Novel transcriptomic panel identifies histologically active eosinophilic oesophagitis.

BACKGROUND AND AIMS: Eosinophilic oesophagitis (EoE) is characterised by symptoms of esophageal dysfunction and oesinophil tissue infiltration. The EoE Diagnostic Panel (EDP) can distinguish between active and non-active EoE using a set of 77 genes. Recently, the existence of distinct EoE variants featuring symptoms similar to EoE, such as oesophageal dysfunction but lacking eosinophil infiltration, had been determined. METHODS: We used oesophageal biopsies from patients with histologically active (n=10) and non-active EoE (n=9) as well as from healthy oesophageal controls (n=5) participating in the Swiss Eosinophilic Esophagitis Cohort Study (SEECS) and analysed the gene expression profile in these biopsies by total RNA-sequencing (RNA-seq). Moreover, we employed the publicly accessible RNA-seq dataset (series GSE148381) as reported by Greuter et al, encompassing a comprehensive genomic profile of patients presenting with EoE variants. RESULTS: A novel, diagnostic gene expression panel that can effectively distinguish patients with histologically active conventional EoE from patients with EoE in histological remission and control individuals, and from three newly discovered EoE variants was identified. Histologically Active EoE Diagnostic Panel (HAEDP) consists of 53 genes that were identified based on differential expression between histologically active EoE, histological remission and controls (p≤0.05). By combining the HAEDP with EDP, we expanded our knowledge about factors that may contribute to the inflammation in EoE and improved our understanding of the underlying mechanisms of the disease. Conversely, we suggested a compact group of genes common to both HAEDP and EDP to create a reliable diagnostic tool that might enhance the accuracy of EoE diagnosis. CONCLUSION: We identified a novel set of 53 dysregulated genes that are closely associated with the histological inflammatory activity of EoE. In combination with EDP, our new panel might be a valuable tool for the accurate diagnosis of patients with EoE as well as for monitoring their disease course.

TiO2 nanoparticles abrogate the protective effect of the Crohn's disease-associated variation within the PTPN22 gene locus.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.

Induction of human regulatory innate lymphoid cells from group 2 innate lymphoid cells by retinoic acid.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play critical roles in induction and exacerbation of allergic airway inflammation. Thus clarification of the mechanisms that underlie regulation of ILC2 activation has received significant attention. Although innate lymphoid cells are divided into 3 major subsets that mirror helper effector T-cell subsets, counterpart subsets of regulatory T cells have not been well characterized. OBJECTIVE: We sought to determine the factors that induce regulatory innate lymphoid cells (ILCregs). METHODS: IL-10+ ILCregs induced from ILC2s by using retinoic acid (RA) were analyzed with RNA-sequencing and flow cytometry. ILCregs were evaluated in human nasal tissue from healthy subjects and patients with chronic rhinosinusitis with nasal polyps and lung tissue from house dust mite- or saline-treated mice. RESULTS: RA induced IL-10 secretion by human ILC2s but not type 2 cytokines. IL-10+ ILCregs, which were converted from ILC2s by means of RA stimulation, expressed a regulatory T cell-like signature with expression of IL-10, cytotoxic T lymphocyte-associated protein 4, and CD25, with downregulated effector type 2-related markers, such as chemoattractant receptor-homologous molecule on TH2 cells and ST2, and suppressed activation of CD4+ T cells and ILC2s. ILCregs were rarely detected in human nasal tissue from healthy subjects or lung tissue from saline-treated mice, but numbers were increased in nasal tissue from patients with chronic rhinosinusitis with nasal polyps and in lung tissue from house dust mite-treated mice. Enzymes for RA synthesis were upregulated in airway epithelial cells during type 2 inflammation in vivo and by IL-13 in vitro. CONCLUSION: We have identified a unique immune regulatory and anti-inflammatory pathway by which RA converts ILC2s to ILCregs. Interactions between airway epithelial cells and ILC2s play an important roles in the generation of ILCregs.

Der p 1-specific regulatory T-cell response during house dust mite allergen immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only available treatment for allergic diseases that can induce specific immune tolerance to allergens. The key mechanisms involved in this process include changes in allergen-specific regulatory T (Treg) cells. METHODS: We studied 25 allergic rhinitis patients undergoing subcutaneous house dust mite-specific immunotherapy. Peripheral blood mononuclear cells were studied before and after 10, 30 weeks, and 3 years of AIT. Der p 1-specific T regulatory cell responses were investigated by characterization of Der p 1-MHC class II tetramer-positive cells and correlated with nasal symptom score. RESULTS: Twelve of 25 AIT patients matched with their MHC class II expression to the Der p 1 peptide-MHC class II tetramers. A significant increase in the numbers of Der p 1-specific FOXP3+ Helios+ CD25+ CD127- Treg cells after 30 weeks was observed, which slightly decreased after 3 years of AIT. In contrast, Der p 1-specific immunoglobulin-like transcript 3 (ILT3)+ CD25+ Treg cells decreased substantially from baseline after 3 years of AIT. ILT3+ Treg cells displayed compromised suppressive function and low FOXP3 expression. In addition, Der p 1-specific IL-10 and IL-22 responses have increased after 30 weeks, but only IL-10+ Der p 1-specific Treg cells remained present at high frequency after 3 years of AIT. Increased number of FOXP3+ Helios+ and IL-10+ and decreased ILT3+ Treg cell responses correlated with improved allergic symptoms. CONCLUSION: The results indicate that AIT involves upregulation of the activated allergen-specific Treg cells and downregulation of dysfunctional allergen-specific Treg cell subset. Correction of dysregulated Treg cells responses during AIT is associated with improved clinical response.

Bacterial secretion of histamine within the gut influences immune responses within the lung.

BACKGROUND: Histamine is an important immunomodulator influencing both the innate and adaptive immune system. Certain host cells express the histidine decarboxylase enzyme (HDC), which is responsible for catalysing the decarboxylation of histidine to histamine. We and others have shown that bacterial strains can also express HDC and secrete histamine; however, the influence of bacterial-derived histamine on the host immune responses distant to the gut is unclear. METHODS: The Escherichia coli BL21 (E coli BL21) strain was genetically modified to express the Morganella morganii (M morganii)-derived HDC gene (E coli BL21_HTW). E coli BL21 and E coli BL21_HTW were gavaged to ovalbumin (OVA) sensitized and challenged mice to investigate the effect of bacterial-derived histamine on lung inflammatory responses. RESULTS: Oral administration of E coli BL21_HTW, which is able to secrete histamine, to wild-type mice reduced lung eosinophilia and suppressed ex vivo OVA-stimulated cytokine secretion from lung cells in the OVA respiratory inflammation mouse model. In histamine receptor 2 (H2R)-deficient mice, administration of histamine-secreting bacteria also reduced inflammatory cell numbers in bronchoalveolar lavage (BAL). However, the suppressive effect of bacterial-derived histamine on BAL inflammation was lost in HDC-deficient mice. This loss of activity was associated with increased expression of histamine degrading enzymes and reduced histamine receptor expression. CONCLUSION: Histamine secretion from bacteria within the gut can have immunological consequences at distant mucosal sites, such as within the lung. These effects are influenced by host histamine receptor expression and the expression of histamine degrading enzymes.

High levels of butyrate and propionate in early life are associated with protection against atopy.

BACKGROUND: Dietary changes are suggested to play a role in the increasing prevalence of allergic diseases and asthma. Short-chain fatty acids (SCFAs) are metabolites present in certain foods and are produced by microbes in the gut following fermentation of fibers. SCFAs have been shown to have anti-inflammatory properties in animal models. Our objective was to investigate the potential role of SCFAs in the prevention of allergy and asthma. METHODS: We analyzed SCFA levels by high-performance liquid chromatography (HPLC) in fecal samples from 301 one-year-old children from a birth cohort and examined their association with early life exposures, especially diet, and allergy and asthma later in life. Data on exposures and allergic diseases were collected by questionnaires. In addition, we treated mice with SCFAs to examine their effect on allergic airway inflammation. RESULTS: Significant associations between the levels of SCFAs and the infant's diet were identified. Children with the highest levels of butyrate and propionate (≥95th percentile) in feces at the age of one year had significantly less atopic sensitization and were less likely to have asthma between 3 and 6 years. Children with the highest levels of butyrate were also less likely to have a reported diagnosis of food allergy or allergic rhinitis. Oral administration of SCFAs to mice significantly reduced the severity of allergic airway inflammation. CONCLUSION: Our results suggest that strategies to increase SCFA levels could be a new dietary preventive option for allergic diseases in children.

Human CD40 ligand-expressing type 3 innate lymphoid cells induce IL-10-producing immature transitional regulatory B cells.

BACKGROUND: Type 3 innate lymphoid cells (ILC3s) are involved in maintenance of mucosal homeostasis; however, their role in immunoregulation has been unknown. Immature transitional regulatory B (itBreg) cells are innate-like B cells with immunosuppressive properties, and the in vivo mechanisms by which they are induced have not been fully clarified. OBJECTIVE: We aimed to investigate the ILC3-B-cell interaction that probably takes place in human tonsils. METHODS: ILC3s were isolated from peripheral blood and palatine tonsils, expanded, and cocultured with naive B cells. Tonsillar ILC3s and regulatory B cells were visualized with immunofluorescence histology. ILC3 frequencies were measured in tonsil tissue of allergic and nonallergic patients and in peripheral blood of allergic asthmatic patients and healthy control subjects. RESULTS: A mutually beneficial relationship was revealed between ILC3s and B cells: ILC3s induced IL-15 production in B cells through B cell-activating factor receptor, whereas IL-15, a potent growth factor for ILC3s, induced CD40 ligand (CD40L) expression on circulating and tonsillar ILC3s. IL-15-activated CD40L+ ILC3s helped B-cell survival, proliferation, and differentiation of IL-10-secreting, PD-L1-expressing functional itBreg cells in a CD40L- and B cell-activating factor receptor-dependent manner. ILC3s and regulatory B cells were in close connection with each other in palatine tonsils. ILC3 frequency was reduced in tonsil tissue of allergic patients and in peripheral blood of allergic asthmatic patients. CONCLUSION: Human CD40L+ ILC3s provide innate B-cell help and are involved in an innate immunoregulatory mechanism through induction of itBreg cell differentiation, which takes place in palatine tonsils in vivo. This mechanism, which can contribute to maintenance of immune tolerance, becomes insufficient in allergic diseases.

Exposure to nonmicrobial N-glycolylneuraminic acid protects farmers' children against airway inflammation and colitis.

BACKGROUND: Childhood exposure to a farm environment has been shown to protect against the development of inflammatory diseases, such as allergy, asthma, and inflammatory bowel disease. OBJECTIVE: We sought to investigate whether both exposure to microbes and exposure to structures of nonmicrobial origin, such as the sialic acid N-glycolylneuraminic acid (Neu5Gc), might play a significant role. METHODS: Exposure to Neu5Gc was evaluated by quantifying anti-Neu5Gc antibody levels in sera of children enrolled in 2 farm studies: the Prevention of Allergy Risk factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n = 299) and the Protection Against Allergy Study in Rural Environments (PASTURE) birth cohort (cord blood [n = 836], 1 year [n = 734], 4.5 years [n = 700], and 6 years [n = 728]), and we associated them with asthma and wheeze. The effect of Neu5Gc was examined in murine airway inflammation and colitis models, and the role of Neu5Gc in regulating immune activation was assessed based on helper T-cell and regulatory T-cell activation in mice. RESULTS: In children anti-Neu5Gc IgG levels correlated positively with living on a farm and increased peripheral blood forkhead box protein 3 expression and correlated inversely with wheezing and asthma in nonatopic subjects. Exposure to Neu5Gc in mice resulted in reduced airway hyperresponsiveness and inflammatory cell recruitment to the lung. Furthermore, Neu5Gc administration to mice reduced the severity of a colitis model. Mechanistically, we found that Neu5Gc exposure reduced IL-17+ T-cell numbers and supported differentiation of regulatory T cells. CONCLUSIONS: In addition to microbial exposure, increased exposure to non-microbial-derived Neu5Gc might contribute to the protective effects associated with the farm environment.

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients.

BACKGROUND: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. OBJECTIVE: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. METHODS: The expression, regulation, and function of TJs were determined in air-liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. RESULTS: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL-4 and IL-13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens-1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL-4 and IL-13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. CONCLUSION: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL-4, and IL-13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.

Interleukins (from IL-1 to IL-38), interferons, transforming growth factor ß, and TNF-α: Receptors, functions, and roles in diseases.

There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-α, and TGF-ß offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene-deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-α, TGF-ß, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.

The effects of cryopreservation on the expression of canine regulatory T-cell markers.

BACKGROUND: Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. HYPOTHESIS/OBJECTIVES: The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. ANIMALS: Nine privately owned dogs. METHODS: Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations were calculated. RESULTS: A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations, respectively, was observed. CONCLUSIONS: This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.

A wide diversity of bacteria from the human gut produces and degrades biogenic amines.

Background: Biogenic amines (BAs) are metabolites produced by the decarboxylation of amino acids with significant physiological functions in eukaryotic and prokaryotic cells. BAs can be produced by bacteria in fermented foods, but little is known concerning the potential for microbes within the human gut microbiota to produce or degrade BAs. Objective: To isolate and identify BA-producing and BA-degrading microbes from the human gastrointestinal tract. Design: Fecal samples from human volunteers were screened on multiple growth media, under multiple growth conditions. Bacterial species were identified using 16S rRNA sequencing and BA production or degradation was assessed using ultra-performance liquid chromatography. Results: In total, 74 BA-producing or BA-degrading strains were isolated from the human gut. These isolates belong to the genera Bifidobacterium, Clostridium, Enterococcus, Lactobacillus, Pediococcus, Streptococcus, Enterobacter, Escherichia, Klebsiella, Morganella and Proteus. While differences in production or degradation of specific BAs were observed at the strain level, our results suggest that these metabolic activities are widely spread across different taxa present within the human gut microbiota. Conclusions: The isolation and identification of microbes from the human gut with BA-producing and BA-degrading metabolic activity is an important first step in developing a better understanding of how these metabolites influence health and disease.

Functional anatomy of neural circuits regulating fear and extinction.

The memory of fear extinction is context dependent: fear that is suppressed in one context readily renews in another. Understanding of the underlying neuronal circuits is, therefore, of considerable clinical relevance for anxiety disorders. Prefrontal cortical and hippocampal inputs to the amygdala have recently been shown to regulate the retrieval of fear memories, but the cellular organization of these projections remains unclear. By using anterograde tracing in a transgenic rat in which neurons express a dendritically-targeted PSD-95:Venus fusion protein under the control of a c-fos promoter, we found that, during the retrieval of extinction memory, the dominant input to active neurons in the lateral amygdala was from the infralimbic cortex, whereas the retrieval of fear memory was associated with greater hippocampal and prelimbic inputs. This pattern of retrieval-related afferent input was absent in the central nucleus of the amygdala. Our data show functional anatomy of neural circuits regulating fear and extinction, providing a framework for therapeutic manipulations of these circuits.

IgG4 production is confined to human IL-10-producing regulatory B cells that suppress antigen-specific immune responses.

BACKGROUND: IL-10-producing regulatory B cells suppress immune responses, and lack of these cells leads to exacerbated symptoms in mouse models of chronic inflammation, transplantation, and chronic infection. IgG4 is a blocking antibody isotype with anti-inflammatory potential that is induced in human high-dose antigen tolerance models. OBJECTIVE: We sought to characterize human inducible IL-10-secreting B regulatory 1 (BR1) cells and to investigate their immunoregulatory capacity through suppression of cellular immune responses and production of anti-inflammatory immunoglobulins. METHODS: Highly purified IL-10-secreting B cells were phenotypically and functionally characterized by means of whole-genome expression analysis, flow cytometry, suppression assay, and antibody production. B cells specific for the major bee venom allergen phospholipase A2 (PLA) were isolated from beekeepers who displayed tolerance to bee venom antigens and allergic patients before and after specific immunotherapy. RESULTS: Human IL-10+ BR1 cells expressed high surface CD25 and CD71 and low CD73 levels. Sorting of CD73-CD25+CD71+ B cells allowed enrichment of human BR1 cells, which produced high levels of IL-10 and potently suppressed antigen-specific CD4+ T-cell proliferation. IgG4 was selectively confined to human BR1 cells. B cells specific for the major bee venom allergen PLA isolated from nonallergic beekeepers show increased expression of IL-10 and IgG4. Furthermore, the frequency of IL-10+ PLA-specific B cells increased in allergic patients receiving allergen-specific immunotherapy. CONCLUSION: Our data show the characterization of IL-10+ BR1 cells and in vivo evidence for 2 essential features of allergen tolerance: the suppressive B cells and IgG4-expressing B cells that are confined to IL-10+ BR1 cells in human subjects.

Influence of matrix metalloproteinase MMP-9 on dendritic spine morphology.

An increasing body of data has shown that matrix metalloproteinase-9 (MMP-9), an extracellularly acting, Zn(2+)-dependent endopeptidase, is important not only for pathologies of the central nervous system but also for neuronal plasticity. Here, we use three independent experimental models to show that enzymatic activity of MMP-9 causes elongation and thinning of dendritic spines in the hippocampal neurons. These models are: a recently developed transgenic rat overexpressing autoactivating MMP-9, dissociated neuronal cultures, and organotypic neuronal cultures treated with recombinant autoactivating MMP-9. This dendritic effect is mediated by integrin ß1 signalling. MMP-9 treatment also produces a change in the decay time of miniature synaptic currents; however, it does not change the abundance and localization of synaptic markers in dendritic protrusions. Our results, considered together with several recent studies, strongly imply that MMP-9 is functionally involved in synaptic remodelling.

Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity.

BACKGROUND & AIMS: Glycoprotein (GP)96 is an endoplasmic reticulum-resident master chaperone for cell surface receptors including the Wnt co-receptors low-density lipoprotein-receptor-related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. METHODS: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS: Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.

Spermidine Ameliorates Colitis via Induction of Anti-Inflammatory Macrophages and Prevention of Intestinal Dysbiosis.

BACKGROUND AND AIMS: Exacerbated immune activation, intestinal dysbiosis and a disrupted intestinal barrier are common features among inflammatory bowel disease [IBD] patients. The polyamine spermidine, which is naturally present in all living organisms, is an integral component of the human diet, and exerts beneficial effects in human diseases. Here, we investigated whether spermidine treatment ameliorates intestinal inflammation and offers therapeutic potential for IBD treatment. METHODS: We assessed the effect of oral spermidine administration on colitis severity in the T cell transfer colitis model in Rag2-/- mice by endoscopy, histology and analysis of markers of molecular inflammation. The effects on the intestinal microbiome were determined by 16S rDNA sequencing of mouse faeces. The impact on intestinal barrier integrity was evaluated in co-cultures of patient-derived macrophages with intestinal epithelial cells. RESULTS: Spermidine administration protected mice from intestinal inflammation in a dose-dependent manner. While T helper cell subsets remained unaffected, spermidine promoted anti-inflammatory macrophages and prevented the microbiome shift from Firmicutes and Bacteroides to Proteobacteria, maintaining a healthy gut microbiome. Consistent with spermidine as a potent activator of the anti-inflammatory molecule protein tyrosine phosphatase non-receptor type 2 [PTPN2], its colitis-protective effect was dependent on PTPN2 in intestinal epithelial cells and in myeloid cells. The loss of PTPN2 in epithelial and myeloid cells, but not in T cells, abrogated the barrier-protective, anti-inflammatory effect of spermidine and prevented the anti-inflammatory polarization of macrophages. CONCLUSION: Spermidine reduces intestinal inflammation by promoting anti-inflammatory macrophages, maintaining a healthy microbiome and preserving epithelial barrier integrity in a PTPN2-dependent manner.

Crosstalk within peripheral blood mononuclear cells mediates anti-inflammatory effects of n-3 PUFA-rich lipid emulsions in parenteral nutrition.

BACKGROUND AND AIMS: Parenteral nutrition (PN) rich in n-6 and n-3 long-chain fatty acids is used in clinical practice for nourishing patients who are unable to receive adequate nutrition through their digestive systems. In this study, we compare the effect on inflammation of the commonly used lipid emulsions Omegaven (n-3-rich) and Intralipid (n-6-rich) in human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were treated with different doses of n-3-rich Omegaven and n-6-rich Intralipid and the immune cells were characterized by flow cytometry. RESULTS: We show that incubation of PBMCs with n-3-rich Omegaven leads to an increase in expression of CD1d and CD86 in CD14+monocytes. At the same time, an increased number of NKT cells expressing cytotoxic T cell antigen 4 is observed, suggesting immunological synapse formation. Both CD14+monocytes and NKT cells showed an increase in IL-10 production and a reduction in the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-4, which led to an increase in the number of FOXP3+T regulatory cells. In addition, we show that n-3-rich Omegaven reduces the expression of TNFα, IFNγ and IL-4 in CD4+T and CD8+T cells independent of the presented interaction between CD14+monocytes and NKT cells. The described mechanism of n-3 rich lipid emulsions was confirmed in PBMCs from patients with inflammatory bowel disease but not in colorectal cancer patients which seem to lack the interaction between CD14+monocytes and NKT cells. CONCLUSIONS: These results show a mechanism for the beneficial effect of the n-3-rich Omegaven in patients with inflammatory conditions but questions its use in patients with cancer. Hence, our results may assist in choosing the best lipid emulsion for patients who require PN.