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Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet.

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1,2,3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated ‘sensing’ of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4

An important hallmark for the characterisation of Th cells is their capacity for cytokine expression. In this chapter, we describe how Th cells can be restimulated polyclonally to reveal their cytokine-producing potential that can then be analysed by intracellular staining and flow cytometry.

Isobiotic mice, with an identical stable microbiota composition, potentially allow models of host-microbial mutualism to be studied over time and between different laboratories. To understand microbiota evolution in these models, we carried out a 6-year experiment in mice colonized with 12 representative taxa. Increased non-synonymous to synonymous mutation rates indicate positive selection in multiple taxa, particularly for genes annotated for nutrient acquisition or replication. Microbial sub-strains that evolved within a single taxon can stably coexist, consistent with niche partitioning of ecotypes in the complex intestinal environment. Dietary shifts trigger rapid transcriptional adaptation to macronutrient and micronutrient changes in individual taxa and alterations in taxa biomass. The proportions of different sub-strains are also rapidly altered after dietary shift. This indicates that microbial taxa within a mouse colony adapt to changes in the intestinal environment by long-term genomic positive selection and short-term effects of transcriptional reprogramming and adjustments in sub-strain proportions. ISSN:1931-3128 ISSN:1934-6069

An important hallmark for the characterisation of Th cells is their capacity for cytokine expression. In this chapter, we describe how Th cells can be restimulated polyclonally to reveal their cytokine-producing potential that can then be analysed by intracellular staining and flow cytometry.

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4+ T helper cells have been shown to strongly contribute to the disease pathogenesis of IBD. Here, we present a detailed protocol of the preclinical model of T-cell transfer colitis, which can easily be utilized in the laboratory to study T helper cell functions in intestinal inflammation.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZF

© 2020 International Society for Advancement of Cytometry.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the genera- tion and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer’s patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation. © 2017 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.

International audience; Introduction: The microbiota-gut brain (MGB) axis is the bidirectional communication between the intestinal microbiota and the brain. An increasing body of preclinical and clinical evidence has revealed that the gut microbial ecosystem can affect neuropsychiatric health. However, there is still a need of further studies to elucidate the complex gene-environment interactions and the role of the MGB axis in neuropsychiatric diseases, with the aim of identifying biomarkers and new therapeutic targets, to allow early diagnosis and improving treatments. Areas covered: To review the role of MGB axis in neuropsychiatric disorders, prediction and prevention of disease through exploitation, integration, and combination of data from existing gut microbiome/microbiota projects and appropriate other International '-Omics' studies. The authors also evaluated the new technological advances to investigate and modulate, through nutritional and other interventions, the gut microbiota. Expert opinion: The clinical studies have documented an association between alterations in gut microbiota composition and/or function, whereas the preclinical studies support a role for the gut microbiota in impacting behaviors which are of relevance to psychiatry and other central nervous system (CNS) disorders. Targeting MGB axis could be an additional approach for treating CNS disorders and all conditions in which alterations of the gut microbiota are involved.

Miniaturization and modularization are fast growing fields in chemical engineering in recent years. Fast and flexible production processes for microstructured devices are desirable to meet the requirements of rapid prototyping and flexible chip manufacturing. Reactive ion etching provides a structuring process which leads to a highly precise and anisotropic etching behavior. A new manufacturing process for polyimide-based microstructured devices with low surface roughness was developed and applied on reactor geometry for liquid-liquid two-phase-flow. The fabricated chip geometry is designed for creating droplets via flow focusing as the dispersed phase is incised by two continuous phase inlet streams. The droplets are created in the widening channel. In order to keep the pressure loss for the developed reactor geometry and the production time as small as possible, the manufacturing process was optimized with a view to minimize surface roughness and maximizing the etching rate by using Design of Experiments. The corresponding pressure drop was measured for flow rates from 0.05 ml min−1 to 0.5 ml min−1.

Besides the common benefits of micro-structured reactors such as enhanced mass and heat transfer caused by a high specific surface and enhanced mixing, they have also drawbacks due to their tiny dimensions. Non-invasive temperature measurement is an important issue for micro process engineering regarding to process control and safety issues. High resolution and robustness are requirements which should be met by the temperature measuring method. Thin film technology combines the possibility to manufacture micro scaled structures with great flexibility in choosing material and geometry of the structures. Layers of aluminum with a thickness in nanometer range are deposited on flexible polyimide foil and structured lithographically to obtain electrical conductor tracks which are used as temperature sensors based on their electrical resistance. The produced temperature sensors were calibrated in the range from 20 to 70 °C and the accuracy of the sensors was checked.

Background The transcription factor ‘T-box expressed in T cells’ (T-bet) is highly expressed by Th cells isolated from the inflamed tissue in many chronic inflammatory diseases. Here we have analysed in detail the contribution of T-bet in shaping the inflammation in the T-cell transfer induced colitis model as a prototypic model for T cell mediated chronic, progressive inflammation. Methods To induce colitis Tbx21-deficient Th cells were adoptively transferred into Rag-deficient mice. Intestinal inflammation was characterised by quantitative and qualitative analysis of lamina propria-infiltrating cells, Th cell phenotyping and histology. Results Here we show that the requirement for T-bet expression by Th cells for the manifestation of clinical symptoms of T cell-induced colitis is absolutely dependent on the composition of the intestinal microbiota. When colitis develops, T-bet exerts several non-redundant functions in shaping the inflammation. T-bet expressed by Th cells promotes the survival and positioning of the Th cells in the colon and their repertoire of chemokine and chemokine receptor expression, the recruitment of myeloid cells to the inflamed colon, and the differentiation of macrophages/monocytes. Remarkably, in T cell-induced colitis, T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17 upon restimulation. Th cell-derived IFN-γ or IL-17, however, are not required to induce colitis. Conclusions We demonstrate that while T-bet shapes the type of the intestinal inflammatory reaction, it is not an essential pre-requisites for T cell-induced colitis. Our data show that the key cytokines IFN-γ and IL-17 play a subordinate role in colitis induction and suggest that the type of inflammation is determined by the chemokine expression profile of the Th cells. In addition, whether Th cells are pathogenic is largely determined by the composition of the intestinal microbiota. Thus, our data shed light on the heterogeneity of inflammatory bowel diseases and their variable response to therapy depending on the microbiota composition and the Th cells involved.

Since the bona fide Fc receptor for IgM antibody (FcµR) was identified eight years ago, much progress has been made in defining its biochemical nature, cellular distribution, and effector function. However, there are clearly conflicting results, especially about the cellular distribution and function of murine FcµR. In this short article, we will discuss recent findings from us and other investigators along with our interpretations and comments that may help to resolve the existing puzzles and should open new avenues of investigation.

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

Restimulation of memory–effector T helper lymphocytes with PMA/ionomycin is an established technique to determine their imprinting for the expression of cytokine genes, that is their functional potential. Here, we show that for the maximal reexpression of cytokine genes, the Ca2+ concentration of the medium has to be greater than 1.5 mM. For RPMI (Roswell Park Memorial Institute 1640), the most commonly used medium, the Ca2+ concentration has to be increased from the regular 0.42 to 1.5 mM.

Background and objectives T helper type 1 (Th1) cells are involved in rheumatic diseases such as Crohn´s disease or rheumatoid arthritis and have a history of chronic autoantigenic stimulation. We have shown that Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miR)-148a. MiR-148a promotes the survival of chronically activated Th1 cells by regulating the expression of the proapoptotic protein Bim. Thus, we tested the suitability of miR-148a as a therapeutic target for the selective elimination of proinflammatory Th1 cells in a preclinical model of colitis. Methods Chronically activated Th1 cells were transferred into Rag1-deficient mice to induce colitis. Then, mice were intravenously injected with antagomirs that specifically target the microRNA-148a (antagomir-148a) or with control antagomirs (antagomir-scr). For assessing the function of T cell-intrinsic expression of miR-148a in colitis, we transduced Th1 cells with inducible microRNA sponges prior to transfer into Rag1-deficient mice. Colitic inflammation was determined by the weight-to-length ratios of colons. Colonic Th cells were sorted by FACS to measure miR-148a expression by qRT-PCR. The expression of Bim and Bcl-2 as well as numbers of viable Th1 cells, were determined by flow cytometry. Results Systemic inhibition of miR-148a by antagomirs alleviated colitis in mice as measured by reduced weight-to-length ratios of their colons. The numbers of viable Th1 cells were reduced up to 50% in mice that were treated with antagomir-148a when compared to mice that were injected with antagomir-scr. Antagomir-148a injections were efficient and resulted in a 30% reduction of miR-148a in colonic Th1 cells. The expression of the proapoptotic protein Bim was increased up to 30% in Th cells from antagomir-148a treated mice, while the anti-apoptotic protein Bcl-2 was unchanged. Th cell specific inhibition of miR-148a by miR-sponges during colitis led to a 30% reduction of Th cells in the colons of colitic mice and reduced the weight-to-length ratio. Conclusions We suggest that miR-148a controls the survival of proinflammatory Th1 cells in chronic inflammation by inhibiting Bim expression in a Th cell intrinsic fashion. Thus, miR-148a might represent a suitable target for the selective depletion of proinflammatory Th1 cells in chronic inflammation.

Background Proinflammatory T helper (Th) cells are critically involved in the initiation and perpetuation of chronic inflammatory diseases (CID). In these diseases, proinflammatory Th cells persist in inflamed tissues and are refractory to current immunosuppressive therapies. The molecular factors regulating the survival of proinflammatory Th cells are not fully understood. Recently, we have shown that proinflammatory Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miRNA, miR)-148a 1 . MiR-148a promotes the survival of proinflammatory Th1 cells by regulating the expression of the pro-apoptotic protein Bim. Memory Th cells isolated from inflamed synovia of patients with rheumatoid arthritis induce the expression of miR-148a, presumably enhancing their survival. Therefore we hypothesize, that miR-148a could represent a therapeutic target for the selective elimination of proinflammatory Th1 cells in CID. Objectives To test the suitability of miR-148a as a therapeutic target in a preclinical murine model of transfer colitis. Methods We have generated a retroviral based inducible miR-148a inhibitor, containing complementary binding sites for miR-148a, termed “miR-148a sponge”. We transduced transgenic Th cells with the miR-148a sponge or a scrambled (scr) control sponge. Induction of sponge expression was initiated by tamoxifen treatment. Expression of miRNA sponges was detected by GFP reporter gene expression and the functionality of miR148a inhibition was determined by real-time PCR for the miR-148a target Bim. MiR-148a sponge-transduced CD4 + Th1 cells were FACS-sorted and transferred into Rag1 -/- mice. After manifestation of colitis, mice were sacrificed and T cells were re-isolated from the spleens and colons for determining their phenotype and number by FACS analysis. Results Expression of miR-148a sponges resulted in a 2 fold increase of Bim expression in activated Th1 cells in vitro . In addition, we found reduced numbers of miR-148a-sponge expressing Th cells as compared to scr-sponge expressing Th cells in the spleens and colons of colitic mice. Finally, miR-148a sponge-expressing Th cells from spleens and colons of colitic mice showed higher levels of Bim expression per cell than scr-sponge expressing cells. Conclusions Our data suggest that miR-148a controls the survival of pro-inflammatory Th1 cells in chronic inflammation by inhibiting Bim expression. Thus, therapeutic targeting of miR-148a probably is suitable for the selective depletion of proinflammatory Th1 cells in chronic inflammation. References Haftmann, C. et al. miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim. European journal of immunology, doi:10.1002/eji.201444633 (2014). Acknowledgements Patrick Maschmeyer is supported by EUTRAIN, a FP7 Marie Curie Initial Training Network for Early Stage Researchers funded by the European Union. Disclosure of Interest None declared