Acute graft-versus-host disease (aGVHD) is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT) and patients with steroid-refractory aGVHD have a dismal prognosis. We have previously shown that the enteroendocrine hormone glucagon-like peptide-2 (GLP-2) has tissue regenerative activity in the lower GI in mice and patients with steroid-refractory aGVHD. Here we explored the tissue protective effect of the enteroendocrine hormone gastrin for aGVHD of the stomach. We observed that aGVHD caused a loss of gastrin-producing G-cells and parietal cells (PCs) and an increase of pH in the stomach, while allogeneic T cells infiltrated the stomach wall. Pentagastrin treatment of aGVHD mice rescued the loss of PCs, normalized the pH in the stomach, increased stomach stem cell marker expression and abundance of LGR5+ cells, and changes in the stomach microbiome. Gastrin also increased the viability of stomach and small intestine organoids in vitro. Gast-/- mice experienced more severe aGVHD in the intestine and liver compared to WT mice, which was rescued by pentagastrin-treatment. In patients developing aGVHD, low gastrin levels in stomach biopsies were connected to reduced survival. Moreover, gastrin expression in the stomach correlated with aGVHD severity and tissue damage scores in independent patient cohorts. This study delineates the protective role of gastrin in aGVHD of the stomach in mice and patients and provides a rationale for therapeutic use of pentagastrin in a clinical trial for patients with aGVHD.

Abstract The aryl hydrocarbon receptor (AHR) is recognised as an important mediator of inflammatory processes, including those in kidney health. Patients suffering from kidney diseases have an increased risk of cardiovascular diseases, such as atherosclerosis. Since the AHR can be activated in the kidney by, e.g., tryptophan metabolites and contribute to inflammatory processes, we hypothesized that a deficiency of the Ahr in the kidney epithelium in mice fed a high-fat diet could impact the development of atherosclerosis by altering renal function. We analysed Apoe −/− Cdh16 Cre Ahr fl/fl mice in comparison to Apoe −/− Ahr fl/fl after feeding a 12-week high-fat diet in terms of systemic inflammation, plaque, and kidney phenotype. Accordingly, we found that the absence of Ahr in the kidney epithelium did not affect atherosclerotic outcomes and, surprisingly, did not alter the kidney phenotype with respect to inflammation or fibrosis. We therefore concluded that a high-fat diet does not sufficiently drive an AHR-mediated response in kidney epithelium, which could contribute to plaque development.

Autosomal dominant polycystic kidney disease (ADPKD), the leading genetic cause of kidney failure, results from loss-of-function mutations in PKD1, encoding polycystin-1 (PC1). PC1 localizes to the primary cilium. In the absence of PC1, adverse signaling from the primary cilium orchestrates cyst formation, but the biomechanical underpinnings of this cilia-dependent cyst activation (CDCA) remain unclear. Combining tubule-specific orthologous mouse models with a tubule-on-chip platform, we show that PC1 and cilia govern the composition, mechanical properties and shape of the tubular basement membrane (TBM), the principal rigid determinant of tubule geometry. PC1 loss triggers TBM thinning, heparan sulfate enrichment and deformation, leading to distension, preferentially of the distal nephron. These changes are driven by a cilia-dependent transcriptional program, with GLIS2 - a key CDCA effector - participating as a downstream mediator. Reduction of TBM stiffness amplifies Pkd1-/- tubule-on-chip dilation and increases cyst formation in vivo. Conversely, increasing luminal pressure through ureteral obstruction induces disproportionate distension of Pkd1-deficient tubules and triggers an irreversible cystogenic program. Together, these findings establish a TBM-centered biomechanical model of ADPKD in which tubule deformation is governed by both basolateral and luminal mechanical factors, and identify the cilium-TBM axis, operating in part through GLIS2, as a central driver of cystogenesis.

Granulomas are disease-defining heterocellular tissue structures in mycobacterial infections and play ambiguous roles ranging from pathogen containment to tissue destruction. Here, we established a mature peritoneal granuloma model in C57BL/6 mice to investigate dynamic cell-cell-interactions during mycobacterial infection, including long-term immune alterations within serous cavities as important disease manifestation sites. We show that mycobacteria reside in stromal cells, which modulate the local tissue milieu and shape macrophage responses, particularly through chemokine and colony-stimulating factor 1 production. Chronic infection induces sustained reprogramming and diversification of stromal cells toward specialized, immune-like states including active transfer of mycobacteria to macrophages and a pronounced interferon response. Consequently, stromal cells acquire immunoregulatory properties and support pathogen handling, monocyte recruitment and macrophage maturation thereby critically shaping granuloma formation and contributing to containment of mycobacteria. ### Competing Interest Statement The authors have declared no competing interest. University of Freiburg, https://ror.org/0245cg223, 2023/A2-Fol; 2021/B3-Fol Mertelsmann Foundation German Research Foundation, DFG, Project ID 450392965, Project ID 256073931, Project ID 413517907, Project ID 259373024, HE3127/12 and HE3127/16, Project ID 491676693 European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Individual Research Grant 2024 German Federal Ministry of Research, Technology and Space (BMFTR), DZIF; TTU-TB, grant number 02.717 German Federal Ministry of Education and Research BMFTR, 031 A538A de.NBI-RBC, 01EO2103 Ministry of Science, Research and the Arts Baden-Württemberg (MWK), LIBIS/de.NBI Freiburg Fritz Thyssen Foundation

Abstract CTLA-4 (haplo)insufficiency displays incomplete penetrance and phenotypic heterogeneity, indicating the involvement of additional disease modifiers beyond the genetic defect. Microbiome analyses reveal a positive association between disease severity and intestinal dysbiosis, highlighting the microbiome as a critical contributor. To investigate this relationship mechanistically, we generated Ctla4⁺ / ⁻ wildlings harboring a natural microbiota. Unlike specific pathogen free (SPF) counterparts, which remain healthy, Ctla4⁺ / ⁻ wildlings spontaneously develop disease phenotypes resembling human CTLA-4 haploinsufficiency. Disease onset is followed by reduced microbial diversity and expansion of pathobionts. Integrative immunophenotyping shows that the natural microbiota synergizes with Ctla4 haploinsufficiency to reshape innate and adaptive immune compartments, generating a sustained pro-inflammatory milieu and reduced CTLA-4 expression in the cecum. Furthermore, microbiota-derived metabolites promote inflammatory cytokine production in both murine and human primary T cells via NF-κB activation. Collectively, Ctla4⁺ / ⁻ wildlings constitute an effective model for dissecting microbiome–immune crosstalk in CTLA-4 (haplo)insufficiency and for exploring therapeutic strategies.

BACKGROUND & AIMS:Kupffer cells (KCs), the prenatally seeded macrophages of the liver, display unique functional and immunophenotypic features among tissue-resident macrophages. They are considered terminally differentiated with negligible postnatal cellular input. Their adaptability in disease has thus been attributed to recruited, monocyte-derived KCs. Here, we explored KC plasticity and the impact of their ontogeny in persisting mycobacterial infections, which primarily target macrophages. METHODS:Models of systemic chronic infection with Mycobacterium bovis or Mycobacterium avium were combined with genetic monocyte deficiency (Ccr2-/-, Irf8-/-), KC fate-mapping mice, bone marrow transplantation, high-resolution imaging and immunophenotyping. In addition, ATAC, bulk RNA, and single-cell RNA sequencing were conducted to specifically delineate origin, heterogeneity, and adaptations of KCs during infections. RESULTS:Mycobacterial infections induced the emergence of a unique KC subset, which lacked the signature markers CLEC4F and VSIG4 ("KClow"). KClow were derived from embryonically seeded KCs and accumulated exclusively within hepatic granuloma cores. In contrast, monocyte-derived macrophages were localized at the granuloma periphery, yet contributed to the tissue reaction. We identified KClow as specialized antimycobacterial effector cells with a heightened ability to produce iNOS, adapted to a hypoxic microenvironment, and able to modulate adaptive immune responses. Despite their fundamental deviation from the classical KC phenotype, KClow showed remarkable plasticity. Monocytes, on the other hand, were crucial for granuloma initiation. CONCLUSIONS:Mycobacterial infections reveal KCs as highly plastic cells that are critical for responding to extreme environmental changes. IMPACT AND IMPLICATIONS:Kupffer cells (KCs) are traditionally regarded as terminally differentiated macrophages with limited plasticity, reliant on monocyte-derived KCs during liver infections or injury. In contrast, using a chronic mycobacterial infection model, we found tissue-resident KCs to be very plastic cells, which are strongly engaged in the antimycobacterial response and adopt a specific spatial distribution. These findings suggest that KCs respond with a unique and organ-specific program to chronic mycobacterial infections.

INTRODUCTION:Pathogenic variants in the CLCN5 gene encoding the chloride-hydrogen exchanger ClC-5 cause Dent's disease type 1, a genetic disorder of the endolysosomal pathway in the proximal tubules of the kidneys. A hallmark of this disease is the downregulation of the protein uptake receptor consisting of megalin, cubilin and amnionless, causing low-molecular-weight proteinuria. Why these receptors are downregulated is not fully understood. METHODS:To clarify this, we established an in vivo model for Dent's disease using Drosophila nephrocytes that share similarities with podocytes and proximal tubule cells. Dissected nephrocytes were subjected to immunostaining, uptake tracer studies and transmission electron microscopy. Additionally, histological analysis, immunostainings and Western blotting were performed on Clcn5Y/- mice. RESULTS:Upon depletion of ClC-c, the fly ortholog of CLCN5, Cubilin was lost from the plasma membrane of nephrocytes, leading to a strong decrease in albumin uptake and ectopic slit diaphragms. Importantly, Cubilin exhibited a strong accumulation in the endoplasmic reticulum, while its binding partner Amnionless was overall reduced. This was accompanied by a fragmentation of the endoplasmic reticulum (ER) morphology and an increase in ER exit sites and associated Golgi stacks. Additionally, the actin and microtubular cytoskeleton as well as recycling endosomes showed a strong cortical accumulation, whereas cholesterol-enriched autophagic compartments emerged in the perinuclear area. Similar phenotypes were observed upon silencing of the C subunit of the vacuolar proton-ATPase (V-ATPase), suggesting they might depend on defects in acidification and glycosylation in the Golgi apparatus. Amnionless loss and ER retention of cubilin was confirmed in ClC-5 knockout mice, underscoring the relevance of this pathomechanism for Dent's disease. CONCLUSIONS:Our findings suggest that in addition to its endosomal role ClC-c/ClC-5 acts in the secretory pathway to promote surface trafficking of the Cubilin/Amnionless complex. This function may partly explain low-molecular-weight proteinuria in patients with Dent's disease.

Base editors enable precise correction of point mutations without requiring DNA double-strand breaks, yet platform-and cell type-specific genotoxicities remain incompletely characterized. Here, we applied cytosine base editing (CBE) to disrupt a cryptic splice-site mutation in the Unc13d locus of Jinx mice, a model of familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Efficient editing (62%-89%) in fibroblasts, T cells, and hematopoietic stem cells (HSCs) restored Unc13d splicing, reconstituted cytotoxic T cell function, and protected mice from virus-triggered hyperinflammation after transplantation of edited HSCs. Comparative genotoxicity profiling revealed distinct platform-and cell type-specific patterns: hyperactive CBE induced broader off-target activity and more structural variants than CRISPR-Cas9. Although off-target sequence edits persisted, the stability of CBE-induced chromosomal translocations differed between cell types. These findings establish base editing as a therapeutic strategy for a genetically predisposed hyperinflammatory syndrome and underscore the importance of context-specific safety profiling to guide the clinical translation of genome editors.

Abstract The importance of the extracellular matrix (ECM) influencing tumor biology in stroma-rich tumors is well established. However, the relevance of individual ECM proteins in rather stroma-poor cancers such as clear cell renal cell carcinoma (ccRCC) is ill-defined. Using bulk proteomics, spatial imaging, and single-cell transcriptomics, we identify collagen VI (COL6) as a predominant ECM component of the ccRCC interstitial stroma, synthesized primarily by fibroblasts and pericytes. Using cell-derived matrix (CDM) models, we demonstrate that COL6 is essential for maintaining an isotropic ECM network architecture and governs the broader matrisomal composition, with direct pro-proliferative consequences for tumor cells both in vitro and in situ. Granular spatial analysis reveals that COL6-rich stromal septa constrain tumor-infiltrating T cells to boundary zones, where CD8+PD1+ phenotypes predominate. Importantly, tyrosine kinase inhibition (TKI) with cabozantinib suppresses COL6 expression in fibroblasts in vitro and in ex vivo tumor models, mirroring COL6-depleted CDM phenotypes. Our findings establish COL6 as a central stromal regulator of ccRCC tumor biology and immune contexture, revealing ECM remodeling as an underappreciated mechanism of TKI action, with implications for combination immunotherapy strategies.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent renal malignancy with a poor prognosis when metastasized. The invasive growth of cancer cells relates to membrane-damaging forces, but the relevance of plasma membrane repair machinery in ccRCC remains incompletely understood. Employing proteomics, analysis of scRNA-sequencing data, and multiplex imaging, we identified ANXA4 as selectively expressed in ccRCC, with distinct localization patterns at the plasma and nuclear membranes. Genetic titration studies demonstrated that reduced ANXA4 expression impairs membrane repair and invasive capabilities. Further segmentation analysis of ANXA4-low tumors showed a distinct composition of the tumor microenvironment, with increased tumor-infiltrating lymphocytes and acellular extracellular matrix deposition. Transcriptomic analysis demonstrated alterations in epithelial-mesenchymal transition and immune signaling signatures in ANXA4-low tumors. Transcription factor enrichment analysis identified ELF3 as a regulator of invasive properties. Our integrative approach uncovered multiple roles for ANXA4 in modulating membrane repair, transcriptional regulation, and shaping the ccRCC tumor microenvironment composition.

Many metastatic clear cell renal cell carcinomas (ccRCC) are resistant to immune checkpoint inhibitor therapies, however the mechanisms underlying sensitivity or resistance remain incompletely characterised. We demonstrate that ccRCCs in the Vhl/Trp53/Rb1 mutant mouse model are resistant to combined anti-PD-1/anti-CTLA-4 therapy alone and in combination with additional therapeutic agents that reflect current ccRCC clinical trials. However, in some animals in vivo checkpoint therapy allowed isolated splenic T cells to recognise cultured ccRCC cells from the same animal, implicating the tumour microenvironment in suppression of T cell activation. We identified putative immunosuppressive myeloid cell populations with features similar to myeloid cells in the microenvironment of human ccRCC. The expression patterns of immune checkpoint ligands in both the mouse model and in human ccRCC suggests that several checkpoint systems other than PD-1 and CTLA-4 are likely to represent the dominant T cell suppressive forces in ccRCC. Our findings characterise an autochthonous mouse ccRCC model of immune checkpoint inhibitor therapy resistance and pave the way for a systematic functional dissection of the identified potential molecular barriers to effective immune therapy of ccRCC.

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement, and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet α-granules and released from activated endothelial cells, interacts with factor H (FH) and vWF. However, to our knowledge, direct regulatory interaction with the complement cascade has not yet been described. Our study shows that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins and inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. We validated complement-regulatory function in blood samples from patients with primary complement defects. The physiological relevance of TSP-1 was demonstrated in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis after TSP-1 deficiency in an in vitro and in vivo model of AAV. The complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia and complement. In particular, the interplay between released TSP-1 and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

ABSTRACT:The controlled development of cellular intestinal immunity in the face of dynamic microbiota emergence constitutes a major challenge in very early life and is a bottleneck for sustained growth and well-being. Early-onset inflammatory bowel disease (IBD) represents an extreme disturbance of intestinal immunity. It is a hallmark and often the first manifestation of chronic granulomatous disease (CGD), caused by inborn defects in the nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) in phagocytes and thus the failure to produce reactive oxygen species (ROS). However, in contrast to the known role of ROS in antimicrobial defense, the mechanisms underlying intestinal immunopathology in CGD remain enigmatic. This is partly due to the incomplete recapitulation of the CGD-IBD phenotype in established mouse models. We found that mice deficient in the NOX2 subunits p47phox or gp91phox showed similar baseline disturbances in lamina propria macrophage differentiation but responded differently to chemically induced colitis. Although p47phox- and gp91phox-deficient mice differed markedly in microbiota composition, crossfostering failed to equalize discrepant IBD phenotypes and microbiota, pointing at extremely early and functionally important microbiota fixation under specific pathogen-free housing conditions. In contrast, neonatal acquisition of a complex wild-mouse microbiota triggered spontaneous IBD, granuloma formation, and secondary sepsis with intestinal pathogens in both NOX2-deficient mouse lines, which was in part dependent on NOX2 in intestinal macrophages. Thus, in experimental CGD, the aberrant development of tissue immunity and microbiota are closely intertwined immediately after birth.

Conventional laboratory mice housed under specific pathogen-free (SPF) conditions are the standard model in biomedical research. However, in recent years, many rodent-based studies have been deemed irreproducible, raising questions about the suitability of mice as model organisms. Emerging evidence indicates that variability in SPF microbiota plays a significant role in data inconsistencies across laboratories. Although efforts have been made to standardize microbiota, existing microbial consortia lack the complexity and resilience necessary to replicate interactions in free-living mammals. We present a robust, feasible and standardizable approach for transplanting natural gut microbiota from wildlings into laboratory mice. Following engraftment, these TXwildlings adopt a structural and functional wildling-like microbiota and host physiology toward a more mature immune system, with characteristics similar to those of adult humans. We anticipate that adopting wild mouse-derived microbiota as standard for laboratory mouse models will improve the reproducibility and generalizability of basic and preclinical biomedical research.

DNA repair is essential for preserving genome integrity. Podocytes, postmitotic epithelial cells of the kidney filtration unit, bear limited regenerative capacity, yet their survival is indispensable for kidney health. Podocyte loss is a hallmark of the aging process and of many diseases, but the underlying factors remain unclear. We investigated the consequences of DNA damage in a podocyte-specific knockout mouse model for DNA excision repair protein Ercc1 and in cultured podocytes under genomic stress. Furthermore, we characterized DNA damage-related alterations in mouse and human renal tissue of different ages and patients with minimal change disease and focal segmental glomerulosclerosis. Ercc1 knockout resulted in accumulation of DNA damage and ensuing albuminuria and kidney disease. Podocytes reacted to genomic stress by activating mTOR complex 1 (mTORC1) signaling in vitro and in vivo. This was abrogated by inhibiting DNA damage signaling through DNA-dependent protein kinase (DNA-PK) and ataxia teleangiectasia mutated (ATM) kinases, and inhibition of mTORC1 modulated the development of glomerulosclerosis. Perturbed DNA repair gene expression and genomic stress in podocytes were also detected in focal segmental glomerulosclerosis. Beyond that, DNA damage signaling occurred in podocytes of healthy aging mice and humans. We provide evidence that genome maintenance in podocytes is linked to the mTORC1 pathway and is involved in the aging process as well as the development of glomerulosclerosis.

Bona fide Kupffer cells (KCs) are prenatally seeded and show unique functional and immunophenotypic features among tissue macrophages. They are considered as terminally differentiated, and adaptability in disease is attributed to recruited, monocyte-derived KCs. Here, we investigated the extent of KC plasticity and the impact of origin in mycobacterial infections that target macrophages and can persist for months. Fate-mapping combined with high-resolution imaging revealed the emergence of a unique, infection specific KC subset which downregulated the signature markers CLEC4F and VSIG4 (″KClow″). KClow were derived from bona fide KCs and located exclusively to granuloma cores. In contrast, monocyte-derived macrophages were contained at the granuloma borders and contributed to this tissue reaction. ATAC and single-cell RNA sequencing identified a specific signature of KClow with high antimycobacterial activity and specialization to a hypoxic microenvironment. Despite their fundamental deviation from the classical KC phenotype, KClow showed remarkable adaptability, and were capable to return to a homeostatic-like KC state. Accordingly, mycobacterial infections unmask KCs as highly plastic cells, capable of responding to extreme environmental changes. ### Competing Interest Statement The authors have declared no competing interest.

Complement-associated disorders are caused by the dysregulation and disbalance of the complement system, especially excessive activation. Most drugs that target the complement system are designed to inhibit the complement pathway at either the proximal or terminal levels. The use of a natural complement regulator such as factor H (FH) could provide a superior treatment option by restoring balance to an overactive complement system. We recently reported the moss-based production of an analog of human FH with an optimized glycan profile (CPV-104), which showed in vitro and in vivo characteristics comparable to its human counterpart. Here, we follow up our previous work, focusing in more detail on the time course and long-term efficacy of CPV-104 treatment in FH-deficient (FH–/–) mice. The analysis of long-term treatment effects following multiple injections of human FH into mice was previously hindered by the immune response, so we developed a protocol for the sustained depletion of CD20+ B-cells and CD4+ T-cells, preventing antibody formation without influencing the C3G phenotype. Using this dual-depletion method, we were able to complete dosing interval experiments in FH–/– mice, administering up to three injections of CPV-104 at different intervals. Repeated CPV-104 administration was able to lastingly resolve C3 deposits, offering additional rationale for the clinical testing of CPV-104 in human C3G patients. Moreover, our novel dual-depletion method has the potential for adaptation to different mouse models, allowing the testing of multiple doses of other therapeutic proteins.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease. Limited treatment options lead to renal failure in the vast majority of affected individuals. Novel therapeutic approaches are needed. Recent evidence has identified inflammation as an important driver of ADPKD. We analyzed transcriptional profiles in an orthologous Pkd1 mouse model and found a strong upregulation of the inflammasome pathway. To investigate the role of inflammasomes on cyst formation and kidney function, we modulated inflammasome activity through genetic targeting of the essential inflammasome component Pycard/Asc or treatment with the inflammasome inhibitor MCC950. Genetic deletion of Pycard/Asc in Pkd1 mutant mice significantly reduced cyst formation, and kidney function was improved. Reductions were seen in inflammation, fibrosis, and urinary excretion of IL-18. Analogous results were obtained through tubule-specific inactivation of Pycard/Asc or treatment of Pkd1 mutant mice with the inflammasome inhibitor MCC950. These findings demonstrate that inflammasomes act as drivers of disease severity in an orthologous mouse model of ADPKD. We pinpoint a separate, epithelial pool of inflammasomes in the diseased kidney and identify inflammasome inhibition as a promising strategy for the treatment of ADPKD.

Proteinuric kidney disease substantially affects renal tubules through incompletely understood mechanisms. We identify elongation of primary cilia in distal renal tubules in the context of glomerular nephropathy. In renal biopsies and mouse models, tubular injury correlates with ciliary elongation, tubule dilation, and disruption of the cortical actin cytoskeleton. In vitro studies implicate biophysical cues of the glomerular filtrate and subsequent dysregulation of the actin cytoskeleton as contributing factors, confirmed by conditional deletion of N-WASP and Arp2/3 in vivo and in vitro. Electron and fluorescence microscopy revealed enlarged ciliary pockets, basal body mislocalization, and intracellular cilia formation in Arp3 knockout conditions. Transcriptome analysis identifies the essential role of cilia in maintaining adaptive tubular cell states, while persistent activation leads to disease progression through extracellular matrix remodeling, exemplified by Tenascin-C. Our findings establish cilia as central mediators of tubular adaptation to injury and identify the Arp2/3-dependent actin cytoskeleton as a critical regulator, providing essential insights into the pathogenesis of chronic kidney disease.