Primary human intestinal epithelial cells (IECs) require microenvironments that reproduce various in vivo cues to maintain survival, differentiation, and function in vitro. In this study, we investigated how intestinal stem cell (ISC)-derived monolayers respond to biomimetic substrates and shear stress using 3D-printed hydrogels based on bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA) integrated into a custom millifluidic system. Extensive bulk RNA sequencing experiments revealed that, compared with Matrigel-coated tissue culture plastic, dSIS-MA hydrogels supported survival- and differentiation-related signaling, stabilized gene expression over time, and promoted absorptive lineage maturation while reducing crypt-associated signatures. By applying dynamic culture conditions to the hydrogel system, IECs underwent transcriptional remodeling, characterized by activation of metabolic and immune pathways. Longitudinal analysis further indicated that shear stress enhanced metabolic pathway-associated gene expression and promoted differentiation toward absorptive lineages. These findings establish dSIS-MA hydrogels with controlled fluid flow as a biomimetic in vitro model that supports survival, maturation of human IECs and enables transcriptional adaptation to defined biochemical and mechanical cues, supporting future applications in disease modelling, drug testing, and regenerative medicine.

Acquired generalized lipodystrophy (AGL) is a rare metabolic disorder frequently associated with autoimmunity. Its etiology is incompletely understood, and the effect of adipose tissue loss on intestinal inflammation in AGL remains unclear. Using mass cytometry and single-cell RNA-seq, we observed an oligoclonal expansion of T cells in the periphery and inflamed intestine in a patient with AGL and Crohn's disease (AGLCD). To explore if loss of adipose tissue triggers lymphoproliferation, we studied lipodystrophic mice as a model for AGL. Unexpectedly, lipodystrophic mice did not show T cell expansion, were protected from colitis, and displayed a defect in the development of proinflammatory T cells, which could be reversed by allogeneic fat transplantations, indicating that clonal T cell expansion in AGLCD is not primarily caused by lipodystrophy. Instead, gene sequencing revealed a T cell-intrinsic de novo neuroblastoma RAS viral oncogene homolog (NRAS) mutation, implicating somatic mosaicism as a facilitator of clonal T cell expansion and intestinal inflammation in AGLCD.

Class II histone deacetylases (HDAC) orchestrate T cell-dependent immune responses via the epigenetic control of genes and via the post-translational modification of cytoplasmic and nuclear proteins. However, the contribution of single HDAC family members to the differentiation and function of peripheral CD8+ T cells remains elusive. We here demonstrate that HDAC7-deficiency leads to the upregulation of immune checkpoint molecules, increased apoptosis and disturbed glutamine homeostasis of peripheral murine CD8+ T cells, which we could link to a MEF2D-dependent induction of FasL expression ultimately deterring the survival of HDAC7-deficient CD8+ T cells. Likewise, we observed in mouse models of lymphoma, that mice with a T cell-specific deletion of Hdac7 harbor impaired anti-tumor immune responses in syngeneic transfer models of lymphoma and we found that HDAC7 is required for CD8+ T cell-dependent memory recall responses in models of lymphocytic choriomeningitis virus infection. Taken together, we identify HDAC7 as a central regulator of cellular exhaustion and apoptosis of peripheral CD8+ T cells, controlling CD8+ T cell dependent anti-tumor and anti-viral immunity in mice.

Lymphoid follicles in the human gut are critical immune hubs, yet their role in Crohn's disease pathogenesis remains poorly understood. Here, we apply multiplexed imaging mass cytometry to spatially profile Peyer's patches and lymphoid follicles in biopsies from healthy controls and Crohn's disease patients with ileitis, isolated colonic involvement, or in remission. Despite tissue heterogeneity, our optimized preprocessing pipeline enabled robust tissue annotation, single-cell phenotyping, and neighbourhood-level analysis. While conventional analysis based on cell frequencies did not distinguish disease states, spatial analysis revealed disease-associated remodelling of lymphoid architecture. Biopsies from colonic Crohn's disease patients showed, within follicles, increased frequencies of activated CD8⁺ T cells and a reduction in naïve T cells, alongside enrichment of B cell-T cell interaction neighbourhoods. These alterations were most pronounced in smaller B-cell patches, suggesting more functionally dynamic immune-cell interactions in compact lymphoid structures. In contrast, Crohn's disease ileitis samples closely resembled healthy tissue, with minimal structural or immune cell perturbations. Our data support a model in which Peyer's patches and lymphoid follicles undergo structural and functional remodelling in response to colonic inflammation. These findings underscore the value of spatially resolved immune profiling to uncover tissue-specific immune dynamics in inflammatory bowel disease.

Critical illness often leads to the development of intestinal dysbiosis, which can have a significant impact on disease outcome. Intestinal barrier dysfunction is a common problem in intensive care unit patients, particularly those with sepsis. Despite its importance, early and reliable diagnosis of barrier dysfunction and evaluation of therapeutic options remain lacking in clinical practice. Given that intestinal hyperpermeability is associated with increased translocation of luminal antigens and subsequent priming of naïve T cells, we hypothesized that analysis of circulating peripheral antigen-reactive T cells could provide insight into the functionality of the intestinal barrier. To test this hypothesis, 70 ICU patients were enrolled, including those with sepsis, those not meeting sepsis criteria, and COVID-19 patients, as well as 20 healthy volunteers. We identified a sepsis-specific T-helper cell signature in peripheral blood using the antigen-reactive T-cell enrichment (ARTE) technique followed by flow cytometric analysis. This signature was characterized by an expansion of gut trophic Bifidobacterium longum-reactive T-helper cells, indicating significant intestinal barrier dysfunction during sepsis. This approach allows the study of intestinal barrier functionality and provides a means to monitor the effects of potential therapeutic interventions over time using blood samples.

Abstract Background The intestinal epithelium is protected by a vital mucus layer which is essential for maintaining gut homeostasis and regulating host-microbial interactions. However, studying this mucus layer in vitro has been challenging due to the lack of physiologically relevant models. Methods We hypothesized that culturing human adult intestinal stem cells (ISCs) under flow conditions on a biomimetic scaffold could better recapitulate in vivo epithelial differentiation and mucus production. To test this, we developed a hydrogel-integrated millifluidic chamber, where ISCs were cultured on decellularized and methacrylated porcine small intestinal submucosa (dSIS-MA) under both static and dynamic (laminar flow) conditions. RNA bulk sequencing was used later to explore gene expression profiles across early and late time points to assess the impact of dSIS-MA and fluid flow on ISC differentiation and function over time. Results Our results reveal significant changes in pathways related to epithelial lineage commitment and intestinal barrier function on dSIS-MA compared to tissue culture plastic and in dynamic compared to static conditions, highlighting the critical influence of matrix structure and fluid flow. Conclusion In summary, we have successfully established a model that mimics in vivo conditions and reveals that surface and shear stress strongly impact intestinal epithelial cell development and function. This advanced in vitro approach enhances our understanding of molecular processes underlying epithelial cell behavior in conditions closely resembling the gut environment, with applications in gut health and disease research.

Acquired generalized lipodystrophy (AGL) is a rare metabolic disorder frequently associated with autoimmunity. Its etiology is incompletely understood and the impact of adipose tissue loss on autoimmunity and intestinal inflammation in AGL remains unclear. Using mass cytometry and single-cell RNA sequencing, we observed an oligoclonal expansion of T cells in the periphery and inflamed intestine in a patient with AGL and Crohns disease (AGLCD). To explore if loss of adipose tissue triggers lymphoproliferation, we studied lipodystrophic mice as a model for AGL. Unexpectedly, lipodystrophic mice did not show T-cell expansion, were protected from colitis and displayed a defect in the development of pro-inflammatory T cells, which could be reversed by allogeneic fat transplantations, indicating that clonal T-cell expansion is not primarily caused by lipodystrophy. Instead, gene sequencing revealed a T cell-intrinsic de-novo NRAS mutation, pointing towards somatic mosaicism as a driver of clonal T-cell expansion and systemic autoimmunity in AGLCD. ### Competing Interest Statement B.S. received grant support by Pfizer, served as consultant for Abbvie, BMS, Boehringer, Endpoint Health, Falk, Galapagos, Gilead, Janssen, Johnson & Johnson, Landos, Lilly, MSD, Pfizer, Takeda (BS served as representative of the Charité) and received speaker's fees from Abbvie, AlfaSigma, BMS, CED Service GmbH, Falk, Ferring, Galapagos, Janssen, Lilly, Pfizer, Takeda (BS served as representative of the Charité). C.W. received grant support by Pfizer, served as consultant for Pfizer and received speaker's fees from Falk, Ferring, Janssen. The other authors declare to not have any competing interests. ### Funding Statement This work was funded by the German Research Foundation (We 5303/3‐2 to CW, SFB‐TRR 241 (project-ID 375876048) B01 to BS and CW, A09 to AS and CW, A03 to CB and Z02 to BS, CRU 5023 (project-ID: 50474582), CRC 1449-B04 (project-ID: 431232613); CRC 1340-B06 (project-ID 372486779), SI749/14-1 (project-ID: 418055832) all to BS. CW and JZ received funding by the Clinician Scientist Program of the Berlin Institute of Health. CW received funding by the Fritz-Thyssen Foundation (10.19.2.028MN). Prargfl/fl mice were a kind gift of Prof. Dr. Ulrich Kintscher, Charite, Berlin. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Study was approved by the institutional review board of Charite - Universitatsmedizin Berlin (Approval number EA1/200/17). Written informed consent was obtained from all healthy volunteers and patients as approved by the institutional review board of Charite - Universitatsmedizin Berlin. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Patient data supporting the human findings of this study are available from the corresponding authors upon reasonable request. scRNA-seq and mouse bulk seq data for this project will be uploaded to the NCBI database upon acceptance of this paper.

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disease, which can affect any part of the gastrointestinal tract. It is characterized by alterations in the innate and adaptive immune systems. Changes in both monocyte and macrophage (MΦ) populations have been reported in CD patients. However, the precise contribution of these cells to disease pathogenesis and progression remains unclear. Our hypothesis is that in CD, the local environment affects the metabolism of monocytes and MΦ. Furthermore, we believe that the cell’s metabolism is essential for the phenotypic and functional identity of these cells. The aim of this study is to analyze and compare the metabolic signatures of peripheral blood monocyte subsets and tissue MΦ in CD patients with ileitis (iCD), deep remission (rCD) and healthy controls (HC). Methods A total of 28 adults from the IBDome5 cohort were analyzed, consisting of 10 individuals with iCD, 8 with rCD and 10 HC. Suspension mass cytometry was utilized to analyze peripheral blood mononuclear cells (PBMCs) using a 34-marker panel targeting key proteins and rate-limiting enzymes across multiple metabolic pathways. In addition, we conducted further analysis on intestinal tissue obtained during ileocolonoscopy using imaging mass cytometry. This allowed for the application of a similar marker panel to compare the metabolic states of tissue-resident MΦ (ongoing analysis) with the identified signatures found in peripheral blood. Results Peripheral blood samples of CD patients and HC were analyzed. FlowSOM clustering of CD45+ events discriminated 11 major clusters of PBMCs. Reclustering based on CD14 and CD16 allowed the identification of 4 monocyte clusters, including classical monocytes. Classical monocytes exhibited unique metabolic signatures that distinguished them from other monocyte populations. Ongoing investigations targeting metabolic signatures in the monocyte-MΦ compartment will determine whether disease states differ significantly between the single disease entities. Conclusion Single-cell phenotyping using suspension and imaging mass cytometry can assess metabolic states of the monocyte-MΦ compartment in CD and HC. Such insights may provide novel markers for monitoring disease activity and reveal potential therapeutic targets.

The IL-6–gp130–STAT3 signaling axis is a major regulator of inflammation. Activating mutations in the gene encoding gp130 and germline gain-of-function mutations in STAT3 (STAT3 GOF ) are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis. To dissect crucial cellular subsets and disease biology involved in activated gp130 signaling, the gp130-JAK-STAT3 axis was constitutively activated using a transgene, L-gp130 , specifically targeted to T cells. Activating gp130 signaling in T cells in vivo resulted in fatal, early onset, multi-organ autoimmunity in mice that resembled human STAT3 GOF disease. Female mice had more rapid disease progression than male mice. On a cellular level, gp130 signaling induced the activation and effector cell differentiation of T cells, promoted the expansion of T helper type 17 (T H 17) cells, and impaired the activity of regulatory T cells. Transcriptomic profiling of CD4 + and CD8 + T cells from these mice revealed commonly dysregulated genes and a gene signature that, when applied to human transcriptomic data, improved the segregation of patients with transcriptionally diverse STAT3 GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to T H 17-driven autoimmunity that phenotypically resembles human STAT3 GOF disease.

Abstract Background Crohn’s disease (CD) is a chronic transmural disease that can affect any part of the gastrointestinal tract. Typically, the terminal ileum is impacted. The pathogenesis of this disease results from a complicated interaction of multiple factors, with the intestinal barrier playing a critical role. The intestinal barrier in the ileum is comprised of epithelial cells that are coated with a mucus layer. This barrier is permeable to nutrient absorption and microbiota. Detailed analyses of human ileal mucus are currently absent. Our study aimed to comprehend the function of mucus and the biophysical and biochemical stimuli that exist in the mucus layer during ileal inflammation, in contrast to the healthy state. Methods We enrolled 28 patients diagnosed with Crohn’s disease (CD) and 27 healthy individuals as part of our study. Mucus and biopsies from the terminal ileum were collected during a colonoscopy. The extent of clinical, endoscopic and histologic inflammation in the terminal ileum at the time of collection was evaluated using the Harvey-Bradshaw Index, Simple Endoscopic Score, and modified Naini and Cortina score. The disease trajectory and dietary behaviors of all patients and healthy controls were documented. Samples were processed following protocols for various analyses, including rheology, proteomics and scanning electron microscopy (SEM). Results Our study presents the first data on the rheology and viscoelastic properties, of human ileal mucus. Interestingly, we discovered notable variations in the viscoelastic properties of mucus in CD patients compared to healthy individuals. Furthermore, the mucus mesh size and structure differed in CD patients in comparison to healthy controls. The SEM pictures also displayed these quantitative changes. We further assessed clinical characteristics, encompassing duration of disease, and observed certain variations, although their significance is not yet statistically established. A distinct profile was detected in CD patients compared to healthy controls using proteomic analysis. The ileal mucus of CD patients demonstrated elevated levels of ITGB2 and S100 proteins, which are involved in inflammatory pathways. Moreover, a correlation was found with histologic inflammation at the time of the collection. Conclusion In our study, significant changes in mucus composition and structure were found in the terminal ileum of CD patients, compared to healthy controls. This preliminary analysis provides a basis for further investigations to establish correlations between mucus composition and viscoelastic properties, and the subsequent effects on the underlying epithelial barrier.

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

Crohn's disease (CD) is an inflammatory bowel disease that can affect any part of the gastrointestinal tract, frequently involving the terminal ileum. While colonic mucus alterations in CD patients have been described, terminal ileal mucus and its mechanobiological properties have been neglected. Our study is the first of its kind to decipher the viscoelastic and network properties of ileal mucus. With that aim, oscillatory rheological shear measurements based on an airway mucus protocol that was thoroughly validated for ileal mucus were performed. Our pilot study analyzed terminal ileum mucus from controls (n = 14) and CD patients (n = 14). Mucus network structure was visualized by scanning electron microscopy. Interestingly, a statistically significant increase in viscoelasticity as well as a decrease in mesh size was observed in ileal mucus from CD patients compared to controls. Furthermore, rheological data were analyzed in relation to study participants' clinical characteristics, revealing a noteworthy trend between non-smokers and smokers. In conclusion, this study provides the first data on the viscoelastic properties and structure of human ileal mucus in the healthy state and Crohn's disease, demonstrating significant alterations between groups and highlighting the need for further research on mucus and its effect on the underlying epithelial barrier.

To demonstrate and analyze the specific T-cell response following barrier disruption and antigen translocation, circulating food antigen-specific effector T-cells isolated from peripheral blood were analyzed in patients suffering from celiac disease (CeD) as well as inflammatory bowel disease (IBD). We applied the antigen-reactive T-cell enrichment (ARTE) technique allowing for phenotypical and functional flow cytometric analyses of rare nutritional antigen-specific T-cells, including the celiac disease-causing gliadin (gluten). For CeD, patient groups, including treatment-refractory cases, differ significantly from healthy controls. Even symptom-free patients on a gluten-free diet were distinguishable from healthy controls, without being previously challenged with gluten. Moreover, frequency and phenotype of nutritional antigen-specific T-cells of IBD patients directly correlated to the presence of small intestinal inflammation. Specifically, the frequency of antigen specific T-cells as well as pro-inflammatory cytokines was increased in patients with active CeD or Crohn's disease, respectively. These results suggest active small intestinal inflammation as key for the development of a peripheral food antigen-specific T-cell response in Crohn's disease and celiac disease.

Inflammatory bowel diseases are medically intractable and require constant therapy in many cases. While a growing number of biologicals and small molecules is available for treatment, a substantial portion of patients experiences primary non-response to these compounds and head-to-head evidence for therapy selection is scarce. Thus, approaches to predict treatment success in individual patients are a huge unmet need.We had previously suggested that the expression and function of α4β7 integrin on T cells in the peripheral blood correlate to outcomes of therapy with the anti-α4β7 integrin antibody vedolizumab. Here, we conducted a translational multicenter trial to prospectively evaluate this hypothesis.In a cohort of 89 patients with inflammatory bowel disease undergoing regular therapy with vedolizumab, lower baseline expression of α4β7 was associated with short-term clinical response. Consistently, low α4β7 expression in patients achieving remission predicted sustained remission in week 30. Moreover, high dynamic adhesion of CD4+ T cells to MAdCAM-1 and high reduction of adhesion by vedolizumab in vitro at baseline were associated with clinical remission.These data substantiate the potential of α4β7 integrin function and expression to forecast outcomes of vedolizumab therapy. Further translational efforts are necessary to improve the performance of the assays and to implement the concept in clinical practice.

Relapse of transplanted MCA205 tumors after chemotherapy.Blood vessels were less covered by pericytes during tumor relapse. FACS analysis of Tie2+CD11b+ cells in DOX-treated tumor and peripheral blood.Generation and genotyping of Tie2 conditional knockout mice.CD11b+Ly6C+ MDSC accumulated in tumor microenvironment after chemotherapy.DOX induced higher level of hypoxia in tumor microenvironment.DOX did not directly induce Tie2 expression on CD11b+Ly6C+ MDSC in vitro.No Tie2+EGFP+ cells in spleen and blood of DOX-treated mice.Identification of Tie2 expression on transfected macrophages.Tie2 sustains the viability of macrophages in stressed condition.Tie2 gene deletion in CD11b+Ly6C+ MDSC did not impair their viability in spleens. Schematic diagram of tumor blood vessel reconstruction after chemotherapy.

Abstract Background Crohn’s disease (CD) is a chronic inflammatory condition that can affect all parts of the intestine. Commonly, there is inflammation in the terminal ileum. An intestinal barrier consisting of epithelial cells covered by a mucus layer is on the one hand preventing pathogen invasion while on the other hand allowing the uptake of nutrients. However, despite the significance of the terminal ileum in CD, little is known about the mucus composition and viscoelastic properties in the terminal ileum. In our study, we wanted to do a comprehensive analysis of the mucus in the terminal ileum of CD patients compared to healthy controls. Thus, we aspire to gain a better understanding of the role of mucus and biophysical and biochemical triggers contained in the mucus layer during the course of ileal inflammation. Methods During colonoscopy, native mucus and saline washes containing mucus as well as biopsies from the terminal ileum of 33 CD patients and 25 healthy controls were collected. The disease course and dietary habits of all patients and healthy controls were recorded. Samples were treated according to protocol for different types of analysis such as rheology, proteomics, metabolomics, and microbiome. Furthermore, human intestinal organoids were generated from the biopsies. Additionally, we analysed bulk RNA sequencing data from intestinal mucosal biopsies of a cohort of 128 patients, consisting of non-IBD individuals and CD patients. Results We here present preliminary data for the different types of analyses. Regarding the viscoelastic properties, preliminary rheology data suggests that there is no major difference between CD patients and healthy controls. As for proteomics, the first cohort is currently being analysed. Concerning the microbiome, we could show that saline washes containing mucus are more suitable than biopsies for analysing bacterial as well as host DNA. Although not statistically significant, healthy controls showed a tendency for higher alpha- diversity in microbiota. Also there were some differences in the taxonomy. Intestinal organoids were grown as cultures. In the end, the results of the analyses mentioned above will be interpreted together with the clinical data. Concerning the gene expression, we found that 12 mucin and mucin-like genes were expressed both in colon and ileum tissue. Interestingly, a subset of CD patients showed a de-novo expression of MUC5AC, MUC6, MUCL1 and MUCL3 in the ileum. Conclusion All in all, in our multidisciplinary study we hope to achieve a thorough analysis of the mucus layer in the terminal ileum, thus gaining new insight into the pathogenetic mechanisms in intestinal inflammation that could prospectively be used for therapeutic means.