging (DNAge) and asthma severity. The relationship between IL-6, DNAge and sex differences in asthma remains unclear.

We aim to investigate DNAge differences between asthmatic males and females, focusing on IL-6 DNA methylation (IL6m).

Nasal brushings from the ALLIANCE cohort (Pediatric:n=60F/92M; asthma (A)=74, healthy (H)=43, wheeze=35; avg_age =10yr, IQR=4–14yr; Adult:n=117F/97M; A=172, H=42; avg_age =55yr, IQR=42–65yr) were collected. DNA methylation (DNAm) analysed by Illumina EPIC 850K array using R (v4.5.1). Using skinHorvath clock, DNAge acceleration was calculated relative to chronological age. Based on symptoms reported over the previous 4 weeks, patients were classified as controlled, partially controlled, or uncontrolled. IL6m was assessed via Ward hierarchical clustering to stratify patients into high and low IL6m clusters. Patient distribution between IL6m status and DNAge association was assessed by Chi-square testing.

Chronological age correlated strongly with DNAge (pediatric R=0.92; adult R=0.81). Pediatric, uncontrolled females showed higher DNAge than males (0.99yr;p<0.05) further increased post-puberty (1.52yr;p<0.05). High IL6m was associated with decelerated DNAge in female children (p<0.05) but not in males. In adults, this association was observed regardless of sex (p<0.001).

HS pediatric asthmatic females exhibited higher DNAge than males specifically post-puberty. IL6m and DNAge demonstrated sex and age specific associations. Further research is needed to determine how the relationship between asthma, sex and DNAge contributes to asthma heterogeneity.

P11: Targeting IL-13 signalling pathway as a potential therapeutic approach in experimental models of pulmonary arterial hypertension.

Jakia Khan^a, Lifang Wen^a, Christoph Hölscher^b, Jacqueline Wax^a, Xiaoyang Yue^a, Muzamil Majid Khan^c, Torsten Goldmann^d, Gabriela Riemekasten^e, Xinhua Yu^a, Frank Petersen^a

¹Division of Pulmonary Immune Diseases, Research Center Borstel, Leibniz Lung Center, Priority Area Chronic Lung Diseases, Borstel, Germany,²Division of Infection Immunology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,³Department of Rheumatology and Clinical Immunology, University Clinic of Schleswig Holstein, University of Lübeck, 23538 Lübeck, Germany

Systemic sclerosis (SSc) is a severe autoimmune disease characterized by inflammation, fibrosis, and vasculopathy. Pulmonary arterial hypertension (PAH), one of its most fatal complications, is driven by progressive pulmonary vascular remodeling and vascular occlusion. Current therapies improve symptoms but do not adequately prevent disease progression because the molecular mechanisms underlying vascular remodeling remain incompletely understood. Based on our previous findings, we propose a Two-Hit Hypothesis, in which autoimmune activation provides the initial inflammatory response, while IL-13 overexpression acts as a second hit driving pulmonary vascular remodeling. This study aimed to define the molecular mechanisms underlying IL-13-induced pulmonary vascular remodeling and to establish ex-vivo experimental models for therapeutic evaluation. Our previous in vivo studies demonstrated that IL-13 transgenic (IL-13tg) mice develop severe occlusive pulmonary vasculopathy independent of AT1R immunization, characterized by pulmonary arterial smooth muscle cell (PASMC) hyperplasia. Building on these findings, we investigated the signaling pathway mediating IL-13-induced proliferation in human pulmonary arterial smooth muscle cells (HPASMCs). IL-13 induced dose-dependent HPASMC proliferation, which was significantly inhibited by IL-4Rα blockade, whereas IL-13Rα2 blockade had minimal effect, identifying the IL-13Rα1/IL-4Rα receptor complex as the predominant signaling pathway mediating this response. To complement these studies, we established a long-term mouse precision-cut lung slice (PCLS) model by optimizing culture conditions to maintain tissue viability for more than 14 days. Following IL-13 stimulation, H&E and α-smooth muscle actin (α-SMA) staining demonstrated smooth muscle cell hyperplasia and vascular occlusion in small pulmonary arteries, partially recapitulating the phenotype observed in IL-13tg mice. However, these findings require further validation. Overall, this multi-system approach identifies the IL-13 signaling pathway as a key driver of pulmonary vascular remodeling, provides a translational platform for testing targeted inhibitors, and highlights IL-13 signaling as a promising therapeutic target in SSc-associated PAH.

P12: Atomic Force Microscopy Reveals Cytoadhesion Mechanisms of Plasmodium Falciparum Infected Red Blood Cells

Rieke Bruns ^1,2, David Danicic-Rauchberger ³, Lara Zirwes ³, Johannes Allweier ³, Iris Bruchhaus ³, Thomas Gutsmann ^1,2

¹Research Center Borstel, Leibniz Lung Center, Division of Biophysics, Borstel, Germany,²Centre for Structural Systems Biology, Hamburg, Germany,³Bernhard Nocht Institute for Tropical Medicine, Host-Parasite Interaction, Hamburg, Germany

Malaria remains among the deadliest infectious diseases worldwide, with most fatal cases caused by Plasmodium falciparum. Severe disease results from parasite–host interactions, most notably the cytoadhesion of infected red blood cells (iRBCs) to the vascular endothelium of vital organs. This process is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, large transmembrane proteins concentrated in membrane protrusions termed knobs. Although cytoadhesion is central to malaria pathogenesis, the biophysical mechanisms linking PfEMP1 binding properties, knob architecture, endothelial cell receptor (ECR) profiles, and disease severity remain poorly understood.

In this project, the molecular binding properties between iRBCs and ECRs will be quantified using single-cell force spectroscopy (SCFS), including measurements of binding events, rupture forces, bond lifetimes, and dissociation kinetics. Second, the influence of PfEMP1 on iRBC morphology and mechanics will be characterized, focusing on knob size and elasticity. PfEMP1 distribution will be analyzed by molecular recognition imaging with different binding partners. Third, synthetic peptides derived from ECR binding epitopes will be iteratively optimized and tested as potential inhibitors of cytoadhesion.

Preliminary observations revealed distinct adhesion behaviors, with rolling interactions via CD36 and stable adhesion to other ECRs such as ICAM-1. SCFS demonstrated receptor-specific differences in binding behavior, indicating distinct force regimes and bond stabilities. These findings support the hypothesis that PfEMP1 variant, endothelial cell origin, and physical conditions determine cytoadhesion and influence disease severity. This project provide insight into how cytoadhesion contribute to malaria pathogenesis at molecular level, and aims to generate therapeutically applicable active substances against malaria.

P13: Smoke and Adiposity: Transgenerational Consequences for Respiratory and Systemic Health

Ann-Cathrin Hofacker ^1,2, Thomas Roeder ¹, Susanne Krauss-Etschmann ²

¹Dept. Zoology, Molecular Physiology, CAU Kiel, Kiel, Germany,²PRA Chronic Lung diseases, FZ Borstel, Borstel, Germany

Lifestyle factors such as high-calorie diets and cigarette smoke are major drivers of metabolic and physiological dysfunction, contributing to chronic diseases including respiratory disorders. Yet, their combined impact on organismal physiology and tissue homeostasis remains poorly understood. To address this, we use the fruit fly Drosophila as a model to investigate how these lifestyle-related exposures affect systemic physiology and airway homeostasis.

In intragenerational experiments, flies were exposed to a high-calorie diet and cigarette smoke either individually or in combination. Both interventions significantly shortened lifespan (p<0.0001), with the combination producing the strongest effect (p<0.0001). At the tissue level, both interventions increased intestinal stem cell proliferation and compromised gut integrity (p<0.0001). We also observed structural abnormalities in the intestinal tracheal network, suggesting that lifestyle-related stressors disrupt local airway organization at the gut–trachea interface. Notably, flies exposed to both stressors showed increased susceptibility to airborne challenges (p=0.0002), indicating reduced respiratory resilience.

Building on these findings, we are currently analyzing how these exposures affect airway structure and airway progenitor cells, as well as physiology and metabolism of offsprings: maternal exposure reduced offspring’s lifespan (p<0.0001). Parental detrimental lifestyle significantly impaired tracheal coverage of the intestinal epithelium and affected lipids, and protein metabolism. Interestingly, effects on body composition extended to the subsequent ‘grand-children’-generation, suggesting long-lasting effects of parental lifestyle.

P14: Deciphering the Metabolic Potential of CF Sputum Commensals: An LC-HRMS Guided Approach to Novel Biofunctional Metabolites.

Keshab Bhattarai ¹, Andrew Tony-Odigie ², Alexander Dalpke ², Katarzyna A. Duda ¹

¹Research Group of Biofunctional Metabolites and Structures, Priority Research Area Chronic Lung Diseases, Research Center Borstel, Leibniz Lung Center, Member of German Center for Lung Research (DZL), Airway Research Center North (ARCN), Borstel, Germany

²Dept. of Infectious Diseases, Medical Microbiology and Hygiene, Medical Faculty Heidelberg, Heidelberg University, Heidelberg, Germany, German Center for Lung Research (DZL), Translational Lung Research Center Heidelberg (TLRC), Heidelberg, Germany

Background: The respiratory microbiome plays a vital role in preventing opportunistic infections. Pseudomonas aeruginosa remains a major priority pathogen causing chronic lung infection and high mortality in cystic fibrosis (CF) patients. While certain resident commensal bacteria exhibit antagonistic interactions against P. aeruginosa, identifying and retrieving the specific underlying chemical drivers remains a major natural products bottleneck due to low physiological expression levels and complex matrix masking effects.

Methods: In this study, 20 distinct commensal bacterial strains isolated from sputum samples of CF patient were evaluated. Whole-genome sequencing paired with antiSMASH genomic mining was employed to map biosynthetic gene clusters (BGCs). Untargeted metabolomics was executed via cell-free culture supernatant extraction followed by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). Structural annotations of bacterial metabolites were resolved utilizing mass spectral libraries and database of chemical structures.

Results: Genomic mining revealed rich BGC topographies primarily dominated by Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs) alongside Non-Ribosomal Peptide Synthetase (NRPS) assemblies. While crude cell-free supernatant extracts tested up to 5 mg/mL did not display dominant standalone antimicrobial activity against P. aeruginosa, LC-HRMS profiling successfully dereplicated 31 known metabolites and resolved 8 abundant, highly promising unknown candidate compounds predicted as novel peptides.

Conclusion and Outlook: To circumvent matrix dilution effects, we aimed to fractionate the crude extracts, isolate candidate metabolites, and subsequently verify targeted bioactivity. Detected candidate compounds were computationally annotated as novel peptides with high structural prediction scores. This workflow underscores a high-resolution, metabolomic pipeline to understand the mechanism of Pseudomonas inhibition in CF patients.

P15: Haemophilus Influenza-derived Metabolite May Activate Rhinovirus Induced Trained Immunity in Respiratory Epithelial Cells.

Xiaohua Chu ^1,2, Wasifa Nurieva ^1,2,3, Karosham Diren Reddy ^1,2,3, Fabien Beaufils ^4,5,6, Thomas Trian ^4,5, Laurence Delhaes ^4,5,7, Patrick Berger ^4,5,6, Bianca Schaub ^8,9,10, Matthias Kopp ^1,2,11, Erika von Mutius ^8,12 , Nicole Maison ^8,9,12, Thomas Bahmer ^2,13, Klaus F. Rabe ^2,13,14, Carsten B. Schmidt-Weber ^8,15, Constanze  A. Jackwerth ^8,15, Gesine Hansen ^16,17,18, Anne-Marie Dittrich ^16,17,18, Aleksandra Sarnowicz ¹⁹, Folke Brinkmann ^1,2, Katarzyna A. Duda ¹⁹ & Markus Weckmann ^1,2,3 & ALLIANCE Study Group

¹Division of Paediatric Pneumology & Allergology, University Medical Center Schleswig-Holstein, Luebeck, Germany;²Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany;³Priority Research Area Chronic Lung Diseases: Epigenetic of chronic respiratory diseases, Leibniz Lung Research Center Borstel;⁴Centre de Recherche Cardio-Thoracique de Bordeaux, University of Bordeaux, Bordeaux, France;⁵Centre de Recherche Cardio-Thoracique de Bordeaux, INSERM, Bordeaux, France,⁶Service d’Explorations Fonctionnelles Respiratoires, Centre Hospitalier, Universitaire de Bordeaux, Bordeaux, France, ⁷Laboratoire de Parasitologie-Mycologie, Bordeaux University Hospital, Bordeaux, France,⁸Comprehensive Pneumology Centre, Munich (CPC-M), Germany; German Centre for Lung Research (DZL);⁹Dr. von Hauner Children's Hospital, University Hospital, Ludwig Maximilian's University (LMU), Munich, Germany;¹⁰DZL, Member of German Center for Child and Adolescent Health (DZKJ), Dr von Hauner Children's Hospital, LMU, Munich, Germany;¹¹Department of Paediatric Respiratory Medicine, Inselspital, University Children's Hospital of Bern, University of Bern, Bern, Switzerland;¹²Institute for Asthma and Allergy Prevention (IAP), Helmholtz Centre Munich, German Research Centre for Environmental Health (GmbH), Neuherberg, Germany; ¹³University Hospital Schleswig-Holstein Campus Kiel, Department for Internal Medicine I, Kiel, Germany;¹⁴Department of Pneumology, LungenClinic Grosshansdorf, Grosshansdorf, Germany;¹⁵Centre of Allergy and Environment (ZAUM), Technische Universität and Helmholtz Zentrum München, Munich, Germany;¹⁶Biomedical Research in End Stage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre of Lung Research (DZL), Hannover, Germany;¹⁷Cluster of Excellence RESIST (EXC 2155), German Research Foundation (DFG), Hannover Medical School, Hannover, Germany;¹⁸Cluster of Excellence RESIST (EXC 2155), German Research Foundation (DFG), Hannover Medical School, Hannover, Germany;¹⁹Research Group of Biofunctional Metabolites and Structures, Research Center Borstel, Leibniz Lung Center, Member of German Center for Lung Research (DZL), Airway Research Center North, Borstel, Germany.

Background: Childhood asthma exacerbations are mainly caused by human rhinovirus (HRV) infection and linked to airway microbiome changes. Repeated HRV infections induce epigenetic training in bronchial epithelial cells (Beas-2B), reduce DNA methylation (DNAm) of IL6, which is linked to persistent asthma phenotype. Early-life colonisation with non-typeable Haemophilus influenza (NTHi) increases asthma risk, with an abundant inflammation-related NTHi metabolite hypoxanthine. However, interactions between respiratory infections, DNAm, and hypoxanthine in asthma remain unknown.

Objective: Investigation of whether NTHi-derived hypoxanthine upregulates IL6 gene expression and potentially activates trained immunity induced by prior viral infection.

Methods: To model recurrent viral infections, Beas-2B cells were infected five times with HRV and DNAm was detected by Illumina 450k arrays. Differential methylation was analysed in R (version 4.5.1, limma). Nasal brushings from ALLIANCE children (asthmatics=34/wheezers=19) were analysed using Illumina 850k arrays. Patients were stratified by IL6 DNAm status using Ward hierarchical clustering, followed by trajectory analysis of exacerbations and medication use. To investigate inflammatory effects of hypoxanthine, Beas-2B cells were treated with hypoxanthine followed by detecting inflammatory markers by RT-qPCR.

Results: In Beas-2B cells, 33,826 CpGs (FDR p<0.01) changed with five recurrent HRV infections. A persistent asthma phenotype revealed overlapping methylation sites among its differentially methylated 10,140 CpGs (FDR p<0.01). Hypoxanthine(25 M) induced a 2.5-fold increase in IL6 expression after 3 hours versus control (adjusted p-value < 0.05, n=3).

Conclusion: These findings suggest that recurrent viral exposure and NTHi-derived hypoxanthine promote persistent inflammatory responses in respiratory cells, requiring further study of the underlying interplay of viral and hypoxanthine exposure.

P16: Immunoproteasomes in feto-maternal immune responses and immune priming

Jinyuan Wang ¹, Silke Meiners ²

¹Research Center Borstel/Leibniz Lung Center, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel (Germany),²Institute of Experimental Medicine, Christian-Alberts-University Kiel (Germany)

The immunoproteasome is a specialized type of proteasome constitutively expressed in immune and medullary thymic epithelial cells but inducible in non-immune cells by interferon signalling. It plays a crucial role in establishing self-tolerance, autoimmunity, and infection responses by degrading intracellular, including viral, proteins into MHC class I antigens for CD8+ T cell activation. We propose that the immunoproteasome will be regulated by maternal immune activation (MIA) in response to viral infections. The focus will be on delineating the role of the immunoproteasome in anti-viral immune responses in the pregnant host. We will investigate this by comprehensive analysis of immunoproteasome activation, MHC class I antigen presentation, CD8+ T cell activation in influenza-infected allogenically pregnant mice with wildtype (WT) and constitutive and cell-specific immunoproteasome knockout (KO) background. Moreover, as the immunoproteasome is also crucial for T cell selection in the thymus, we speculate that MIA during pregnancy impacts immune priming and development of tolerance in the offspring by affecting thymic selection processes. The effects of MIA on the offspring will be determined by analysis of the thymus focusing on the regulation of the thymoproteasome and its consequences on CD8+ T cell selection and a/b TCR repertoire. The project positions the immunoproteasome at the crossroads of viral infection and autoimmune responses and provides a potential biomarker to identify pregnant women at risk of severe viral infections.

P17: Association of systemic sclerosis-related interstitial lung disease with tumor-associated antigens and their autoantibodies

Jiaxi Guo ¹, Aiping Ma ², Heqing Huang ³, Shaowei Lin ⁴, Biqi Fu ¹, Anja Kerstein-Stähle ⁵, Frank Petersen ¹, Xinhua Yu ¹

¹Department of Pulmonary Immune Diseases, Research Center Borstel, Borstel, Germany, ²Department of Respiratory and Critical Medicine, The First Affiliated Hospital of Xiamen University, Xiamen, China,³Department of Rheumatology and Clinical Immunology, The First Affiliated Hospital of Xiamen University, Xiamen, China,⁴Department of Nuclear Medicine, The First Affiliated Hospital of Xiamen University, Xiamen, China,⁵Department of Rheumatology and Clinical Immunology, University Clinic of Schleswig Holstein, University of Lübeck, Lübeck, Germany

Background: Interstitial lung disease (ILD) is the leading cause of systemic sclerosis (SSc) patients’ morbidity and mortality. The role of tumor-associated antigens (TAAs) in SSc-ILD remains unclear.

Methods: A total of 106 SSc patients were enrolled at the First Affiliated Hospital of Xiamen University, China. Clinical and serological data, including serum levels of TAAs, were retrospectively collected from medical records. Single-cell RNA sequencing (scRNA-seq) data were obtained from the public GEO database (GSE128169) to evaluate the expression of TAAs in SSc-Fibrosing-ILD (SSc-F-ILD) patients lung tissue. Additionally, immunohistochemical staining was performed to validate carcinoembryonic antigen (CEA) protein expression in SSc-F-ILD patients lung tissue.

Results: Compared with healthy controls, patients with SSc showed significantly elevated serum levels of CEA, cytokeratin 19 fragment (CYFRA 21-1), neuron-specific enolase (NSE), carbohydrate antigen 125 (CA125) and carbohydrate antigen 153 (CA153). Among those SSc-associated TAAs, serum CEA, CYFRA 21-1 and CA153 levels were positively correlated with high-resolution computed tomography (HRCT)-derived alveolar and fibrosis scores. Patients with SSc-ILD showed significantly higher serum levels of CEA, CYFRA 21-1, CA153 and pro-gastrin-releasing peptide (proGRP) than those without ILD. Furthermore, patients with SSc-F-ILD exhibited significantly higher serum levels of CEA, NSE and CA153 than those with SSc-non-F-ILD. scRNA-seq analysis revealed that genes encoding CEA, CYFRA 21-1 and CA153 were predominantly expressed in lung epithelial cells and were significantly upregulated in multiple epithelial subtypes in patients with SSc-F-ILD. Immunohistochemical analysis further confirmed predominant CEA protein expression in lung epithelial cells and increased CEA protein levels in lung tissues from patients with SSc-F-ILD compared with healthy donors.

Conclusions: Serum levels of multiple TAAs, particularly CEA, CYFRA 21-1 and CA153, are elevated in SSc and associated with pulmonary involvement, particularly fibrosing ILD, with lung epithelial cells representing a plausible pulmonary source of these TAAs.

P18: Pangenome Graph-Based Genotyping Identifies Structural Variants Linked to Asthma

Lennart Bartels ^1,2, Tim Hasenbein ^1,2, Daniel Meißner ^1,2,3, Michael Olbrich ⁴, Habiba Alsafar ⁴, Saleh Ibrahim ^4,5, Susanne Krauss-Etschmann ^2,3,6, Inken Wohlers ^1,2,5,7

¹Biomolecular Data Science in Pneumology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,²Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany,³Early Life Origins of Chronic Lung Diseases, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,⁴Center for Biotechnology, Khalifa University of Science and Technology, Abu Dhabi, UAE,⁵College of Medicine & Health Sciences, Khalifa University of Science and Technology, Abu Dhabi, UAE,⁶Institute of Experimental Medicine, University of Kiel, Kiel, Germany,⁷University of Lübeck, Lübeck, Germany

Structural variants (SVs), genomic alterations larger than 50 bp, can substantially affect gene regulation and disease risk. In contrast to single nucleotide polymorphisms (SNPs), which have been extensively studied, SVs have been underexplored. Recent advances in long-read sequencing enable haplotype-resolved genome assemblies capturing variations from SNPs to complex SVs. In parallel, pangenome graphs integrate multiple genomes into a single reference, reducing bias inherent to linear references. The Human Pangenome Reference Consortium (HPRC) has released a high-quality pangenome graph resource based on diverse human genomes.

We used PanGenie, a graph-based genotyping tool, together with the HPRC pangenome graph to genotype SNPs and common SVs from short-read sequencing data of 99 European-ancestry genomes from the 1000 Genomes Project. We then screened for SVs in linkage disequilibrium (LD) with SNPs associated with asthma and reported at least five times in the genome-wide association studies (GWAS) catalog. Gene expression data and transcription factor binding site annotations were used to assess functional relevance.

We identified ten loci with common SV sites in strong LD with asthma-associated GWAS SNPs. Notably, a 68 bp insertion in high LD with rs12788104 was detected upstream of MUC5AC, a structurally variable gene encoding a mucin produced by goblet cells and known to be upregulated in asthma. Single-cell analysis of IL-13 challenged bronchial epithelial cells confirms strong MUC5AC upregulation in secretory cells, supporting a role in asthma pathology.

P19: Molecular Characterisation of Epithelioid Cells in Human Tuberculosis

Nele Bruske ¹, Natalia Shubladze ², Sergo Vashakidze ^2,3, Tobias Dallenga ^4,5, Christiane Kümpers ^6,7, Torsten Goldmann ^1,6, Sebastian Marwitz ^1,6

¹Histology, Research Center Borstel – Leibniz Lung Center, Borstel, Germany,²The National Center for Tuberculosis and Lung Diseases, Tbilisi, Republic of Georgia,³Department of Medicine, The University of Georgia, Tbilisi, Georgia,⁴Cellular Microbiology, Research Center Borstel - Leibniz Lung Center, Borstel, Germany,⁵German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany,⁶Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL),⁷Institute of Pathology, University Hospital Schleswig-Holstein, Campus Luebeck, Luebeck, Germany

Infections with mycobacteria from the Mycobacterium tuberculosis complex are characterised by the formation of granulomas, organised immune structures that recruit macrophages, which undergo a distinct morphological transformation. Resembling epithelial cells with euchromatic nuclei and connections to neighbouring cells, these macrophages have been termed “epithelioid cells”. Animal models have shown that reprogramming events resembling the mesenchymal epithelial reprogramming of embryonic development occur in these cells. While this reprogramming has been observed in human tuberculosis, the spatial organisation within granulomas and possible differences across lesion types remain unclear. This study investigates and quantifies the magnitude of mesenchymal-epithelial transition (MET) of macrophage populations within different lesions of surgical samples from TB patients by multiplexed immunofluorescence (mIF) staining and downstream analysis of cellular neighbourhoods. The panel consisted of antibodies against CD206, the mannose receptor of mature macrophages, and CD68, a glycoprotein of monocytes and macrophages to detect macrophage populations. To investigate mesenchymal epithelial reprogramming, we included an antibody against E-cadherin, which is a membrane protein essential for epithelial cells. The fourth antibody in the panel was directed against ZO-1, which is a component of tight junctions. Preliminary results showed quadruple-positive cells were significantly more abundant within granulomas than in adjacent non-granulomatous tissue or uninfected control regions suggesting increased numbers of activated macrophage populations that can perform an epithelial-like barrier function in granulomas. This indicates that the MET is significantly upregulated in granuloma-associated macrophages, with distinct spatial distribution across different granuloma types, suggesting MET as a driver of granuloma organisation and maturation.

P20: Spatial Profiling of Langhans Giant Cells and their Cellular Neighbourhoods in Human Tuberculosis

Neele Dunst ¹, Natalia Shubladze ³, Sergo Vashakidze ^3,4, Torsten Goldmann ^1,2, Tobias Dallenga ^5,6, Sebastian Marwitz ^1,2

¹Histology, Research Center Borstel - Leibniz Lung Center, Borstel, Germany,²Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany,³National Center for Tuberculosis and Lung Diseases (NCTLD), Tbilisi, Georgia,⁴The University of Georgia, Tbilisi, Georgia,⁵Cellular Microbiology, Program Area Infections, Research Center Borstel - Leibniz Lung Center, Borstel, Germany,⁶German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany.

Tuberculosis (TB) is characterized by the formation of granulomas, highly organized structures composed of diverse immune and stromal cell populations. Langhans giant cells (LGCs) are multinucleated macrophages observed in TB lesions and are thought to contribute to the organization and immune regulation in granulomas. There is little known about LGCs in TB pathology and their cellular neighborhoods remain poorly understood. To investigate the local immune environment of LGCs in TB, 46 formalin-fixed, paraffin-embedded human patient samples were analyzed using multiplex immunofluorescence targeting fibroblasts (FAP), V-ATPase (ATP6V1B2), T cells (CD3), macrophages (CD68) and B cells (CD20). LGCs within whole-slide images were identified based on their characteristic morphology and expression of CD68 and classified according to their specific localization within TB lesions as granuloma-associated and non-granuloma-associated. Cell-type-specific classifiers and the single-cell-resolved spatial microenvironment of LGCs were computed using QuPath and downstream analyses using R package imcRtools, focusing on the abundance and spatial distribution of T cells, B cells and fibroblasts near to LGCs and their V-ATPase expression, as a surrogate of lysosomal acidification. This study provides a thorough investigation of the cellular environment surrounding LGCs in human TB, while also providing insights into their overall spatial distribution in TB lesions. Comparing LGCs according to their association with granulomatous structures and their level of ATP6V1B2 expression may reveal differences in their local cellular microenvironment and provide further insights into the spatial organization and potential heterogeneity of LGCs in human TB.

P21: Transgenerational Consequences of Parental Lifestyle: Individual Effects of Parental Smoking and Obesity on Offspring Systemic Health

Ann-Kathrin Ebeling ¹, Stina Ahrens ¹, Ann-Cathrin Hofacker ^1,2, Susanne Krauss-Etschmann ², Thomas Roeder ¹

¹Dept. Zoology, Molecular Physiology, CAU Kiel, Kiel, Germany,²PRA Chronic Lung diseases, FZ Borstel, Borstel, Germany

Lifestyle in Western societies is characterized by a caloric dense diet with highly processed food containing large amounts of fat and sugar. Additionally, the consumption of stimulants, such as tobacco impacts respiratory and systemic health. However, the transgenerational consequences of such lifestyle habits remain poorly understood although they potentially comprise serious health issues for offsprings.

This project aims to disentangle the impacts of parental diet and smoke exposure on offspring health including the relative contributions of maternal and paternal lifestyle.

Using the model organism Drosophila, we exposed the parental generation either to a Western Diet or to Cigarette smoke. Parental diet was closely related to offsprings body composition and feeding behavior as lipid accumulation was significantly increased (p<0.0001) in offspring who had one parent on Western Diet. Additionally, also the bodyweight of offspring whose mother was on Western Diet showed a significant increase (p<0.0001).

Parental smoke exposure seriously comprised offsprings health as the lifespan was significantly reduced in male and female offsprings (p<0.0001). Besides, female offspring fecundity was impaired (p<0.0001) and bodyweight was increased (p<0.0001).

Furthermore, the human homolog to tracheoles named terminal trachea cells that cover the intestinal tissue were impaired by both stressors individually, highlighting the systemic consequences of both respiratory and intestinal stressors.

P22: Advanced Imaging and AI Analysis Reveal Sex-Specific B Cell Responses During Mycobacterium tuberculosis Infection

David Hertz ¹, Ali A. Tuz ², Sebastian Marwitz ³, Jochen Behrends ⁴, Matthias Gunzer ², Torsten Goldmann ³, Bianca E. Schneider ¹

¹Host determinants in lung infections, Research Center Borstel,²Institute for Experimental Immunology and Imaging, University Hospital Essen,³Core Facility Histology, Research Center Borstel,⁴Core Facility Fluorescence Cytometry, Research Center Borstel

Recent studies have highlighted the importance of B cells in tuberculosis (TB) immunity, challenging the traditional view that these cells are non-essential for TB defense. B cells accumulate at infection sites within organized structures called B cell follicles in the lungs of experimental animals and humans, where they are associated with protective immunity. However, the functional role of B cells in TB remains poorly defined. Beyond antibody production, B cells serve as antigen-presenting cells and secrete cytokines that profoundly shape the local immune environment. Our previous work revealed a sex bias in pulmonary B cell follicle formation, with males developing significantly smaller follicles. This finding unveils a previously unrecognized structural basis for sex-biased TB susceptibility. To analyze these complex structures and elucidate the role of B cell follicles and B cells during Mtb infection, advanced analytical techniques are required. We employed light sheet microscopy, multiplex immunohistochemistry, and artificial intelligence-based flow data analysis to investigate the role of B cells in TB. These techniques provide unprecedented spatial and phenotypic resolution, enabling detailed characterization of cellular interactions and immune responses within the pulmonary microenvironment. Importantly, these approaches are not limited to animal models but can be applied to patient samples, offering translational potential. Our findings contribute to understanding the immunological mechanisms underlying sex-specific differences in TB pathogenesis and may inform the development of targeted therapeutic strategies. By revealing how B cells and their organizational structures influence TB outcomes differently between sexes, this work opens new avenues for personalized approaches to TB prevention and treatment.

P23: Establishment of a human bronchial epithelial barrier model to study mucosal immune responses

Felix Jarecki ^1,3, Marco van Ham ¹, Malena Bos ^2,4, Hortense Slevogt ^2,4, Lothar Jänsch ^1,3

¹CPRO (Cellular Proteome Research), Helmholtz Centre for Infection Research, Braunschweig, Germany,²DINF (Dynamics of Respiratory Infections), Helmholtz Centre for Infection Research, Braunschweig, Germany,³TU Braunschweig, Braunschweig, Germany,⁴Hannover Medical School MHH, Hannover, Germany

Mucosal-associated invariant T (MAIT) cells are multifunctional immune cells contributing to cytotoxic, inflammatory and regulatory responses, wound healing and antiviral defense. Their canonical activation relies on antigen presentation by the MHC-I-related molecule MR1 and recognition via their invariant T cell receptor within an immunological synapse, requiring cell-cell contact. In contrast, this project focuses on perception at the respiratory epithelium of stimuli with contact-independent signaling. We use a Normal human bronchial epithelial (NHBE) cell air-liquid interface (ALI) model to study how apically applied pathogen-associated stimuli are perceived by primary human MAIT cells placed basally. The distance between epithelial barrier and MAIT cells excludes direct contact and thereby isolates contact-independent signaling. Preliminary tests (stimulation with the MR1-ligand 5-OP-RU) have already shown that, despite an increased surface expression of MR1 on NHBE cells, MAIT cell activation (CD69 surface expression) was not observed. First, we define value ranges for quality and physiological relevance of NHBE barrier cultures, combining software-assisted light microscopy analysis (confluence / cell density), TEER measurements and H&E histology with flow cytometry and fluorescence microscopy of differentiation markers (EpCAM, acTub, MUC5AC, CD271, CD66c). Suitable cultures are then stimulated apically and responses are analyzed by mass spectrometry-based proteomics and flow cytometry of activation markers (MR1, CD274, CD279, CD69). Next to pathogen-like stimuli (LPS, Poly(I:C), LTA, MALP-2), also more physiologically relevant models (Staphylococcus aureus, GFP-tagged Herpes simplex) will follow up.

References:

[1] J. Jakob et al., Translatome analyses by bio-orthogonal non-canonical amino acid labeling reveal that MR1-activated MAIT cells induce an M1 phenotype and antiviral programming in antigen-presenting monocytes. 2023, Front. Immunol.14:1091837.

[2] R. Lehmann et al., Differential regulation of the transcriptomic and secretomic landscape of sensor and effector functions of human airway epithelial cells. 2018, Mucosal Immunology, 11, 627-642.

P24: The Drosophila mirtrons miR-1006 and miR-1011 regulate tracheal development and organismal fitness

Fjolla Kadiri ¹, Leizhi Shi ¹, Susanne Krauss-Etschmann ^1,2

¹Division of Early Life Origins of Chronic Lung Diseases, Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,²Institute of Experimental Medicine, Christian-Albrechts University Kiel, Kiel, Germany

Background: Tobacco-smoke exposure disrupts airway epithelial homeostasis and is a major risk factor for chronic lung diseases, including COPD, yet the post-transcriptional regulators involved remain poorly defined. In smoke-exposed Drosophila airways, transcriptomics revealed dysregulated EGFR, WNT and Notch signaling pathways. TargetScan predicted 25 microRNAs targeting all three pathways, and a tracheal screen prioritized two uncharacterized Drosophila-specific mirtrons, miR-1006 and miR-1011. [1].

Methods: Using the btl-GAL4/UAS system, miR-1006 and miR-1011 were inhibited by miRNA sponges or overexpressed specifically in the airway system. Developmental timing, body size and weight, locomotor activity, lifespan, intestinal barrier integrity, hypoxia tolerance and tracheoblast phenotypes were assessed in both sexes. Whole-trachea and tracheoblast transcriptomes were analyzed by RNA sequencing (RNAseq).

Results: Airway-specific inhibition or overexpression of miR-1006 and miR-1011 delayed development and produced distinct, sex-dependent changes in body size, locomotor activity, lifespan, intestinal barrier integrity and hypoxia tolerance. Impairment of barrier integrity and hypoxia tolerance was more pronounced in females. Overexpression reduced tracheoblast area, while miR-1006 overexpression increased apoptosis specifically in females. Whole-trachea RNAseq revealed recurrent dysregulation of ion transport (ppk13) and extracellular matrix-related genes. miR-1011 tracheoblast RNAseq further identified altered metabolic, immune and epithelial migration pathways.

Conclusions: miR-1006 and miR-1011 are dosage-sensitive regulators of airway development and organismal fitness. Their recurrent association with DEG/ENaC family member ppk13 suggests a potential miRNA–ion-channel regulatory axis; the human counterpart ENaC governs airway liquid clearance and is implicated in cystic fibrosis and COPD. Ongoing genetic rescue experiments will test whether ppk13 inhibition rescues the developmental and functional defects.

References:

[1] K. T. Sirocko et al., Early-life exposure to tobacco smoke alters airway signaling pathways and later mortality

in D. melanogaster. Environ. Pollut. 2022, 309, 119696.

P25: From Host–Pathogen Interactions to Polymicrobial Lung Infection: Investigating Stenotrophomonas maltophilia Pathogenesis at the Polarized Airway Epithelium

Alicia Konrath ^{1, 2}, Uwe Mamat ¹, Claire Taylor ¹, Lisann Doose, Thomas Gutsmann ², Ulrich Schaible ^{1, 3}

¹Department of Cellular Microbiology, Program Area Infections, Research Center Borstel, Leibniz Lung Center Institution, Germany,²Department of Biophysics, Program Area Infections, Research Center Borstel, Leibniz Lung Center Institution, Germany,³Department of Immunochemistry and Biochemical Microbiology, University of Lübeck, Germany

Stenotrophomonas maltophilia is an emerging opportunistic pathogen of increasing relevance in chronic respiratory diseases. Its high intrinsic antimicrobial resistance and capacity to form biofilms on different surfaces make it a considerable concern in clinical settings. Yet its interactions with the human airway epithelium remain poorly understood. Moreover, chronic airway infections often are shaped by complex microbial communities, highlighting the need to investigate the pathogenicity of S. maltophilia in a polymicrobial context. One particularly intriguing partner is the environmental fungus Aspergillus fumigatus: both opportunists can be found in the airways of patients with chronic respiratory conditions, yet little is known about how their interaction shapes infection.

Here, we use polarized Calu-3 airway epithelial cells grown at the air-liquid interface (ALI) to investigate host-pathogen interactions of the clinical S. maltophilia isolate Sm314. Microscopy revealed that Sm314 invades the intercellular space of the airway epithelium. This involves the putative proteases DegQ and DegP, which contribute to cleavage of the adherens junction protein E-cadherin. A degQ/degP double-knockout mutant failed to colonize the intercellular space, indicating their importance for epithelial invasion. In addition, electron and fluorescence microscopy demonstrated intracellular bacteria, with a subset localizing to LAMP1-positive compartments, suggesting entry into the endolysosomal pathway.

Building on these findings, we are extending the ALI model to investigate polymicrobial infection with A. fumigatus. Both microorganisms colonize diseased airways and are co-detected in chronic respiratory conditions, especially in cystic fibrosis. We aim to determine how bacterial-fungal interactions influence microbial growth and antimicrobial susceptibility, as well as epithelial integrity and host responses.

P26: Analysis of lipoteichoic acids and glycosyldiacylglycerols of Streptococcus canis

Anna Nehrmann ¹, Miriam Katsburg ², Ursula Schombel ¹, Dominik Schwudke ^1,3,4, Marcus Fulde ^2,5, Nicolas Gisch ¹

¹Division of Bioanalytical Chemistry, Priority Area Infections, Research Center Borstel, Borstel, Germany,²Institute of Microbiology and Epizootics, Centre for Infection Medicine, Consulting Laboratory for beta-haemolytic streptococci in Veterinary Clinics and Practice, Freie Universität Berlin, Berlin, Germany,³German Center for Infection Research, Thematic Translational Unit Tuberculosis, Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany,⁴German Center for Lung Research (DZL), Airway Research Center North (ARCN), Research Center Borstel, Leibniz Lung Center, Borstel, Germany,⁵Institute of Microbiology, University of Veterinary Medicine Hannover, Germany

Streptococcus canis is an opportunistic pathogen observed in animals, especially dogs and cats, but occasionally also in humans. Usually, this pathogen is classified into the Lancefield group G. Recently, two strains of S. canis belonging to Lancefield group C (IMT52870 and IMT56394) were identified. Whole genome sequencing analysis pointed towards a horizontal gene transfer between S. canis and Streptococcus dysgalactiae.^[1]

Besides the Lancefield assignment, little is known about cell wall components of S. canis on the molecular level. The aim of this study is to close this gap of knowledge by structural analysis of the lipoteichoic acid (LTA) and glycosyldiacylglycerols (GLs) from six selected S. canis strains: four group G strains (G361, IMT49926, AGF1074 and AGF1293) and the two group C strains. For this, we applied our expertise for the isolation and structural characterization of Gram-positive bacterial cell wall components by mass spectrometry and nuclear magnetic resonance spectroscopy.^[2,3]

All examined strains express two GLs, α-D-glucose-3-(1,2-diacyl)-glycerol and α-D-glucose-(1→2)-α-D-glucose-3-(1,2-diacyl)-glycerol. They also produce a type I LTA with the diglucosyl-DG as GL anchor and poly-glycerolphosphate chains that are partially substituted with alanine. The conserved structure of GLs and LTAs in S. canis is shared with several other streptococci. Based on this, it was possible to predict the members of the biosynthetic pathway for LTA and GL synthesis in S. canis using BLAST analysis.

References:

[1] Katsburg et al., Vet. Microbiol. 2023

[2] Heß, Waldow, Kohler et al., Nat. Commun. 2017

[3] Gisch, Auger et al., J. Biol. Chem. 2018

P27: Optimization of the microfluidic platform SynerChip for analyzing synergies between antibiotics and antimicrobial peptides on giant lipid vesicles and bacteria.

Henrik Osewold ¹, Janina Nandy ¹, Christian Nehls ¹

¹Division of Biophysics, Research Center Borstel, Leibniz Lung Center, Borstel, Germany

The combination of antimicrobial peptides (AMPs) with antibiotics (ABs) is a promising therapeutic strategy against multi-drug-resistant bacteria. These drug combinations can act synergistic, additively or antagonistically. To identify synergistic combinations, a fast and resource efficient method for screening of various combinations of concentration is therefore desired. For that, we developed the microfluidic Platform SynerChip, which allows us to generate and investigate various combinations of concentrations of two analytes, AMPs and ABs, simultaneously according to their membrane permeabilization efficiency. Taking advantage of microfluidic methods enables us to investigate these interactions resource-efficient and fast. Vesicles filled with fluorescent dye are produced microfluidically by octanol-assisted liposome assembly, hundreds trapped individually in the SynerChip and exposed to the specific titrated concentration-combinations. This allows us to draw a conclusion about the synergistic effects of analytes (AMP-AB) on the vesicle membrane, using a dye release assay.

This work focuses on developing the SynerChip by increasing the number of concentration-combinations, encapsulating bacteria in water-in-oil droplets, and evaluating different mixing strategies of analytes in microfluidic systems. Due to laminar flow in microfluidics, combined flows only mix by diffusion. At the flow rates needed this is not sufficient for ideal mixing of solutes, producing undesired concentration-gradients, notable at the investigated vesicle membranes – a mixing strategy is needed. To minimize the effect of undesired concentration-gradients, the mixing geometry of the microfluidic platform were iteratively optimized by computer-aided-design (CAD), chip fabrication using in-house lithography and experimental testing using fluorescent dyes. Tested mixing strategies are passive advection-based micromixers with intersecting channels.

P28: Airborne Microbiomes and Antimicrobial Resistance Genes in Agricultural Dust

P. Paulsen ¹, S. Pennuttis ², T. Kabelitz ³, T. Gutsmann ², H. Schulenburg ⁴, M. Merker ¹

¹Evolution des Resistoms, Forschungszentrum Borstel,²Biophysik, Forschungszentrum Borstel,³Infektionen und AMR bei Nutztieren, Leibniz-Institut Agrartechnik und Bioökonomie,⁴Evolutionsökologie und Genetik, Christian-Albrechts-Universität zu Kiel

Background: Antimicrobial resistance (AMR) is one of the greatest global health threat, and has been recognized by the United Nations as a major One Health challenge. By 2050, the mortality associated with AMR is predicted to reach 10 Million deaths per year. Livestock production represents an important environmental reservoir, where antimicrobial use selects for resistant bacteria and antimicrobial resistance genes (ARGs). Airborne dust can serve as a transmission vehicle dispersing microbial communities and clinically relevant ARGs beyond farm boundaries.

Methods: This PhD-Project characterizes the microbiome and resistome of dust from pig farms in Germany and Denmark. Farm dust will be analysed using shotgun metagenomic sequencing and targeted qPCR. Taxonomic and resistome profiles will be determined using established bioinformatic tools, to identify farm-specific microbial and resistance signatures and establish a microbiome and resistome atlas of pig-farm dust.

Results: First dust specimens from a pig farm have been collected and processed. A workflow to extract DNA from settled dust samples has been established, and initial DNA samples are currently undergoing sequencing. In parallel, bioinformatic workflows are being established and optimized for subsequent analysis.

Conclusion: The project will provide insights into the airborne dissemination of AMR in livestock production systems an generate genomic resources for environmental AMR surveillance. By integrating environmental metagenomics with advanced resistome analyses, it will improve our understanding of AMR transmission at the human-animal-environment interface. As part of the AiRisk consortium, the project complements biophysical, epidemiological, and quantitative microbial risk assessment studies thereby strengthening interdisciplinary One Health approaches to mitigate the spread of AMR.

P29: Methanogenic Archaea in Human Alveolar Macrophage Models

Peiling. Zhou ^{1, 2, 3} Fan. Xu ^{1, 2}, Holger. Heine ^{1, 2 *}

¹Division of Innate Immunity, Research Center Borstel-Leibniz Lung Center, Borstel, Germany,²Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,³The Second Affiliated Hospital of Zunyi Medical University, Department of Respiratory Medicine, Zunyi, Guizhou, China

*Correspondence: Diese E-Mail-Adresse ist vor Spambots geschützt! Zur Anzeige muss JavaScript eingeschaltet sein. (Holger Heine)

Background: Methanogenic archaea are well established in the human gut and have also been detected in respiratory samples, including bronchoalveolar lavage. However, their biological relevance in the lung remains unclear. Human alveolar macrophages (AMs) are specialized immune cells that maintain pulmonary homeostasis and respond to inhaled microorganisms. Although methanogenic archaea can modulate human immune cells, it is unknown whether their effects depend on macrophage differentiation and lung-specific identity. We therefore investigated archaeal responses across macrophage models and primary human AMs.

Methods: Primary human AMs were exposed to Methanobrevibacter smithii or Methanosphaera stadtmanae and analysed by RNA-seq. In parallel, peripheral blood monocytes were differentiated into conventional monocyte-derived macrophages (MDMs) or alveolar macrophage-like (AML) cells. Cytokine responses to M. stadtmanae, M. smithii, and Methanobrevibacter woesei were assessed by ELISA. AML phenotype was evaluated by flow cytometry, with transcriptional validation ongoing.

Results: In primary AMs from one donor, M. smithii and M. stadtmanae induced distinct transcriptional responses, with upregulated genes linked to transcriptional regulation and chromatin-related functions rather than canonical inflammatory pathways. Across multiple donors, preliminary flow cytometry showed increased expression of selected alveolar-associated markers, including CD64, MARCO, and CD206, with donor-dependent variation. Preliminary ELISA analyses across five donors showed cytokine responses to methanogenic archaea in both MDM and AML models, with variation across donors and archaeal species.

Conclusions: These findings support further investigation of how macrophage differentiation and lung-specific identity shape responses to methanogenic archaea. The AML model is being further validated as a controlled system for studying archaeal interactions with lung macrophages.

References:

[1] T. Vierbuchen, C. Bang, H. Rosigkeit, R.A. Schmitz, H. Heine. The Human-Associated Archaeon Methanosphaera stadtmanae Is Recognized through Its RNA and Induces TLR8-Dependent NLRP3 Inflammasome Activation. Front Immunol. 2017, 8, 1535.

[2] S. Pahari, E. Arnett, J. Simper, et al. A human alveolar macrophage-like cell model for studying innate immune responses to inhaled pathogens. mBio 2023, 14, e00834-23.

[3] S. Pahari, A.L. Neehus, B.C. Trapnell, et al. Generation of human alveolar macrophage-like cells from peripheral blood monocytes. STAR Protoc. 2024, 5, 103061.

[4] P. Blais-Lecours, C. Duchaine, M. Taillefer, et al. Immunogenic properties of archaeal species found in bioaerosols. PLoS ONE 2011, 6, e23326.

[5] K. Koskinen, M.R. Pausan, A.K. Perras, et al. First insights into the diverse human archaeome. mBio 2017, 8, e00824-17.

[6] D.J.M. Barnett, M. Mommers, J. Penders, I.C.W. Arts, C. Thijs. Early-life gut microbiota and risk of childhood asthma. J Allergy Clin Immunol. 2019, 143, 2305–2307.

P30: Increasing the effectiveness of antimicrobial therapy and reducing resistance evolution in Mycobacterium tuberculosis through drug cycling

Nico Rabold ¹, Stefan Niemann ^1,2,3,4, Lindsay Sonnenkalb ^1,2

¹Molecular and Experimental Mycobacteriology, Research Center Borstel Leibniz Lung Center, Borstel, German,²German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel- Riems, Borstel, Germany,³EPHE, PSL University, Paris, France,⁴Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France

Tuberculosis (TB) remains a global health crisis, particularly due to rifampicin- and isoniazid-resistant strains, which are classified as multidrug-resistant (MDR) TB. Current regimens such as BpaLM (bedaquiline, pretomanid, linezolid, and moxifloxacin) have markedly improved outcomes, yet their effectiveness is often limited by treatment-related toxicity. Adverse effects compromise patient adherence, lead to treatment interruptions, and ultimately reduce treatment success. Optimizing how existing drugs are administered, rather than developing new ones alone, offers a promising strategy to address this challenge. Drug cycling, i.e. the intermittent, time-limited administration of individual drugs within a combination regimen, could reduce cumulative toxicity without compromising antimicrobial efficacy. A successful cycling model is expected to lower the incidence of adverse effects, decrease treatment discontinuation, and enhance overall regimen performance. Ultimately, such optimized dosing strategies could improve the quality of care for patients with MDR-TB and contribute to global TB control efforts. This poster will examine existing research on the BPaLM regimen and discuss how its administration could be optimized to improve treatment outcomes in MDR-TB. Building on these findings, it will outline possible in vitro approaches to pilot such strategies.

P31: Soluble Antigen Arrays: Precision, Infection-Sparing Immunotherapy for Autoimmune Disease

Saransh Sharma ¹, Ketan Chandra ¹

¹Molecular Life Sciences, University of Lübeck, Lübeck, Germany

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Autoimmune diseases like type 1 diabetes (T1D) and lupus are still managed with broadly immunosuppressive therapies that shut down the immune system indiscriminately, leaving patients vulnerable to infection. This review examines antigen-specific immunotherapy (ASIT) as a precision alternative and synthesizes recent evidence for soluble antigen arrays (SAgAs) as the platform now closing ASIT's long-standing efficacy gap. Unlike free peptides, which engage T cells weakly and carry anaphylaxis risk, SAgAs tether antigenic peptides multivalently to a hyaluronic acid backbone via hydrolysable or click-chemistry linkers. The result functions as an inverse vaccine, retraining only the disease-driving T cells toward tolerance rather than triggering an immune attack. Preclinical evidence in the non-obese diabetic mouse model shows that SAgA-delivered peptide combinations, but not the same peptides given free, arrest spontaneous T1D onset, sustain antigen-specific T-cell engagement, and upregulate tolerance markers including CD73, IL-10, and PD-1, while lowering anaphylaxis risk. Mechanistic follow-up work shows SAgAs steer antigen-specific T cells toward regulatory or anergic/exhausted states, in contrast to the effector phenotype driven by free peptide. Together, this literature positions SAgAs as a credible precision-immunotherapy platform capable of halting autoimmune disease while sparing the T-cell repertoire needed for infection defence. Extending this linker-tunable approach to human autoantigens relevant to T1D, lupus, and multiple sclerosis is the next translational step toward antigen-specific immunotherapy as a genuine alternative to blanket immunosuppression.

P32: Reprogramming of human macrophages in response to Mycobacterium tuberculosis complex (MTBC) infection

J Schönfeld ¹, K Reddy ^2,3, C Utpatel ¹, M Weckmann ^2,3, S Niemann ^1,4,5,6

¹Molecular and Experimental Mycobacteriology, Research Center Borstel – Leibniz Lung Center, Borstel, Germany,²Epigenetics of Chronic Lung Diseases, Research Center Borstel – Leibniz Lung Center, Borstel, Germany,³Department of Paediatric Pneumology and Allergology, University Clinic Schleswig-Holstein (UKSH), Lübeck, Germany,⁴German Center for Infection Research, Partner site Hamburg-Lübeck-Borstel-Riems, Germany,⁵Laboratoire Biologie Intégrative des Populations, Evolution Moléculaire, EPHE, PSL University, Paris, France,⁶Institut de Systématique, Evolution, Biodiversité, ISYEB, Muséum national d’Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France.

Mycobacterium tuberculosis complex (MTBC) bacteria have evolved diverse strategies to persist within the human host, including potential adaptation to specific human populations. One such strategy involves epigenetic reprogramming of host cells, particularly cells of the innate immune system. DNA methylation, a key epigenetic mechanism, has been shown to be altered in individuals with TB.

We investigate the epigenetic response to MTBC infection using an early-stage human macrophage infection model. We also examine long-term epigenetic changes in TB patients from a Gambian cohort. Specifically, we explore how MTBC lineages L4 and L6 influence DNA methylation and gene expression patterns in host cells. These findings are then correlated with DNA methylation profiles of peripheral blood mononuclear cells (PBMCs) and clinical outcomes, including lung function.

By integrating in vitro and patient-derived data, we aim to identify lineage-specific host responses and epigenetic signatures associated with disease outcome. Our results can offer new insights into how MTBC modulates the host immune response through epigenetic mechanisms, advancing our understanding of TB pathophysiology and host-pathogen interactions.

P33: Cigarette Smoke and HRV-16 Infection Induce Lipocalin-2 Secretion from Airway Epithelium

Bruno Alejandro Steimbrüch ^1,6, Martin Wolff ², Natalia El-Merhie ², Holger Heine ^1,3,6, Torsten Goldmann ^4,6 and Susanne Krauss-Etschmann ^1,2,5,6

¹DZL Laboratory, Experimental Microbiome Research, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,²Early Life Origins of CLD, Priority Area Asthma and Allergies, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,³Research group Innate Immunity, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,⁴Histology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany,⁵Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany,⁶Member of the Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany.

Background: Cigarette smoke (CS) is the main risk factor for COPD. Viral infections are more frequent and more severe in smokers. Our previous findings showed that lipocalin-2 (LCN2), a mediator of innate immune defense, was strongly upregulated in murine cell-free bronchoalveolar lavage fluid (BALF) upon short-term smoke exposure. Here, we investigated whether airway epithelial cells secrete LCN2 in response to CS and viral infection.

Methods: Public scRNA-seq datasets from non-smokers, smokers, and COPD patients were analysed to assess LCN2 expression. Primary bronchial epithelial cells from healthy (NHBE) and COPD (CHBE) donors were cultured at air-liquid interface (ALI) and exposed to smoke (1R6F at 1 puff/min, ExpoCube) for 14 days, followed by infection with human rhinovirus (HRV-16) for 4 hours. Analyses were performed 24 hours post-infection.

Results: In silico LCN2 expression was significantly higher in ciliated and goblet cells of smokers and COPD patients than in non-smokers. Experimentally HRV-16 infection significantly induced LCN2 release in ALI cultures. Moreover, CS further exacerbated LCN2 levels from CHBE, compared with air-exposed controls. In contrast, no significant difference in LCN2 release was observed between CS-exposed and air-exposed NHBE cells after HRV-16 infection.

Conclusions: Together, our findings suggest that LCN2 is secreted by the airway epithelium in smokers and COPD patients. Furthermore, CS exacerbates LCN2 secretion in HRV-infected CHBE cells, potentially

P34: Comparative Analysis of Lipid Droplet Formation in Murine C3HeB/FeJ and C57BL/6 Macrophages During Mycobacterium tuberculosis Infection

Linda Zemke ¹, Nadine Aboutara ², Dominik Schwudke ^2,3,4, Gareth Prosser ^1,4, Norbert Reiling ^1,4

¹Microbial Interface Biology, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany.,²Bioanalytical Chemistry, Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany,³Research Center Borstel, Airway Research North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,⁴German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany.

Mycobacterium tuberculosis (Mtb), the intracellular bacterium causing tuberculosis (TB), infects about one-fourth of the global population, with 5–10% progressing to active disease. Lung granulomas, a hallmark of TB, comprise epithelial, innate, and adaptive immune cells centred around infected macrophages. Macrophages serve as primary host cells for Mtb and often exhibit a foamy phenotype due to lipid droplet (LD) accumulation. LDs contain neutral lipids such as cholesterol esters and triacylglycerols. Although lipids are a key carbon source for Mtb, LD biogenesis, dynamics, and composition in primary macrophages remain poorly studied.

Animal models remain crucial for studying disease mechanisms. No model fully replicates human TB pathology, and mouse strains differ markedly in susceptibility and pulmonary responses. C57BL/6 mice are relatively resistant and develop pathology differing from that observed in humans, whereas C3HeB/FeJ mice are highly susceptible, showing increased bacterial numbers associated with necrotic granulomas, hypoxia, and cavity formation.

In this study, we compared primary macrophages from C3HeB/FeJ and C57BL/6 mice following Mtb infection. Early macrophage activation was closely linked to increased neutral lipid accumulation within one day, while prolonged infection at lower bacterial burdens also promoted LD accumulation. High-content imaging revealed that C3HeB/FeJ macrophages inherently contained more neutral lipids than C57BL/6 cells. Shotgun lipidomics further identified distinct global lipidomic profiles. Ongoing work focuses on phenotypic and metabolomic characterization to define mechanisms underlying strain-specific differences in lipid metabolism and their potential impact on Mtb pathogenesis.

P35: Paternal preconception e-nicotine exposure alters offspring development and airway-associated phenotypes in D. melanogaster

Xijie Zhang ¹, Göksel Dogan ^1,2, Leizhi Shi ^1,3, Qiaoyan Tan ^1,4, Birte Kemper-Ehrhardt ¹, Beate Hoeschler ¹, Jülide Altigan ^1,2, Cecilie Svanes ^5,6, Klaus Rabe ^7,8_, Thomas Roeder ¹⁰, Susanne Krauss-Etschmann ^1,11,12

¹Division of Early Life Origins of Chronic Lung Diseases, Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,²Department of Histology-Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey,³Department of Thoracic Surgery, Linyi People's Hospital, Linyi, China,⁴Department of Obstetrics and Gynecology, Chongqing Health Center for Women and Children, Women and Children´s Hospital of Chongqing Medical University, Chongqing Municipal Health Commission Key Laboratory of Perinatal Medicine, Chongqing, China,⁵Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Bergen, Norway,⁶Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway,⁷Department of Pneumology, LungenClinic, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Grosshansdorf, Germany,⁸Department of Medicine, Christian Albrechts University, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Kiel, Germany,⁹Division of Molecular Physiology, Institute of Zoology, Christian-Albrechts University Kiel, Kiel, Germany,¹⁰Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,¹¹DZL-Laboratory for Experimental Microbiome Research, Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, Germany,¹²Institute of Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany

Background: Electronic cigarette use is increasing among adolescents, but its potential effects on their offspring remain unclear. Previous studies have focused mainly on maternal exposure during pregnancy, whereas the consequences of paternal exposure before conception are poorly understood. We therefore investigated whether paternal preconception e-nicotine exposure affects offspring development, airway morphology and physiological fitness in Drosophila.

Methods: Newly eclosed male Canton-S wild-type Drosophila melanogaster were exposed to e-nicotine for three days and then mated with 5-day-old unexposed virgin females. Survival was monitored in F0 males. F1 offspring were assessed for development, body size, locomotion, hypoxia-related phenotypes and survival. Larvae tracheal morphology and F1 airway transcriptomes were also analyzed.

Results: Paternal e-nicotine exposure reduced survival in F0 males and impaired airway development in F1 offspring, as indicated by reduced tracheal terminal cell length. F1 also exhibited reduced body length at the L1 stage, in male L3 larvae and in adults, together with sex- and stage-dependent alterations in larval crawling, adult locomotor activity and survival. Whole-trachea RNA-seq revealed sex-specific transcriptional changes in fatty acid metabolism and biosynthesis, while FASN2 expression was increased in offspring of both sexes, suggesting FASN2 as a potential contributor to the tracheal developmental defects.

Conclusion & Outlook: Paternal preconception e-nicotine exposure induces intergenerational alterations in offspring growth, behavior and airway-associated phenotypes in Drosophila. Airway transcriptomic analysis identifies altered expression of genes involved in lipid metabolism, particularly increased FASN2 expression, providing potential molecular pathways to the observed tracheal phenotypes. Functional studies are required to determine the underlying mechanisms.

P36: Lineage-dependent Rifampicin tolerance in Mycobacterium tuberculosis complex strains

Annemarie Hintz-Rüter ¹, Lindsay Sonnenkalb ¹, Stefan Niemann ¹

¹Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany

Drug resistance in Mycobacterium tuberculosis complex (MTBC) threatens global TB (tuberculosis) control, with antibiotic tolerance— increased bacterial survival under antibiotic exposure—acting as a key precursor to resistance. Tolerant bacteria prolong treatment, contributing to the 6-month regimens that drive poor adherence and resistance emergence. Differences in resistance profiles between MTBC lineages 2 and 4 suggest potential variations in tolerance, highlighting the need for shorter, more effective therapies tailored to lineage-specific responses.

In this work, the tolerance capacity between clinical strains of L2 and L4 was compared. The strain panel was assessed by determining growth parameters and rifampicin (RIF) minimal inhibitory concentration (MIC), which confirmed homogeneous growth and RIF susceptibility. Tolerance was measured by time-kill assays following quantification by colony forming unit (CFU), a direct measure of bacterial viability. At 0.5 µg/mL RIF (15 × MIC), strains of L4 showed a significantly higher tolerance compared to strains of L2. To further elucidate the underlying cause for this difference the Transcriptomic profile of theses strains were investigated.

These results demonstrate a clear lineage-dependent difference in RIF tolerance. This study reveals a previously underappreciated potential for using tolerance profiling in precision medicine approaches to shorten TB treatment regimens. It highlights the critical role of antibiotic tolerance—not only as a precursor to resistance but also as a key factor in treatment failure across genetically diverse MTBC strains.

P37: Insights into mechanisms relevant to asthma: β-glucan-induced trained immunity in airway epithelial cells alters responses to house dust mite allergens

Nathalie Martin ^1,2, Marua Abu Risha ^2,3, Tobias Dallenga ^4,5, Markus Weckmann ^3,6,7, Uta Jappe ^2,3,8

¹University of Lübeck, Lübeck, Germany,²Clinical and Molecular Allergology, PA Chronic Lung Diseases, Research Center Borstel, Borstel, Germany,³German Center for Lung Research (DZL), Airway Research Center North (ARCN), Borstel, Germany,⁴Cellular Microbiology, PA Infections, Research Center Borstel, Borstel, Germany,⁵German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany,⁶Epigenetics of Chronic Lung Diseases, PA Chronic Lung Diseases, Research Center Borstel, Borstel, Germany,⁷Department of Paediatric Pneumology & Allergology, University Clinics Schleswig Holstein, Lübeck, Germany,⁸Interdisciplinary Allergy Outpatient Clinic, Department of Pneumology, University of Lübeck, Lübeck, Germany

Background: Asthma remains a complex disease, the mechanisms initiating its development are not fully understood. Evidence suggests that airway epithelium can acquire a persistent memory after microbial exposures. This may contribute to asthma progression through long-lasting alterations in barrier function and epigenetic regulation. β‑glucan, a microbial cell wall component with immunomodulatory properties is widely used in trained immunity research and has been implicated in occupational asthma.

Objective: Investigating whether β‑glucan shapes epithelial responses to later allergen exposure.

Method: Calu-3 airway epithelial cells were exposed to β‑glucan for 24 h, rested for 5 days, and challenged with the recombinant house dust mite allergen rDer p 5. Transcriptional changes were assessed using RNA sequencing. The affected epigenetic changes upon β‑glucan training were captured as a chronic effect (3 repeated β‑glucan exposures), including both histone modifications and DNA methylation. Barrier integrity and cytokine release (IL-6 and IL-8) were also measured.

Results: Exposure to the allergen mainly induced significant transcriptional changes after the training. The training resulted in persistent transcriptional reprogramming. Trained cells exhibited an altered epigenetic profile (reduction of H3 modifications, increased H4 modifications), changes in epithelial barrier function and reduced IL-6 and IL-8 release following the allergen challenge.

Conclusion: Our results support the hypothesis that β‑glucan acts as a priming agent, inducing epigenetic and functional changes in the airway epithelium. The modulation of allergen responsiveness is consistent with trained immunity and may support the hygiene hypothesis. However, our in vitro system does not directly test the clinical outcome nor prove.

P38: Paleogenomics reveals leprosy at the Late Antique burial site of Fallward in northern Germany

Daniel Anton Myburgh ¹, Nicolas Antonio da Silva ¹, Almut Nebel ¹, Ben Krause-Kyora ¹

¹Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany

Human mobility was a major factor which shaped cultural and societal transformations in Late Antiquity, driving the exchange of people, ideas and disease across Europe. Yet, despite this understanding, the genetic and pathogen landscape of many communities from this period remain uncharacterised. Here we present the first palaeogenomic investigation of inhabitants of the coastal settlement of Fallward (3rd–5th century AD) in present-day northwestern Germany. Genomic data from three individuals revealed heterogeneous ancestries: two aligned closely with northwestern European Iron Age populations, while one adult male exhibited affinities with Roman frontier groups from central-eastern Europe. Metagenomic screening further identified that this man was infected with Mycobacterium leprae, with the recovered genome belonging to an early lineage that may be associated with the Mediterranean region. Combined with the Roman-influenced grave goods accompanying his elaborate burial, these findings suggest he or his recent ancestors may have had close connections to the populations living at the frontier of the Roman Empire. Overall, the results highlight how pathogens could spread across considerable distances in the Late Antiquity through complex mobility networks and cross-cultural interactions, reaching even small and remote communities such as Fallward.

P39: Keratinocyte-targeting functional autoantibodies in systemic sclerosis are associated with interstitial lung disease

Jacqueline K. Wax ¹, Helene S. Radloff ², Gabriela Riemekasten ², Frank Petersen ¹, Xinhua Yu ¹

¹Priority Research Area Chronic Lung Diseases, Department of Pulmonary Immune Diseases, Research Center Borstel – Leibniz Lung Center, Airway Research Center North (ARCN), Members of the German Center for Lung Research (DZL), Borstel, Germany,²Department of Rheumatology and Clinical Immunology, University Clinic of Schleswig Holstein, University of Lübeck, Lübeck, Germany

Introduction: Systemic sclerosis (SSc) is a chronic autoimmune connective tissue disease characterized by multiple organ involvement and heterogeneous clinical manifestations. Interstitial lung disease is a common complication and a major cause of SSc-related mortality. In addition to autoantibodies directed against nuclear antigens, SSc patients also produce autoantibodies targeting cell surface receptors, which may serve not only as biomarkers but also as mediators of disease pathogenesis. This study aimed to identify novel functional autoantibodies that contribute to the development of SSc.

Material and Methods: Serum samples from SSc patients and healthy controls were analyzed for IgG binding to the surface of normal human epidermal keratinocytes (NHEKs) using a cell-based immunofluorescence assay. Associations between anti-NHEK IgG levels, disease susceptibility, and clinical manifestations were evaluated using appropriate statistical analyses. The functional effects of these autoantibodies were assessed by cytokine array analysis.

Results: IgG autoantibodies against NHEKs were detected in human serum samples, with significantly higher levels in SSc patients compared with healthy controls. Notably, SSc patients with pulmonary fibrosis exhibited higher levels of these autoantibodies than those without, suggesting a potential link to fibrotic progression. Furthermore, stimulation of NHEKs with purified IgG isolated from anti-NHEK-positive SSc sera induced a marked increase in the release of pro-fibrotic cytokines, including PDGF-BB.

Conclusion: These findings support the presence of functional autoantibodies in SSc that target keratinocytes and promote the secretion of pro-fibrotic mediators. This mechanism may contribute to fibroblast activation, myofibroblast differentiation, and the development of fibrosis in SSc.