Alzheimer's disease-related presenilins are key to intestinal epithelial cell function and gut immune homoeostasis.

OBJECTIVE: Mutations in presenilin genes are the major cause of Alzheimer's disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis. DESIGN: Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis. RESULTS: Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation. CONCLUSION: Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.

Spatial single cell profiling using imaging mass cytometry - inflammatory versus penetrating Crohn's disease.

BACKGROUND AND AIMS: Fistula formation is a major complication in Crohn's disease (CD) and the role of the immune cell compartment remains to be elucidated. Thus, we compared the immune-cell compartment of CD fistula to inflammatory CD colitis using imaging mass cytometry and immunofluorescence. METHODS: A 36-marker panel including structural, functional and lineage markers for use in imaging mass cytometry (IMC) was established. This panel was applied to analyze paraffin-embedded CD fistula tract (n=11), CD colitis (n=10), and colon samples from non-inflamed controls (n=12). Computational methods for cell segmentation, dimensionality reduction and cell type clustering were used to define cell populations for cell frequency, marker distribution and spatial neighborhood analysis. Multiplex immunofluorescence was used for higher resolution spatial analysis. RESULTS: Analysis of cell frequencies in CD fistulas compared to CD colitis and control colonic samples revealed a significant increase in neutrophils, effector cytotoxic T cells and inflammatory macrophages in CD fistula samples, whereas regulatory T cells were decreased. Neutrophils in CD fistula expressed significantly more matrix metalloproteinase 9 (MMP9), correlating to extracellular matrix remodeling. Neighborhood analysis revealed a strong association between MMP9+ neutrophils and effector cytotoxic T cells in both CD fistulas and colitis. CONCLUSIONS: This study presents the first highly multiplexed single cell analysis of the immune-cell compartment of CD fistulas and their spatial context. It links immune cell dynamics, particularly MMP9+ neutrophils, to extracellular matrix remodeling in CD fistulas, offering insights into the complex network of cellular interactions and potential therapeutic targets for CD complications.

Predictive role of intracranial PD-L1 expression in a real-world cohort of NSCLC patients treated with immune checkpoint inhibition following brain metastasis resection.

BACKGROUND: Emerging evidence suggests that treatment of NSCLC brain metastases with immune checkpoint inhibitors (ICIs) is associated with response rates similar to those of extracranial disease. Programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) serves as a predictive biomarker for ICI response. However, the predictive value of brain metastasis-specific (intracranial) PD-L1 TPS is not established. We investigated the role of intra- and extracranial PD-L1 TPS in NSCLC patients treated with ICI following brain metastasis resection. METHODS: Clinical data from NSCLC patients treated with ICI following brain metastasis resection (n = 64) were analyzed. PD-L1 TPS of brain metastases (n = 64) and available matched extracranial tumor tissue (n = 44) were assessed via immunohistochemistry. Statistical analyses included cut point estimation via maximally selected rank statistics, Kaplan-Meier estimates, and multivariable Cox regression analysis for intracranial progression-free survival (icPFS), extracranial progression-free survival (ecPFS), and overall survival (OS). RESULTS: PD-L1 expression was found in 54.7% of brain metastases and 68.2% of extracranial tumor tissues, with a median intra- and extracranial PD-L1 TPS of 7.5% (0 - 50%, IQR) and 15.0% (0 - 80%, IQR), respectively. In matched tissue samples, extracranial PD-L1 TPS was significantly higher than intracranial PD-L1 TPS (p = 0.013). Optimal cut points for intracranial and extracranial PD-L1 TPS varied according to outcome parameter assessed. Notably, patients with a high intracranial PD-L1 TPS (> 40%) exhibited significantly longer icPFS as compared to patients with a low intracranial PD-L1 TPS (≤ 40%). The cut point of 40% for intracranial PD-L1 TPS was independently associated with OS, icPFS and ecPFS in multivariable analyses. CONCLUSION: Our study highlights the potential role of intracranial PD-L1 TPS in NSCLC, which could be used to predict ICI response in cases where extracranial tissue is not available for PD-L1 assessment as well as to specifically predict intracranial response.

Activation of gp130 signaling in T cells drives TH17-mediated multi-organ autoimmunity.

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 (STAT3GOF) 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 STAT3GOF 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 (TH17) 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 STAT3GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to TH17-driven autoimmunity that phenotypically resembles human STAT3GOF disease.

Cortical matrix remodeling as a hallmark of relapsing-remitting neuroinflammation in MR elastography and quantitative MRI.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease that involves both white and gray matter. Although gray matter damage is a major contributor to disability in MS patients, conventional clinical magnetic resonance imaging (MRI) fails to accurately detect gray matter pathology and establish a clear correlation with clinical symptoms. Using magnetic resonance elastography (MRE), we previously reported global brain softening in MS and experimental autoimmune encephalomyelitis (EAE). However, it needs to be established if changes of the spatiotemporal patterns of brain tissue mechanics constitute a marker of neuroinflammation. Here, we use advanced multifrequency MRE with tomoelastography postprocessing to investigate longitudinal and regional inflammation-induced tissue changes in EAE and in a small group of MS patients. Surprisingly, we found reversible softening in synchrony with the EAE disease course predominantly in the cortex of the mouse brain. This cortical softening was associated neither with a shift of tissue water compartments as quantified by T2-mapping and diffusion-weighted MRI, nor with leukocyte infiltration as seen by histopathology. Instead, cortical softening correlated with transient structural remodeling of perineuronal nets (PNNs), which involved abnormal chondroitin sulfate expression and microgliosis. These mechanisms also appear to be critical in humans with MS, where tomoelastography for the first time demonstrated marked cortical softening. Taken together, our study shows that neuroinflammation (i) critically affects the integrity of PNNs in cortical brain tissue, in a reversible process that correlates with disease disability in EAE, (ii) reduces the mechanical integrity of brain tissue rather than leading to water accumulation, and (iii) shows similar spatial patterns in humans and mice. These results raise the prospect of leveraging MRE and quantitative MRI for MS staging and monitoring treatment in affected patients.

Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. METHODS: Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. RESULTS: Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. CONCLUSIONS: In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.

IL-20 controls resolution of experimental colitis by regulating epithelial IFN/STAT2 signalling.

OBJECTIVE: We sought to investigate the role of interleukin (IL)-20 in IBD and experimental colitis. DESIGN: Experimental colitis was induced in mice deficient in components of the IL-20 and signal transducer and activator of transcription (STAT)2 signalling pathways. In vivo imaging, high-resolution mini-endoscopy and histology were used to assess intestinal inflammation. We further used RNA-sequencing (RNA-Seq), RNAScope and Gene Ontology analysis, western blot analysis and co-immunoprecipitation, confocal microscopy and intestinal epithelial cell (IEC)-derived three-dimensional organoids to investigate the underlying molecular mechanisms. Results were validated using samples from patients with IBD and non-IBD control subjects by a combination of RNA-Seq, organoids and immunostainings. RESULTS: In IBD, IL20 levels were induced during remission and were significantly higher in antitumour necrosis factor responders versus non-responders. IL-20RA and IL-20RB were present on IECs from patients with IBD and IL-20-induced STAT3 and suppressed interferon (IFN)-STAT2 signalling in these cells. In IBD, experimental dextran sulfate sodium (DSS)-induced colitis and mucosal healing, IECs were the main producers of IL-20. Compared with wildtype controls, Il20-/-, Il20ra-/- and Il20rb-/- mice were more susceptible to experimental DSS-induced colitis. IL-20 deficiency was associated with increased IFN/STAT2 activity in mice and IFN/STAT2-induced necroptotic cell death in IEC-derived organoids could be markedly blocked by IL-20. Moreover, newly generated Stat2ΔIEC mice, lacking STAT2 in IECs, were less susceptible to experimental colitis compared with wildtype controls and the administration of IL-20 suppressed colitis activity in wildtype animals. CONCLUSION: IL-20 controls colitis and mucosal healing by interfering with the IFN/STAT2 death signalling pathway in IECs. These results indicate new directions for suppressing gut inflammation by modulating IL-20-controlled STAT2 signals.

Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

Predictive Potential of Biomarkers of Intestinal Barrier Function for Therapeutic Management with Teduglutide in Patients with Short Bowel Syndrome.

INTRODUCTION: The human intestinal tract reacts to extensive resection with spontaneous intestinal adaptation. We analyzed whether gene expression analyses or intestinal permeability (IP) testing could provide biomarkers to describe regulation mechanisms in the intestinal barrier in short bowel syndrome (SBS) patients during adaptive response or treatment with the glucagon-like peptide-2 analog teduglutide. METHODS: Relevant regions of the GLP-2 receptor gene were sequenced. Gene expression analyses and immunohistochemistry were performed from mucosal biopsies. IP was assessed using a carbohydrate oral ingestion test. RESULTS: The study includes 59 SBS patients and 19 controls. Increases in gene expression with teduglutide were received for sucrase-isomaltase, sodium/glucose cotransporter 1, and calcium/calmodulin serine protein kinase. Mannitol recovery was decreased in SBS but elevated with teduglutide (Δ 40%), showed a positive correlation with remnant small bowel and an inverse correlation with parenteral support. CONCLUSIONS: Biomarkers predicting clinical and functional features in human SBS are very limited. Altered specific gene expression was shown for genes involved in nutrient transport but not for genes controlling tight junctions. However, mannitol recovery proved useful in describing the absorptive capacity of the gut during adaptation and treatment with teduglutide.

Feasibility of in vivo magnetic resonance elastography of mesenteric adipose tissue in Crohn's disease.

Background: Although there is growing evidence that functional involvement and structural changes of mesenteric adipose tissue (MAT) influence the course of Crohn's disease (CD), its viscoelastic properties remain elusive. Therefore, we aimed to investigate the viscoelastic properties of MAT in CD using magnetic resonance elastography (MRE), providing reference values for CD diagnosis. Methods: In this prospective proof-of-concept study, 31 subjects (CD: n=11; healthy controls: n=20) were consecutively enrolled in a specialized care center for inflammatory bowel diseases (tertiary/quaternary care). Inclusion criteria for the CD patients were a clinically and endoscopically established diagnosis of CD based on the clinical record, absence of other concurrent bowel diseases, scheduled surgery for the following day, and age of at least 18 years. Diagnoses were confirmed by histological analysis of the resected bowel the day after MRE. Subjects were investigated using MRE at 1.5-T with frequencies of 40-70 Hz. To retrieve shear wave speed (SWS), volumes of interest (VOIs) in MAT were drawn adjacent to CD lesions (MATCD) and on the opposite side without adjacent bowel lesions in patients (MATCD_Opp) and controls (MATCTRL). The presented study is not registered in the clinical trial platform. Results: A statistically significant decrease in mean SWS of 7% was found for MATCD_Opp vs. MATCTRL (0.76±0.05 vs. 0.82±0.04 m/s, P=0.012), whereas there was a nonsignificant trend with an 8% increase for MATCD vs. MATCD_Opp (0.82±0.07 vs. 0.76±0.05 m/s, P=0.098) and no difference for MATCD vs. MATCTRL. Preliminary area under the receiver operating characteristic curve (AUC) analysis showed diagnostic accuracy in detecting CD to be excellent for SWS of MATCD_Opp [AUC =0.82; 95% confidence interval (CI): 0.64-0.96] but poor for SWS of MATCD (AUC =0.52; 95% CI: 0.34-0.73). Conclusions: This study demonstrates the feasibility of MRE of MAT and presents preliminary reference values for CD patients and healthy controls. Our results motivate further studies for the biophysical characterization of MAT in inflammatory bowel disease.

Prevention of colitis-induced liver oxidative stress and inflammation in a transgenic mouse model with increased omega-3 polyunsaturated fatty acids.

Inflammatory bowel disease (IBD) is an immune-mediated gut dysfunction, which might also be associated with an inflammatory phenotype in the liver. It is known that the nutritional intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is inversely correlated to the severity and occurrence of IBD. In order to investigate whether n-3 PUFA can also reduce liver inflammation and oxidative liver damage due to colon inflammation, we explored the dextran sulfate sodium (DSS)-induced colitis model in wild-type and fat-1 mice with endogenously increased n-3 PUFA tissue content. Besides confirming previous data of alleviated DSS-induced colitis in the fat-1 mouse model, the increase of n-3 PUFA also resulted in a significant reduction of liver inflammation and oxidative damage in colitis-affected fat-1 mice as compared to wild-type littermates. This was accompanied by a remarkable increase of established inflammation-dampening n-3 PUFA oxylipins, namely docosahexaenoic acid-derived 19,20-epoxydocosapentaenoic acid and eicosapentaenoic acid-derived 15-hydroxyeicosapentaenoic acid and 17,18-epoxyeicosatetraenoic acid. Taken together, these observations demonstrate a strong inverse correlation between the anti-inflammatory lipidome derived from n-3 PUFA and the colitis-triggered inflammatory changes in the liver by reducing oxidative liver stress.

Interleukin 36 receptor-inducible matrix metalloproteinase 13 mediates intestinal fibrosis.

Background: Fibrostenotic disease is a common complication in Crohn's disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis. Methods: We performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson's Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis. Results: Bulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis. Conclusion: Targeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.

Toll-like receptors control the accumulation of neutrophils in lymph nodes that expand CD4+ T cells during experimental autoimmune encephalomyelitis.

Toll-like receptors (TLR) control the activation of dendritic cells that prime CD4+ T cells in draining lymph nodes, where these T cells then undergo massive clonal expansion. The mechanisms controlling this clonal T cell expansion are poorly defined. Using the CD4+ T cell-mediated disease experimental autoimmune encephalomyelitis (EAE), we show here that this process is markedly suppressed when TLR9 signaling is increased, without noticeably affecting the transcriptome of primed T cells, indicating a purely quantitative effect on CD4+ T cell expansion. Addressing the underpinning mechanisms revealed that CD4+ T cell expansion was preceded and depended on the accumulation of neutrophils in lymph nodes a few days after immunization. Underlying the importance of this immune regulation pathway, blocking neutrophil accumulation in lymph nodes by treating mice with a TLR9 agonist inhibited EAE progression in mice with defects in regulatory T cells or regulatory B cells, which otherwise developed a severe chronic disease. Collectively, this study demonstrates the key role of neutrophils in the quantitative regulation of antigen-specific CD4+ T cell expansion in lymph nodes, and the counter-regulatory role of TLR signaling in this process.

General practitioner care in nursing homes during the first wave of the COVID-19 pandemic in Germany: a retrospective survey among nursing home managers.

BACKGROUND: Though evidence on the detrimental impact of the COVID-19 pandemic in nursing homes is vast, research focusing on general practitioners' (GP) care during the pandemic in nursing homes is still scarce. METHODS: A retrospective online survey among 1,010 nursing home managers in Germany was conducted during the first wave of the COVID-19 pandemic between November 2020 and February 2021. Associations between perceived deficits in GP care (routine and acute visits) and both general and COVID-19-related characteristics of nursing homes were analysed using multiple logistic regression analyses. RESULTS: The majority of nursing home managers reported no deficits in GP care (routine visits, 84.3%; acute visits, 92.9%). Logistic regression analyses revealed that deficits in GP care (routine visits) were significantly associated with visiting restrictions for GPs and nursing home size. Small nursing homes (1-50 residents) were significantly more likely to report deficits in GP care (routine visits) compared to medium (51-100 residents) and large nursing homes (> 100 residents). Further, deficits in GP care (acute visits) were significantly associated with dementia as a focus of care and the burden of insufficient testing for SARS-CoV-2 among residents. Moreover, visiting restrictions for GPs were significantly associated with dementia as the focus of care and the COVID-19 incidence at the federal state level. Finally, COVID-19 cases in nursing homes were significantly associated with size of nursing homes, COVID-19-incidence on the federal state level and the burden of insufficient testing capacities for SARS-CoV-2 among residents. CONCLUSION: We found structural factors associated with GP care deficits during the pandemic. New concepts for GP care should be implemented in pandemic preparedness plans to ensure high quality, consistent, and reliable GP care as well as effective infection prevention measures in nursing homes.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles.

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.

A Biodegradable Polymeric Matrix for the Repair of Annulus Fibrosus Defects in Intervertebral Discs.

BACKGROUND: Tissue defects in the annulus fibrosus (AF) due to intervertebral disc (IVD) degeneration or after nucleodiscectomy have little self-healing capacity. To prevent progressive degeneration of the IVD, the AF must be repaired. Biological closure has not yet been achieved and is a challenge for the research community. In this study, a scaffold made of absorbable poly (glycolic acid) (PGA) and hyaluronan (HA) that exhibit excellent biocompatibility and cell colonization properties was used to repair AF defects in an ovine model. METHODS: A partial resection was performed in AF in L3/4 or L4/5 of 10 sheep and PGA-HA scaffolds were implanted on the defects (n = 5), while defects in the control group were left untreated (n = 5). Three months post-operation, the lumbar discs were sectioned and stained with hematoxylin and eosin and safranin-O/fast-green. Histological features including proteoglycan content, annular structure, cellular morphology, blood vessel ingrowth and tear/cleft formation were scored using a modified scoring scheme by 3 investigators and evaluated by a pathologist independently. RESULTS: The treated AF exhibited significantly enhanced repair tissue structure with signs of proteoglycan formation compared to the untreated group. The median scores were 4.3 for the treated and 9.8 for the untreated group. Cystic degeneration, perivascular infiltration, inflammation and necrosis were only present in the untreated group. Blood vessel ingrowth and tear/cleft formation were increased, though not significant, in the untreated group while cell morphology was comparable in both groups. CONCLUSION: PGA-HA scaffolds used for AF closure support repair tissue formation in an ovine lumbar disc defect model.

TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability.

Successful treatment of chronic inflammatory diseases integrates both the cessation of inflammation and the induction of adequate tissue repair processes. Strikingly, targeting a single proinflammatory cytokine, tumor necrosis factor (TNF), induces both processes in a relevant cohort of inflammatory bowel disease (IBD) patients. However, the molecular mechanisms underlying intestinal repair following TNF blockade during IBD remain elusive. Using a novel humanized model of experimental colitis, we demonstrate that TNF interfered with the tissue repair program via induction of a soluble natural antagonist of IL-22 (IL-22Ra2; IL-22BP) in the colon and abrogated IL-22/STAT3-mediated mucosal repair during colitis. Furthermore, membrane-bound TNF expressed by T cells perpetuated colonic inflammation, while soluble TNF produced by epithelial cells (IECs) induced IL-22BP expression in colonic dendritic cells (DCs) and dampened IL-22-driven restitution of colonic epithelial functions. Finally, TNF induced IL-22BP expression in human monocyte-derived DCs and levels of IL22-BP correlated with TNF in sera of IBD patients. Thus, our data can explain how anti-TNF therapy induces mucosal healing by increasing IL-22 availability and implicates new therapeutic opportunities for IBD.

The Host Peritoneal Cavity Harbors Prominent Memory Th2 and Early Recall Responses to an Intestinal Nematode.

Intestinal parasitic nematodes affect a quarter of the world's population, typically eliciting prominent effector Th2-driven host immune responses. As not all infected hosts develop protection against reinfection, our current understanding of nematode-induced memory Th2 responses remains limited. Here, we investigated the activation of memory Th2 cells and the mechanisms driving early recall responses to the enteric nematode Heligmosomoides polygyrus in mice. We show that nematode-cured mice harbor memory Th2 cells in lymphoid and non-lymphoid organs with distinct transcriptional profiles, expressing recirculation markers like CCR7 and CD62-L in the mesenteric lymph nodes (mLN), and costimulatory markers like Ox40, as well as tissue homing and activation markers like CCR2, CD69 and CD40L in the gut and peritoneal cavity (PEC). While memory Th2 cells persist systemically in both lymphoid and non-lymphoid tissues following cure of infection, peritoneal memory Th2 cells in particular displayed an initial prominent expansion and strong parasite-specific Th2 responses during early recall responses to a challenge nematode infection. This effect was paralleled by a significant influx of dendritic cells (DC) and eosinophils, both also appearing exclusively in the peritoneal cavity of reinfected mice. In addition, we show that within the peritoneal membrane lined by peritoneal mesothelial cells (PeM), the gene expression levels of cell adhesion markers VCAM-1 and ICAM-1 decrease significantly in response to a secondary infection. Overall, our findings indicate that the host peritoneal cavity in particular harbors prominent memory Th2 cells and appears to respond directly to H. polygyrus by an early recall response via differential regulation of cell adhesion markers, marking the peritoneal cavity an important site for host immune responses to an enteric pathogen.