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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

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.

Tumor relapse after chemotherapy is a major hurdle for successful cancer therapy. Chemotherapeutic drugs select for resistant tumor cells and reshape tumor microenvironment, including the blood supply system. Using animal models, we observed on macrophages in tumor tissue a close correlation between upregulated Tie2 expression and tumor relapse upon chemotherapy. Conditional deletion of Tie2 expression in macrophages significantly prohibited blood supply and regrowth of tumors. Tie2+ macrophages were derived from tumor-infiltrating Tie2−CD11b+ cells and hypoxia-induced Tie2 expression on these cells. Mechanistically, expression of Tie2 prevented macrophages from apoptosis in stress conditions via the AKT-dependent signaling pathway. Together, these results demonstrate that Tie2 expression by macrophages is necessary and sufficient to promote the reconstruction of blood vessels after chemotherapy, shedding new light on developing novel strategies to inhibit tumor relapse. Cancer Res; 76(23); 6828–38. ©2016 AACR.

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, decreased calcium influx as well as increased apoptosis 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.SignificanceAlthough HDAC7 was identified as an important regulator of thymocyte development and survival, its role in the homeostasis and the functions of adult CD8+ T cells is not fully understood. Here, we identify HDAC7 as a critical regulator of peripheral CD8+ T cells since its deletion impairs anti-tumor and anti-viral immune responses in mouse models of LCMV infection and transfer models of lymphoma. We attribute this phenotype to impaired survival, calcium homeostasis as well as deterred memory function and increased exhaustion of HDAC7-deficient CD8+ T cells. Our findings are of clinical relevance regarding potential immune suppressive side effects of HDAC inhibitors that are currently under clinical trials for the treatment of autoimmune diseases and cancers.

Simple Summary The review article discusses metabolic changes in the tumor microenvironment (TME), which in turn influences the immune cell compartment modulating the phenotype and functionality of immune cells. The main focus is to discuss the influence of increased fatty acid content in the TME, storage of fatty acids in lipid droplet (LDs) organelles in myeloid-derived suppressor cells (MDSCs), macrophages, especially tumor-associated macrophages (TAMs) and resulting functional changes towards an immunosuppressive phenotype. Thus, defining the importance of understanding the role of LD organelles in identifying new therapeutic targets for targeting immunosuppression in cancer. Abstract The tumor microenvironment (TME) comprises various cell types, soluble factors, viz, metabolites or cytokines, which together play in promoting tumor metastasis. Tumor infiltrating immune cells play an important role against cancer, and metabolic switching in immune cells has been shown to affect activation, differentiation, and polarization from tumor suppressive into immune suppressive phenotypes. Macrophages represent one of the major immune infiltrates into TME. Blood monocyte-derived macrophages and myeloid derived suppressor cells (MDSCs) infiltrating into the TME potentiate hostile tumor progression by polarizing into immunosuppressive tumor-associated macrophages (TAMs). Recent studies in the field of immunometabolism focus on metabolic reprogramming at the TME in polarizing tumor-associated macrophages (TAMs). Lipid droplets (LD), detected in almost every eukaryotic cell type, represent the major source for intra-cellular fatty acids. Previously, LDs were mainly described as storage sites for fatty acids. However, LDs are now recognized to play an integral role in cellular signaling and consequently in inflammation and metabolism-mediated phenotypical changes in immune cells. In recent years, the role of LD dependent metabolism in macrophage functionality and phenotype has been being investigated. In this review article, we discuss fatty acids stored in LDs, their role in modulating metabolism of tumor-infiltrating immune cells and, therefore, in shaping the cancer progression.

Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory bowel disease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.

The impact of food antigens on intestinal homeostasis and immune function is poorly understood. Here, we explored the impact of dietary antigens on the phenotype and fate of intestinal T cells. Physiological uptake of dietary proteins generated a highly activated CD44(+)Helios(+)CD4(+) T cell population predominantly in Peyer patches. These cells are distinct from regulatory T cells and develop independently of the microbiota. Alimentation with a protein-free, elemental diet led to an atrophic small intestine with low numbers of activated T cells, including Tfh cells and decreased amounts of intestinal IgA and IL-10. Food-activated CD44(+)Helios(+)CD4(+) T cells in the Peyer patches are controlled by the immune checkpoint molecule PD-1. Blocking the PD-1 pathway rescued these T cells from apoptosis and triggered proinflammatory cytokine production, which in IL-10–deficient mice was associated with intestinal inflammation. In support of these findings, our study of patients with Crohn’s disease revealed significantly reduced frequencies of apoptotic CD4(+) T cells in Peyer patches as compared with healthy controls. These results suggest that apoptosis of diet-activated T cells is a hallmark of the healthy intestine.

Macrophages represent not only the first line of defense against pathogens and are the main drivers of inflammation but are also involved in the initiation, immune evasion as well as metastasis of tumors. Therefore, it has been suggested that diminishing the immune regulatory function of macrophages would support the natural immune surveillance or antitumor therapies, respectively. However, the plasticity of macrophages represents an obstacle in understanding and manipulating the role of macrophages in tumor tissue or the tumor microenvironment. Here, we describe a protocol to differentiate macrophages, based on changing their metabolic environment, from bone marrow precursors to tumor-associated macrophage-like cells of an immune suppressive phenotype. Based on these protocols, the inhibitory functional phenotype of macrophages can be manipulated and therefore further analyzed as described, by interrupting metabolic pathways.

Viral infections with SARS-CoV-2 can cause a multi-facetted disease, which is not only characterized by pneumonia and overwhelming systemic inflammatory immune responses, but which can also directly affect the digestive system and infect intestinal epithelial cells. Here, we review the current understanding of intestinal tropism of SARS-CoV-2 infection, its impact on mucosal function and immunology and summarize the effect of immune-suppression in patients with inflammatory bowel disease (IBD) on disease outcome of COVID-19 and discuss IBD-relevant implications for the clinical management of SARS-CoV-2 infected individuals.

Macrophages represent not only the first line of defense against pathogens and are the main drivers of inflammation but are also involved in the initiation, immune evasion as well as metastasis of tumors. Therefore, it has been suggested that diminishing the immune regulatory function of macrophages would support the natural immune surveillance or antitumor therapies, respectively. However, the plasticity of macrophages represents an obstacle in understanding and manipulating the role of macrophages in tumor tissue or the tumor microenvironment. Here, we describe a protocol to differentiate macrophages, based on changing their metabolic environment, from bone marrow precursors to tumor-associated macrophage-like cells of an immune suppressive phenotype. Based on these protocols, the inhibitory functional phenotype of macrophages can be manipulated and therefore further analyzed as described, by interrupting metabolic pathways.

Background The precise causes and drivers of inflammatory bowel disease (IBD) are still incompletely understood. In recent years, intensive research has shown that not only chronic activation of the immune compartment, but also the integrity of the epithelial barrier plays a central role in disease progression. Inhibitors of histone deacetylases (HDACs) are known for their anti-inflammatory effects and increasing evidence suggests them as promising therapeutics for chronic inflammatory conditions such as IBD. However, the roles of single HDACs in the intestinal epithelium, especially class II HDACs, are still scarcely investigated. In genome wide association studies, HDAC7 has been identified as a risk locus for the development of IBD. Here, we investigated the role of HDAC7 in the intestinal epithelium to evaluate its potential as a therapeutic target. Methods Intestinal epithelial organoids were derived from the ileum and colon of tamoxifen-inducible HDAC7 knockout mice. Hdac7 deletion of organoids was achieved in vitro by tamoxifen treatment over five consecutive days of culture. Morphological changes were monitored via microscopy and gene expression was analysed using qPCR. To examine barrier function, colon organoids were seeded as monolayers into filter inserts and transepithelial electrical resistance (TEER) was measured. Monolayer structure was verified using immunofluorescence. Results Hdac7 silencing in organoids of the small intestine caused a significant morphological shift to a spheroidal shape typical for an undifferentiated phenotype. Gene expression analysis revealed a dramatic downregulation of differentiation markers for all epithelial subtypes and an upregulation of stem cell markers. In colon organoids, undifferentiated HDAC7 knockout spheroids showed a significantly increased expression of goblet cell markers but displayed reduced enterocyte marker expression when subjected to differentiation medium. TEER build-up was consistently slower in colon organoid-derived monolayers after Hdac7 deletion but was higher after differentiation compared to wild type monolayers. Conclusion In the ileal epithelium, HDAC7 seems to play an important role in the differentiation process of all specialized epithelial subtypes while it appears to suppress mainly secretory lineages and promote differentiation into absorptive cells in the colon epithelium. These results suggest HDAC7 as an important regulator of intestinal differentiation and regeneration making it an interesting therapeutic target to treat inflammation-induced injuries in the gut.

Background: The present study aims to detect, quantify and analyze circulating nutritional antigen-specific T-cells in patients with celiac disease (CeD) as well as inflammatory bowel disease (IBD), thus comparing the specific T-cell response following barrier disruption and antigen translocation. Methods: The antigen-reactive T-cell enrichment (ARTE) technique was applied allowing for a phenotypical and functional flow cytometric analysis of rare nutritional antigen-specific T-cells, including the CeD-causing gliadin (gluten), in the peripheral blood. Results: Our study indicates that by applying the ARTE technique, differences of gluten-specific T-cells as well as the differential cytokine expression between the patient groups can be detected, even without the burdening gluten re-exposure of the patients. CeD patients, independent from the presence or absence of gluten exposure in their current diet, featured an increase of the frequency of gliadin-specific T-cells, which were characterized by a pro-inflammatory phenotype. However, only for active CeD and a consecutive small intestinal barrier breach, an increase of distinct nutritional T-cells could be detected. Accordingly, frequency as well as pro-inflammatory phenotype of nutritional antigen-specific T cells were highest in Crohn’s disease patients with small intestinal inflammation whereas no significant increase was observed in ulcerative colitis. Conclusion: In summary, the ARTE method allows not only for detection but also for functional analysis of these rare cells even in healthy subjects. Applying this method, we were able to demonstrate that for non-CeD-related nutritional antigens, small intestinal barrier breach is mandatory for a peripheral antigen-specific T-cell.

IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80(+) macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, β-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, μ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80(+) macrophages from IL-4–treated donors. Together, IL-4–induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.

Leptin has been shown to modulate intestinal inflammation in mice. However, clinical evidence regarding its immune-stimulatory potential in human Crohn’s disease remains sparse. We here describe a patient with the unique combination of acquired generalized lipodystrophy and Crohn’s disease (AGLCD) featuring a lack of adipose tissue, leptin deficiency and intestinal inflammation. Using mass and flow cytometry, immunohistochemistry and functional metabolic analyses, the AGLCD patient was compared to healthy individuals and Crohn’s disease patients regarding immune cell composition, function and metabolism and the effects of recombinant N-methionylleptin (rLeptin) were evaluated. We provide evidence that rLeptin exerts diverse pro-inflammatory effects on immune cell differentiation and function, including the metabolic reprogramming of immune cells and the induction of TNFα, ultimately aggravating Crohn’s disease in the AGLCD patient, which can be reversed by anti-TNFα therapy. Our results indicate that leptin is required for human immune homeostasis and contributes to autoimmunity in a TNFα-dependent manner., The adipokine leptin modulates intestinal inflammation in mice. Here the authors describe a patient with inflammatory bowel disease and lipodystrophy, providing evidence that leptin aggravates intestinal inflammation with proinflammatory effects on leukocytes that are reversible by TNFα blockade.

Background & Aims A substantial proportion patients with inflammatory bowel disease (IBD) have a primary non-response to infliximab; markers are needed to identify patients most likely to respond to treatment. We investigated whether production of tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMCs) can be used as a marker to predict response. Methods We performed a prospective study of 41 adults with IBD (mean age, 38 years; 21 male; 21 with Crohn’s disease and 20 with ulcerative colitis) not treated with a biologic agent within the past 6 months; patients were given their first infusion of infliximab at a hospital or clinic in Berlin, Germany. We collected data on clinical scores, levels of C-reactive protein, and ultrasound results (Limberg scores) at baseline (before the first infusion) and after 6 weeks (3rd infliximab infusion). PMBCs were obtained from patients at baseline and 10 healthy individuals (controls) and incubated with lipopolysaccharide. We measured production of cytokines (TNF, interleukin 1 [IL1], IL6, IL8, IL10, IL12p70, and IL22) by ELISA and performed cytometric bead array and flow cytometry analyses. The primary endpoint was clinical response (decrease in Harvey Bradshaw Index scores of 2 or more or decrease in partial Mayo scores of 3 or more at week 6) in patients with PBMCs that produced high vs low levels of TNF. Results Responders had a shorter median disease duration (P = .018) and higher median Limberg score (P = .021), than nonresponders. Baseline PBMCs from responders produced significantly more TNF (P = .049) and IL6 (P = .028) than from nonresponders; a level of 500 pg/ml TNF identified responders with 82% sensitivity and 78% specificity. In patients with Crohn’s disease, this cutoff value (500 pg/ml TNF) identified responders with 100% sensitivity and 82% specificity; TNF levels above this level were independently associated with response to infliximab in multivariate analysis (odds ratio, 16.2; 95% CI, 1.8–148.7; P = .014). The percentage of TNF-positive cells was higher among CD14+ monocytes than lymphocytes after stimulation. Conclusions Production of a high level of TNF by PBMCs (specifically CD14+ cells) from patients with IBD can identify those most likely to have a clinical response to infliximab therapy. In patients with Crohn’s disease, a cutoff value of 500 pg/ml TNF identified responders with 100% sensitivity and 82% specificity.

Summary Inflammaging is associated with an increased risk of chronic disease. Monocytes are the principal immune cells for the production of inflammatory cytokines and contribute to inflammaging in the elderly. However, the underlying mechanisms remain largely unknown. Here, we found that monocytes from aged individuals contained high levels of lipid droplets (LDs), and this increase was correlated with impaired fatty acid oxidation. Downregulated peroxisome proliferator-activated receptor (PPAR)-α may be responsible for the pro-inflammatory phenotype of monocytes in aged individuals, as it was positively correlated with LD accumulation and increasing TNF-α concentration. Interestingly, interventions that result in PPAR-α upregulation, such as fenofibrate treatment, TNF-α neutralization, or calorie restriction, reversed the effect of aging on monocytes. Thus the downregulation of PPAR-α and LD levels in monocytes represents a novel biomarker for inflammaging. Furthermore, PPAR-α activation in the elderly may also alleviate long-term inflammaging, preventing the development of life-limiting chronic diseases., Graphical abstract, Highlights • Monocytes from aged individuals contained high levels of lipid droplets (LDs) • Downregulated PPAR-α is responsible for the aged monocytes profiles • TNF-α might accelerate monocyte aging by downregulating PPAR-α expression • PPAR-α activation in the elderly may also alleviate long-term inflammaging, Human metabolism; Immunology; Cell biology

Background Inflammatory bowel disease (IBD) represents a dysregulation of the mucosal immune system. The pathogenesis of Crohn’s disease (CD) and ulcerative colitis (UC) is linked to the loss of intestinal tolerance and barrier function. The healthy mucosal immune system has previously been shown to be inert against food antigens. Since the small intestine is the main contact surface for antigens and therefore the immunological response, the present study served to analyse food-antigen-specific T cells in the peripheral blood of IBD patients. Methods Peripheral blood mononuclear cells of CD, with an affected small intestine, and UC (colitis) patients, either active or in remission, were stimulated with the following food antigens: gluten, soybean, peanut and ovalbumin. Healthy controls and celiac disease patients were included as controls. Antigen-activated CD4+ T cells in the peripheral blood were analysed by a magnetic enrichment of CD154+ effector T cells and a cytometric antigen-reactive T-cell analysis (‘ARTE’ technology) followed by characterisation of the effector response. Results The effector T-cell response of antigen-specific T cells were compared between CD with small intestinal inflammation and UC where inflammation was restricted to the colon. Among all tested food antigens, the highest frequency of antigen-specific T cells (CD4+CD154+) was found for gluten. Celiac disease patients were included as control, since gluten has been identified as the disease-causing antigen. The highest frequency of gluten antigen-specific T cells was revealed in active CD when compared with UC, celiac disease on a gluten-free diet (GFD) and healthy controls. Ovalbumin-specific T cells were almost undetectable, whereas the reaction to soybean and peanut was slightly higher. But again, the strongest reaction was observed in CD with small intestinal involvement compared with UC. Remarkably, in celiac disease on a GFD only antigen-specific cells for gluten were detected. These gluten-specific T cells were characterised by up-regulation of the pro-inflammatory cytokines IFN-γ, IL-17A and TNF-α. IFN-g was exclusively elevated in CD patients with active disease. Gluten-specific T-cells expressing IL-17A were increased in all IBD patients. Furthermore, T cells of CD patients, independent of disease activity, revealed a high expression of the pro-inflammatory cytokine TNF-α. Conclusion The ‘ARTE’-technique allows to analyse and quantify food antigen specific T cells in the peripheral blood of IBD patients indicating a potential therapeutic insight. These data provide evidence that small intestinal inflammation in CD is key for the development of a systemic pro-inflammatory effector T-cell response driven by food antigens.

Background and Aims Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. Methods Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. Results Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. Conclusions This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.

The synthetic compound dendritic polyglycerol sulfate (dPGS) is a pleiotropic acting molecule but shows a high binding affinity to immunological active molecules as L-/P-selectin or complement proteins leading to well described anti-inflammatory properties in various mouse models. In order to make a comprehensive evaluation of the direct effect on the innate immune system, macrophage polarization is analyzed in the presence of dPGS on a phenotypic but also metabolic level. dPGS administered macrophages show a significant increase of MCP1 production paralleled by a reduction of IL-10 secretion. Metabolic analysis reveals that dPGS could potently enhance the glycolysis and mitochondrial respiration in M0 macrophages as well as decrease the mitochondrial respiration of M2 macrophages. In summary the data indicate that dPGS polarizes macrophages into a pro-inflammatory phenotype in a metabolic pathway-dependent manner.

A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific-pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of "wild immunology" imprintings in detail and comparing it with those of "clean" lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for "wild mice". For this purpose, we developed a 31-antibody panel for murine leukocyte subsets identification and a 35-antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non-SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen-experienced B- and T-cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing. © 2016 International Society for Advancement of Cytometry.

Using high-resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single-cell level.

Background and aims Creeping fat [CF] is a hyperplasia of adipose tissue adjacent to inflamed intestine in Crohn's disease [CD]. Data from genome-wide association studies [GWAS] distinguished Crohn's colitis from ileal CD and ulcerative colitis [UC]. This study analysed the T-cell compartments of ileal and colonic mesenteric fat and corresponding mucosa to provide cellular proof for the suggested GWAS classification. Methods Samples were obtained from 34 CD or UC patients. Cells were analysed by immunohistochemistry and flow cytometry, and tissue cytokine release was assessed by cytometric bead array. Results Only ileal CF revealed the distinct adipocyte hyperplasia combined with dense T-cell infiltration and fibrosis; colonic fat from CD and UC patients lacked these findings. T-cell subpopulations differed between mesenteric fat in ileal CD, colonic CD and UC: ileal CF had nearly 10 times more T-cells than colonic fat. The proportions of regulatory and central memory T-cells were significantly higher in ileal CF compared with colonic fat in CD and UC. In all groups, the mucosal T-cell compartment was distinct from the mesenteric fat. Remarkably, correlation between disease activity and proportion of pro- and anti-inflammatory T-cell subpopulations was inverse, comparing ileal and colonic fat in CD. Conclusions This first in-depth analysis of the T-cell compartment in ileal and colonic mesenteric adipose tissue in CD and UC identifies a unique T-cell niche in the ileal mesenteric fat tissue in CD. From a clinical point of view, our findings underscore the novel concept of colonic and ileal CD as distinct IBD entities.

Intact epithelial barrier function is pivotal for maintaining intestinal homeostasis. Current therapeutic developments aim at restoring the epithelial barrier in inflammatory bowel disease. Histone deacetylase (HDAC) inhibitors are known to modulate immune responses and to ameliorate experimental colitis. However, their direct impact on epithelial barrier function and intestinal wound healing is unknown. In human and murine colonic epithelial cell lines, the presence of the HDAC inhibitors Givinostat and Vorinostat not only improved transepithelial electrical resistance under inflammatory conditions but also attenuated the passage of macromolecules across the epithelial monolayer. Givinostat treatment mediated an accelerated wound closure in scratch assays. In vivo, Givinostat treatment resulted in improved barrier recovery and epithelial wound healing in dextran sodium sulphate-stressed mice. Mechanistically, these regenerative effects could be linked to an increased secretion of transforming growth factor beta1 and interleukin 8, paralleled by differential expression of the tight junction proteins claudin-1, claudin-2 and occludin. Our data reveal a novel tissue regenerative property of the pan-HDAC inhibitors Givinostat and Vorinostat in intestinal inflammation, which may have beneficial implications by repurposing HDAC inhibitors for therapeutic strategies for inflammatory bowel disease.