Biology subjects

Siemes, D.

Publications and source records attributed to Siemes, D..

10 recordsLinked to original sources

bioRxiv2026

Integrated kidney and urine proteomics define encrypted antimicrobial peptides as effectors of host defence in human pyelonephritis

Antimicrobial peptides (AMPs) are key effectors of host defence, however, their functional deployment across renal tissue and urine in pyelonephritis (PN) remains incompletely understood. Here, we integrate kidney and urine proteomics with urinary peptidomics and computational prediction to define AMP organisation and function. Proteomic analysis indicated coordinated induction of multiple AMPs in infected kidneys. These patterns were recapitulated in the urinary proteome, where AMP abundance correlated with leukocyte counts. Multiplex immunofluorescence microscopy localised these AMPs to myeloid cells, identifying them as central effector sources. Importantly, analysis of the urine peptidome revealed multiple encrypted AMPs (EPs), which arise from proteolytic processing of precursor proteins. To systematically assess their relevance for host defence, we applied an ensemble of machine learning-based predictors to prioritise candidates with activity in the urinary environment. This approach identified several potential EPs, among which the S100A12-derived peptide Calcitermin was confirmed in patient urine. Furthermore, it exerts antibacterial activity against uropathogenic E. coli (UPEC) and modulates myeloid cell responses. Together, these findings define a coordinated and compartmentalised AMP defence programme in human PN that extends beyond increased peptide expression, highlighting EPs as functionally relevant effectors with therapeutic potential.

immunology↗

bioRxiv2026

Lactate-dependent metabolic rewiring is associated with CD163+ macrophage polarization in human pyelonephritis

Chronic pyelonephritis is characterized by persistent bacterial infection of the kidney and a dysregulated immune response that promotes disease progression. Macrophages are central regulators of antibacterial immunity in the urinary tract. However, the mechanisms underlying their functional reprogramming in chronic infection remain poorly understood. Here, we identify lactate as a key metabolic determinant of macrophage polarization in human pyelonephritis. Proteomic analysis of human kidney tissue revealed extensive metabolic remodelling, including upregulation of enzymes involved in glycolysis. Consistent with this, lactate levels were significantly elevated in urine and plasma of pyelonephritis patients. Concomitantly, we observed a pronounced accumulation of CD163 macrophages in infected kidneys, representing a distinct macrophage subset with immunomodulatory function. Correlation-based network analysis revealed a strong association between CD163 and lactate dehydrogenase A, supporting a functional association between lactate metabolism and macrophage polarization. Mechanistically, exposure of murine bone marrow-derived macrophages to lactate induced intracellular protein lactylation and promoted polarization toward a CD163 phenotype, defining a metabolically imprinted macrophage state distinct from classical activation paradigms. Proteomic profiling demonstrated extensive remodelling of macrophage protein expression in response to lactate with significant alteration of mediators of phagocytosis, Toll-like receptor signalling, and interferon response. Together, these findings identify lactate as a potent metabolic driver of macrophage reprogramming and establish a foundation for investigating lactylation-dependent immune dysfunction in chronic pyelonephritis.

immunology↗

bioRxiv2026

Histone neutralization protects the ischemic brain against stroke-associated pneumonia

Bacterial pneumonia aggravates ischemic stroke via mechanisms that still remain to be determined. In ischemic stroke patients and mice exposed to middle cerebral artery occlusion, we show that stroke-associated pneumonia markedly worsens clinical stroke outcome. In mice, pneumonia induced 3 days after stroke impaired neurological recovery and increased brain neutrophil infiltrates, blood-brain barrier breakdown, cerebral microvascular thrombosis, and progressive brain atrophy. The antibiotic amoxicillin only partially ameliorated pneumonia-associated neurological deficits and neutrophil infiltrates. Neutrophils were critical mediators of pneumonia-induced blood-brain barrier breakdown and microvascular thrombosis. Notably, administration of a neutralizing anti-histone antibody during pneumonia--unlike degradation or blockade of neutrophil extracellular trap formation or myeloperoxidase inhibition--restored long-term neurological recovery and prevented brain atrophy in stroke-associated pneumonia mice. This study identifies extracellular histones as key drivers of secondary inflammatory brain injury and establishes histone neutralization as a therapeutic strategy with an extended treatment window in the post-acute stroke phase. One Sentence SummaryNeutralizing extracellular histones reverses pneumonia-driven secondary brain injury and restores long-term recovery after ischemic stroke.

neuroscience↗

bioRxiv2025

TSP1/TGF-β1 drives arachidonic acid metabolism to orchestrate neutrophil swarming

Neutrophil swarming has emerged as a conserved multicellular behaviour observed across tissues and pathological contexts. Yet, the molecular cues and the spatially coordinated cellular circuits that drive the process of neutrophil swarming leading to cluster formation remain poorly understood. Here, we combine spatial proteomics and lipid profiling in a model of urinary tract infection to define the epithelial-immune circuits driving neutrophil cluster formation. We identify thrombospondin-1 (TSP1)-mediated activation of transforming growth factor beta 1 (TGF-{beta}1) as key epithelial signal licensing neutrophil clustering and enhancing bacterial control. Spatial lipid analysis further reveals that TSP1/TGF-{beta}1 signalling locally activates arachidonic acid metabolism in epithelial neutrophils, with 5-lipoxygenase dependent leukotriene synthesis required for swarm formation and infection clearance. These findings uncover a spatially coordinated defence mechanism in which epithelial-derived TSP1/TGF-{beta}1 engages neutrophil lipid metabolism to orchestrate neutrophil swarming behaviour and reinforce antibacterial immunity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/688397v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@114ea92org.highwire.dtl.DTLVardef@345244org.highwire.dtl.DTLVardef@10520baorg.highwire.dtl.DTLVardef@1a7c0fe_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

immunology↗

bioRxiv2025

Extrahepatic effects of thyroid hormone and resmetirom override their beneficial hepatic effects in alcohol-associated liver disease in mice

BackgroundAlcohol-associated liver disease (ALD) is a common type of liver disease worldwide. Excessive consumption of ethanol (EtOH) causes fat accumulation leading to hepatic steatosis. Hepatic thyroid hormone (TH) action or liver-specific thyromimetics, e.g. resmetirom, can reduce hepatic triglycerides. We therefore hypothesized that TH treatment could ameliorate ALD. MethodsTo induce ALD, mice were treated with either EtOH or liquid control diet for 10 days followed by a single EtOH or maltose control gavage on day 11. The liquid diets were supplemented with solvent, T3 or Resmetirom. We studied WT and hepatocyte-specific TR{beta} KO mice (hepTR{beta}KO). Effects were measured by clinical chemistry, liver staining, hepatic triglyceride content, and RNA-sequencing. ResultsSurprisingly, resmetirom had no beneficial effect and T3 treatment even aggravated EtOH-induced steatosis (increased liver weight and hepatic triglycerides). The liver phenotype was worsened in hepTR{beta}KO mice, which still suggested beneficial effects of hepatic TR{beta} signaling in WT mice. These seemingly paradoxical results could be explained by extrahepatic effects in WAT: WAT weight and adipocyte size were reduced by EtOH, T3 and resmetirom. These data indicate lipolysis and subsequent fatty acid accumulation in the liver, explaining the more severe ALD phenotype with T3 and attenuated effect of resmetirom. As WAT loss was reduced in hepTR{beta}KO mice, the hepatic TR{beta} mediated the extrahepatic effects of T3 and resmetirom on WAT. ConclusionWe conclude that extrahepatic TH effects in WAT were detrimental in ALD and counteracted beneficial local hepatic TH/TR{beta} action. As WAT loss appeared to originate from the hepatic TR{beta}, this also applied to resmetirom.

pathology↗

bioRxiv2025

SCAMP3 is essential for proper formation and function of neutrophil granules

Host defense functions of neutrophils during infection critically depend on microbicidal and proteolytic proteins stored in primary, secondary and tertiary granules that are released into the phagosome or into the extracellular space upon degranulation. Granules are generated during granulopoiesis and impaired granule production or granule protein sorting has been linked to inefficient pathogen clearance resulting in recurrent bacterial and fungal infections. Here, we studied the role of the membrane protein secretory carrier associated membrane protein 3 (SCAMP3) for neutrophil defense functions. We generated Scamp3 knockout (KO) Hoxb8 cells and found that killing of Escherichia coli by Scamp3 KO Hoxb8 cell-derived neutrophils (dHoxb8 cells) was compromised as compared to control dHoxb8 cells in vitro. Mass spectrometric and Western blot analyses revealed a significant reduction of primary, secondary, and tertiary granule proteins in the genetic absence of Scamp3, resulting in a reduced overall granularity of these cells. Accordingly, degranulation was reduced in Scamp3 KO dHoxb8 cells compared to control dHoxb8 cells. Similarly, SCAMP3 deficiency in zebrafish resulted in reduced neutrophil granularity in comparison to wild-type animals. However, neutrophil migration towards sites of E. coli infection was unaffected in scamp3 KO zebrafish larvae. In summary, SCAMP3 represents an important novel player in granule equipment and degranulation, with key functions in neutrophil defense mechanisms during host-pathogen interactions in vitro. Brief summary sentence: The membrane protein secretory carrier associated membrane protein 3 (SCAMP3) is essential for proper protein equipment of neutrophil granules and Scamp3-deficient neutrophils have an impaired bacterial killing capacity in vitro.

physiology↗

bioRxiv2024

msiFlow: Automated Workflows for Reproducible and Scalable Multimodal Mass Spectrometry Imaging and Immunofluorescence Microscopy Data Processing and Analysis

Multimodal imaging by matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI MSI) and immunofluorescence microscopy holds great potential for understanding pathological mechanisms by mapping molecular signatures from the tissue microenvironment to specific cell populations. However, existing open-source software solutions for analysis of MALDI MSI data are incomplete, require programming skills and contain laborious manual steps, hindering broadly applicable, reproducible, and high-throughput analysis to generate impactful biological discoveries across interdisciplinary research fields. Here we present msiFlow, an accessible open-source, platform-independent and vendor-neutral software for end-to-end, high-throughput, transparent and reproducible analysis of multimodal imaging data. msiFlow integrates all necessary steps from import and pre-processing of raw MALDI MSI data to visual analysis output, as well as registration, along with state-of-the-art and newly developed algorithms, into automated workflows. Using msiFlow, we unravel the molecular heterogeneity of leukocytes in infected tissues by spatial regulation of ether-linked phospholipids containing arachidonic acid. We anticipate that msiFlow will facilitate the broad applicability of MSI in the emerging field of multimodal imaging to uncover context-dependent cellular regulations in disease states.

bioinformatics↗

bioRxiv2023

Canonical and noncanonical contribution of thyroid hormone receptor isoforms alpha and beta to cardiac hypertrophy and heart rate in male mice

BackgroundStimulation of ventricular hypertrophy and heart rate are two major cardiac effects of thyroid hormone (TH). Aim of this study was to determine in vivo which TH receptor (TR), or {beta}, and which mode of TR action, canonical gene expression or DNA-binding independent noncanonical action, mediate these effects. Material and methodsWe compared global TR and TR{beta} knockout mice (TRKO; TR{beta}KO) with WT mice to determine the TR isoform responsible for T3 effects. The relevance of TR DNA- binding was studied in mice with a mutation in the DNA-binding domain that selectively abrogates DNA binding and canonical TR action (TRGS; TR{beta}GS). Hearts were studied with echocardiography at baseline and after seven weeks T3-treatment. Gene expression was measured with real-time PCR. Heart rate was recorded with radiotelemetry transmitters for seven weeks in untreated, hypothyroid and T3-treated mice. ResultsT3 induced ventricular hypertrophy in WT and TR{beta}KO mice, but not in TRKO mice. Hypertrophy was also induced in TRGS mice. Thus, hypertrophy is mostly mediated by noncanonical TR action. Similarly, repression of Mhy7 occurred in WT and TRGS mice. Basal heart rate was largely dependent on canonical TR action. But responsiveness to hypothyroidism and T3-treatment as well as expression of pacemaker gene Hcn2 were still preserved in TRKO mice, demonstrating that TR{beta} could compensate for absence of TR. ConclusionT3-induced cardiac hypertrophy could be attributed to noncanonical TR action, whereas heart rate regulation was mediated by canonical TR action. TR{beta} could substitute for canonical, but not noncanonical TR action.

physiology↗

bioRxiv2023

Proteomic adaptations in the kidney reveal orchestration of local and secreted antimicrobial peptides in human pyelonephritis

Pyelonephritis (PN) is a frequent bacterial infection of the kidney and is often associated with severe diseases, organ loss and sepsis. Antibiotics are the cornerstone of therapy, however, increasing antibiotic resistance threatens therapy success and necessitates novel treatment strategies. Various proteins, such as antimicrobial peptides (AMPs), are key molecules of the innate immune response and insights into their regulation may help overcome multi-drug resistance and severe diseases. Using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS), several cellular, biological, and metabolic processes important for the antimicrobial response were identified, including a significant increase in previously undescribed proteins in human PN with antimicrobial function. Among others, we observed elevation of AMPs, such as calprotectin, azurocidin-1, and cathepsin G in the kidney, which we validated in the urine. Additionally, we observed a negative correlation of azurocidin-1 with plasma levels of C-reactive protein suggesting that the presence in the kidney may protect from severe diseases and systemic inflammation. This study represents the first renal proteomic dataset of human PN, enabling novel insights into the expression of AMPs in the context of PN. Lay SummaryGrowing antimicrobial resistance necessitates a better understanding of the expression of proteins that are critical for the immune response. Using mass spectrometry we identified AMPs in the kidney and urine of PN patients. Elevated levels of the AMP azurocidin-1 was associated with reduced systemic inflammation, indicated by lower C-reactive protein. Overall, this study identified expression of previously undescribed AMPs in the context of human PN. These proteins may play a pivotal role in protection from severe diseases and systemic inflammation.

immunology↗

bioRxiv2022

Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs

Blood vasculature represents a complex network of vessels with varying lengths and diameters that are precisely organized in space to allow proper tissue function. Light-sheet fluorescence microscopy (LSFM) is very useful to generate tomograms of tissue vasculature with high spatial accuracy. Yet, quantitative LSFM analysis is still cumbersome and available methods are restricted to single organs and advanced computing hardware. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ~100 times faster than other existing vessel analysis tools, requires no user interaction, integrates batch processing, and parallelization. Employing an innovative dual Frangi filter approach we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

bioinformatics↗