ABSTRACT Regulatory T (Treg) cells expressing RORγt accumulate in the intestinal mucosa, yet the signals that determine whether they remain suppressive or acquire inflammatory features are incompletely defined. We first reanalyzed human ileal single-cell data and identified Crohn’s disease–enriched FOXP3⁺ states in which RORC, HIF1A, hypoxia-responsive, inflammatory, and metabolic programs converged. We then deleted Hif1a in RORγt-expressing cells and tested acute DSS colitis, T cell transfer colitis, and azoxymethane/DSS-induced colitis-associated colorectal cancer (CAC). ΔHif1a mice were protected in all three settings. In lymphopenic recipients given the same pathogenic naïve T cells, changing only the genotype of the cotransferred Treg population enhanced protection, linking the phenotype to regulatory-cell function in vivo . Reanalysis of mouse colonic Treg single-cell ATAC-seq nominated suppressive and mitochondrial programs for cell-intrinsic testing during low HIF1-α expression. ΔHif1a RORγt⁺ Treg produced more IL-10 and less IL-17A and IFN-γ, limited responder-cell proliferation, contained fewer dysfunctional and mitochondrial-reactive-oxygen-species-high mitochondria, favored fusion-associated transcription, and displayed greater basal and maximal oxygen consumption and reserve capacity. During CAC, HIF-1α loss blunted inflammatory RORγt⁺ Treg accumulation and reduced tumor burden. Human trajectory and gene-regulatory-network analyses further predicted that HIF1A perturbation would oppose selected disease-associated branches. Together, these findings identify HIF-1α as a context-dependent checkpoint that connects hypoxia-responsive transcription to mitochondrial fitness and inflammatory plasticity in intestinal RORγt⁺ Treg.

Intestinal organoids provide physiologically relevant models of the epithelial barrier, but lack the immune compartment that critically shapes host responses to infection. Here, we established a human colon organoid-derived monolayer co-culture system with macrophage-like THP-1 cells positioned directly beneath the epithelial layer. The model enabled controlled apical infection with Listeria monocytogenes and Salmonella Typhimurium while preserving epithelial barrier integrity. PMA-differentiated THP-1 cells reduced intracellular L. monocytogenes burden, whereas additional activation with IFN-γ and LPS resulted in a pronounced reduction of both L. monocytogenes and S. Typhimurium, accompanied by decreased infection-associated cytotoxicity. Bulk RNA sequencing revealed a distinct co-culture transcriptional signature characterized by coordinated changes in inflammatory, antimicrobial, and epithelial lineage-associated programs. These included reduced HLA-D/MHC class II-associated gene expression, altered S100A8/S100A9 expression, and changes in epithelial lineage markers indicating a shift in epithelial cellular composition and differentiation state. Together, these findings establish a versatile human organoid-macrophage platform for dissecting epithelial-immune interactions and macrophage-associated control of invasive bacterial infection.

IntroductionThe intestinal immune system is organized into regionally specialized lymphatic drainage networks that coordinate adaptive responses according to the anatomical site of microbial encounter. During Citrobacter rodentium (C. rodentium) infection in mice, initial colonization occurs in the cecum and proximal colon, where antigen‑presenting cells drain to the corresponding mesenteric lymph nodes (mLN), before the pathogen progresses to dominant attachment in the distal colon at peak infection. These segment‑specific patterns of colonization are matched by segment‑specific lymphatic drainage, such that distinct lymph node compartments support immune priming in different intestinal regions. Understanding how these proximal draining mLN contribute to the development of colonic immunity, despite the later dominance of distal‑colonic infection, remains an important open question.MethodsTo examine the contribution of the colon‑draining mLN, we surgically removed either the entire mLN chain or only the nodes draining the cecum and colon, followed by C. rodentium infection. Immune responses were analyzed at day 10 and day 18, corresponding to phases in which distal‑colonic colonization dominates.ResultsWe found that Th17 effector responses in the colon were maintained independently of the removed mLN, consistent with the fact that local colonic activation is the primary driver of the observed T cell phenotype at these time points. In contrast, B cell differentiation was markedly impaired in mLN‑resected animals: both plasma cell frequencies in the colon and pathogen‑specific serum IgG1 and IgG2a responses were significantly reduced.DiscussionThese findings indicate that, although distal‑colonic T cell activation proceeds locally, effective B cell activation and class switching depend on the presence of the appropriate draining lymph nodes. Overall, this study highlights the segment‑specific organization of intestinal immunity and demonstrates that B cell responses during C. rodentium infection are critically dependent on the lymph nodes draining the cecum and colon, whereas local T cell activation in the distal colon can occur independently of these structures.

Monocyte heterogeneity and plasticity create a spectrum of phagocytes essential for innate immune functions, which is partially regulated by Notch signaling. Using systematic monocyte subset analysis in different compartments, we here confirm that monocyte heterogeneity extends beyond the Ly6Chi and Ly6Clo monocytes and is regulated by Notch. Employing different monocyte-lineage-development-specific Cre-deleter strains in combination with conditional alleles for the receptor Notch2 or the Notch nuclear mediator Rbpj, we also show that subset development is differentially regulated by Notch-signaling components. Deletion of Notch2 broadly affects development of Ly6Clo monocytes, or related monocyte subsets, and alters monocyte phenotypes, while deletion of Rbpj has more restricted effects, mostly on monocyte phenotypes. Furthermore, the developmental plasticity of Ly6Chi monocyte subsets in vitro is regulated by Notch2 but dependent on the context of specific Notch ligand and myeloid growth factor. Thus, Notch signaling components differentially regulate monocyte heterogeneity and plasticity.

Objective: Regulatory T cells (Tregs) are essential in maintaining immune tolerance and controlling inflammation. Treg stability relies on transcriptional and post-translational mechanisms, including histone acetylation at the Foxp3 locus and FoxP3 protein acetylation. Additionally, Tregs depend on specific metabolic programs for differentiation, yet the underlying molecular mechanisms remain elusive. We aimed to investigate the role of acetyl-CoA carboxylase 1 (ACC1) in the differentiation, stability, and function of regulatory T cells (Tregs). Methods: We used either T cell-specific ACC1 knockout mice or ACC1 inhibition via a pharmacological agent to examine the effects on Treg differentiation and stability. The impact of ACC1 inhibition on Treg function was assessed in vivo through adoptive transfer models of Th1/Th17driven inflammatory diseases. Results: Inhibition or genetic deletion of ACC1 led to an increase in acetyl-CoA availability, promoting enhanced histone and protein acetylation, and sustained FoxP3 transcription even under inflammatory conditions. Mice with T cell-specific ACC1 deletion exhibited an enrichment of double positive RORgt+FoxP3+ cells. Moreover, Tregs treated with an ACC1 inhibitor demonstrated superior long-term stability and an enhanced capacity to suppress Th1/Th17-driven inflammatory diseases in adoptive transfer models. Conclusions: We identified ACC1 as a metabolic checkpoint in Treg biology. Our data demonstrate that ACC1 inhibition promotes Treg differentiation and long-term stability in vitro and in vivo. Thus, ACC1 serves as a dual metabolic and epigenetic hub, regulating immune tolerance and inflammation by balancing de novo lipid synthesis and protein acetylation. (c) 2025 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

OBJECTIVE:Neuronal cell death and neuroinflammation are characteristic features of epilepsy, but it remains unclear whether neuronal cell death as such is causative for the development of epileptic seizures. To test this hypothesis, we established a novel mouse line permitting inducible ablation of pyramidal neurons by inserting simian diphtheria toxin (DT) receptor (DTR) cDNA into the Ccl17 locus. The chemokine CCL17 is expressed in pyramidal CA1 neurons in adult mice controlling microglial quiescence. METHODS:Seizure activity in CCL17-DTR mice was analyzed by electroencephalographic recordings following treatment with DT for 3 consecutive days. Neuroinflammation and neuronal cell death were evaluated by (immuno)histochemistry. Pharmacological inhibition of TNFR1 signaling was achieved by treatment with XPro1595, a dominant-negative inhibitor of soluble tumor necrosis factor. RESULTS:Neuronal cell death was detectable 7 days (d7) after the first DT injection in heterozygous CCL17-DTR mice. Spontaneous epileptic seizures were observed in the vast majority of mice, often with an initial peak at d6-9, followed by a period of reduced activity and a gradual increase during the 1-month observation period. Microglial reactivity was overt from d5 after DT administration not only in the CA1 region but also in the CA2/CA3 area, shortly followed by astrogliosis. Reactive microgliosis and astrogliosis persisted until d30 and, together with neuronal loss and stratum radiatum shrinkage, reflected important features of human hippocampal sclerosis. Granule cell dispersion was detectable only 3 months after DT treatment. Application of XPro1595 significantly reduced chronic seizure burden without affecting the development of hippocampal sclerosis. SIGNIFICANCE:In conclusion, our data demonstrate that sterile pyramidal neuronal death is sufficient to cause epilepsy in the absence of other pathological processes. The CCL17-DTR mouse line may thus be a valuable model for further mechanistic studies on epilepsy and assessment of antiseizure medication.

Red pulp macrophages (RPM) and bone marrow macrophages (BMM) are iron-recycling cells involved in iron homeostasis and erythropoiesis. Here, we show, using conditional deletion strategies of Notch signaling components, that the development of RPM and BMM is regulated by canonical Notch2 signaling. Loss of functional Notch2 , or its nuclear mediator Rbpj , causes impairment in RPM and BMM and iron overload in the spleen and bone marrow, while prototypic RPM genes required for iron handling are downregulated. This was accompanied by splenic extramedullary hematopoiesis and changes in splenic microarchitecture. Furthermore, early postnatal transfer of bone marrow and fetal liver progenitors rescued the defects in RPM and BMM and iron overload in a Notch2 -dependent manner, demonstrating the potential to restore defective tissue resident macrophage niches by Notch-competent progenitors. Thus, canonical Notch2 is required for development and function of iron-recycling macrophages. ### Competing Interest Statement The authors have declared no competing interest.

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

Epigenetic modifications such as DNA methylation play an essential role in imprinting specific transcriptional patterns in cells. We performed genome-wide DNA methylation profiling of murine lymph node-derived ILCs, which led to the identification of differentially methylated regions (DMRs) and the definition of epigenetic marker regions in ILCs. Marker regions were located in genes with a described function for ILCs, such as Tbx21, Gata3, or Il23r, but also in genes that have not been related to ILC biology. Methylation levels of the marker regions and expression of the associated genes were strongly correlated, indicating their functional relevance. Comparison with T helper cell methylomes revealed clear lineage differences, despite partial similarities in the methylation of specific ILC marker regions. IL-33-mediated challenge affected methylation of ILC2 epigenetic marker regions in the liver, while remaining relatively stable in the lung. In our study, we identified a set of epigenetic markers that can serve as a tool to study phenotypic and functional properties of ILCs.

BACKGROUND:The cross-talk between the host and its microbiota plays a key role in the promotion of health. The production of metabolites such as polyamines by intestinal-resident bacteria is part of this symbiosis shaping host immunity. The polyamines putrescine, spermine, and spermidine are abundant within the gastrointestinal tract and might substantially contribute to gut immunity.OBJECTIVE:We aimed to characterize the polyamine spermidine as a modulator of T-cell differentiation and function.METHODS:Naive T cells were isolated from wild-type mice or cord blood from healthy donors and submitted to polarizing cytokines, with and without spermidine treatment, to evaluate CD4+ T-cell differentiation in vitro. Moreover, mice were subjected to oral supplementation of spermidine, or its precursor l-arginine, to assess the frequency and total numbers of regulatory T (Treg) cells in vivo.RESULTS:Spermidine modulates CD4+ T-cell differentiation in vitro, preferentially committing naive T cells to a regulatory phenotype. After spermidine treatment, activated T cells lacking the autophagy gene Atg5 fail to upregulate Foxp3 to the same extent as wild-type cells. These results indicate that spermidine's polarizing effect requires an intact autophagic machinery. Furthermore, dietary supplementation with spermidine promotes homeostatic differentiation of Treg cells within the gut and reduces pathology in a model of T-cell transfer-induced colitis.CONCLUSION:Altogether, our results highlight the beneficial effects of spermidine, or l-arginine, on gut immunity by promoting Treg cell development.

Regulating T cell differentiation through the polyamine spermidine 1 Guilhermina M Carriche, MSc1,2, Luís Almeida, MSc1,2, Philipp Stüve, PhD1,2, Lis Velasquez, PhD1,2, , 2 Ayesha Dhillon-LaBrooy, Hons1,2, Urmi Roy, PhD3, Marc Lindenberg, MD1,2, Till Strowig, PhD3,4, Carlos 3 Plaza-Sirvent, PhD5, Ingo Schmitz, PhD5,6,7, Matthias Lochner, PhD1,8, Anna Katharina Simon, PhD9, Tim 4 Sparwasser, MD1,2 5 6 1Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture 7 between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany. 8 2Institute of Medical Microbiology and Hygiene, University Medical Center of the Johannes Gutenberg-University Mainz, 9 Germany 10 3Department of Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany 11 4Hannover Medical School, Hannover, Germany 12 5Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany 13 6Systems-Oriented Immunology and Inflammation Research Group, Department of Experimental Immunology, 14 Helmholtz Center for Infection Research, Braunschweig, Germany 15 7Department of Molecular Immunology, Ruhr-University Bochum, Bochum, Germany 16 8Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Germany 17 9Kennedy Institute of Rheumatology, University of Oxford, UK 18

Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.

CD4+ T cells contribute critically to a protective immune response during intestinal infections, but have also been implicated in the aggravation of intestinal inflammatory pathology. Previous studies suggested that T helper type (Th)1 and Th17 cells depend on de novo fatty acid (FA) synthesis for their development and effector function. Here, we report that T-cell-specific targeting of the enzyme acetyl-CoA carboxylase 1 (ACC1), a major checkpoint controlling FA synthesis, impaired intestinal Th1 and Th17 responses by limiting CD4+ T-cell expansion and infiltration into the lamina propria in murine models of colitis and infection-associated intestinal inflammation. Importantly, pharmacological inhibition of ACC1 by the natural compound soraphen A mirrored the anti-inflammatory effects of T-cell-specific targeting, but also enhanced susceptibility toward infection with C. rodentium. Further analysis revealed that deletion of ACC1 in RORγt+ innate lymphoid cells (ILC), but not dendritic cells or macrophages, decreased resistance to infection by interfering with IL-22 production and intestinal barrier function. Together, our study suggests pharmacological targeting of ACC1 as an effective approach for metabolic immune modulation of T-cell-driven intestinal inflammatory responses, but also reveals an important role of ACC1-mediated lipogenesis for the function of RORγt+ ILC.

Abstract During the last decades, hypervirulent strains of Clostridioides difficile with frequent disease recurrence and increased mortality appeared. Clostridioides difficile DSM 101085 was isolated from a patient who suffered from several recurrent infections and colonizations, likely contributing to a fatal outcome. Analysis of the toxin repertoire revealed the presence of a complete binary toxin locus and an atypical pathogenicity locus consisting of only a tcdA pseudogene and a disrupted tcdC gene sequence. The pathogenicity locus shows upstream a transposon and has been subject to homologous recombination or lateral gene transfer events. Matching the results of the genome analysis, neither TcdA nor TcdB production but the expression of cdtA and cdtB was detected. This highlights a potential role of the binary toxin C. difficile toxin in this recurrent colonization and possibly further in a host-dependent virulence. Compared with the C. difficile metabolic model strains DSM 28645 (630Δerm) and DSM 27147 (R20291), strain DSM 101085 showed a specific metabolic profile, featuring changes in the threonine degradation pathways and alterations in the central carbon metabolism. Moreover, products originating from Stickland pathways processing leucine, aromatic amino acids, and methionine were more abundant in strain DSM 101085, indicating a more efficient use of these substrates. The particular characteristics of strain C. difficile DSM 101085 may represent an adaptation to a low-protein diet in a patient with recurrent infections.