[Effects of biomechanics on colorectal organoid culture].

In recent years, organoids have become a crucial model for studying the physiopathological processes in tissues and organs. The emergence of organoids has promoted the research on the mechanisms of the occurrence and clinical translation of diseases. Among these organoid models, colorectal organoid models are increasingly mature. Colorectal cancer is a common gastrointestinal malignant tumor worldwide, posing a serious threat to human health. Colorectal organoids provide a new model for studying the pathophysiology, drug sensitivity, and precision medicine of colorectal cancer. The conventional culture systems of colorectal organoids focus more on the role of biochemical factors, neglecting the fact that the gut is also influenced by biophysical signals in vivo. Therefore, in this review, we discuss the theories related to colorectal organoids and biomechanics and expound the effects of biomechanics on colorectal organoid culture.

Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons.

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.1.

Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

INTRODUCTION: Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. METHODS: Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 µM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. RESULTS: First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. CONCLUSIONS: During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence.

Interleukin 22 (IL-22) has a non-redundant role in immune defence of the intestinal barrier1-3. T cells, but not innate lymphoid cells, have an indispensable role in sustaining the IL-22 signalling that is required for the protection of colonic crypts against invasion during infection by the enteropathogen Citrobacter rodentium4 (Cr). However, the intestinal epithelial cell (IEC) subsets targeted by T cell-derived IL-22, and how T cell-derived IL-22 sustains activation in IECs, remain undefined. Here we identify a subset of absorptive IECs in the mid-distal colon that are specifically targeted by Cr and are differentially responsive to IL-22 signalling. Major histocompatibility complex class II (MHCII) expression by these colonocytes was required to elicit sustained IL-22 signalling from Cr-specific T cells, which was required to restrain Cr invasion. Our findings explain the basis for the regionalization of the host response to Cr and demonstrate that epithelial cells must elicit MHCII-dependent help from IL-22-producing T cells to orchestrate immune protection in the intestine.

Identifying gene expression programs in single-cell RNA-seq data using linear correlation explanation.

OBJECTIVE: Gene expression analysis through single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of gene regulation in diverse cell types, tissues, and organisms. While existing methods primarily focus on identifying cell type-specific gene expression programs (GEPs), the characterization of GEPs associated with biological processes and stimuli responses remains limited. In this study, we aim to infer biologically meaningful GEPs that are associated with both cellular phenotypes and activity programs directly from scRNA-seq data. METHODS: We applied linear CorEx, a machine-learning-based approach, to infer GEPs by grouping genes based on total correlation optimization function in simulated and real-world scRNA-seq datasets. Additionally, we utilized a transfer learning approach to project CorEx-inferred GEPs to other scRNA-seq datasets. RESULTS: By leveraging total correlation optimization, linear CorEx groups genes and demonstrates superior performance in identifying cell types and activity programs compared to similar methods using simulated data. Furthermore, we apply this same approach to real-world scRNA-seq data from the mouse dentate gyrus and embryonic colon development, uncovering biologically relevant GEPs related to cell types, developmental ages, and cell cycle programs. We also demonstrate the potential for transfer learning by evaluating similar datasets, showcasing the cross-species sensitivity of linear CorEx. CONCLUSION: Our findings validate linear CorEx as a valuable tool for comprehensively analyzing complex signals in scRNA-seq data, leading to deeper insights into gene expression dynamics, cellular heterogeneity, and regulatory mechanisms.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity.

All nucleated cells express major histocompatibility complex I and interferon-γ (IFNγ) receptor1, but an epithelial cell-specific function of IFNγ signalling or antigen presentation by means of major histocompatibility complex I has not been explored. We show here that on sensing IFNγ, colonic epithelial cells productively present pathogen and self-derived antigens to cognate intra-epithelial T cells, which are critically located at the epithelial barrier. Antigen presentation by the epithelial cells confers extracellular ATPase expression in cognate intra-epithelial T cells, which limits the accumulation of extracellular adenosine triphosphate and consequent activation of the NLRP3 inflammasome in tissue macrophages. By contrast, antigen presentation by the tissue macrophages alongside inflammasome-associated interleukin-1α and interleukin-1ß production promotes a pathogenic transformation of CD4+ T cells into granulocyte-macrophage colony-stimulating-factor (GM-CSF)-producing T cells in vivo, which promotes colitis and colorectal cancer. Taken together, our study unravels critical checkpoints requiring IFNγ sensing and antigen presentation by epithelial cells that control the development of pathogenic CD4+ T cell responses in vivo.

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair.

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERß subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERß attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERß promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and ß-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERß were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERß subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

Inference of cell state transitions and cell fate plasticity from single-cell with MARGARET.

Despite recent advances in inferring cellular dynamics using single-cell RNA-seq data, existing trajectory inference (TI) methods face difficulty in accurately reconstructing the cell-state manifold and cell-fate plasticity for complex topologies. Here, we present MARGARET (https://github.com/Zafar-Lab/Margaret) for inferring single-cell trajectory and fate mapping for diverse dynamic cellular processes. MARGARET reconstructs complex trajectory topologies using a deep unsupervised metric learning and a graph-partitioning approach based on a novel connectivity measure, automatically detects terminal cell states, and generalizes the quantification of fate plasticity for complex topologies. On a diverse benchmark consisting of synthetic and real datasets, MARGARET outperformed state-of-the-art methods in recovering global topology and cell pseudotime ordering. For human hematopoiesis, MARGARET accurately identified all major lineages and associated gene expression trends and helped identify transitional progenitors associated with key branching events. For embryoid body differentiation, MARGARET identified novel transitional populations that were validated by bulk sequencing and functionally characterized different precursor populations in the mesoderm lineage. For colon differentiation, MARGARET characterized the lineage for BEST4/OTOP2 cells and the heterogeneity in goblet cell lineage in the colon under normal and inflamed ulcerative colitis conditions. Finally, we demonstrated that MARGARET can scale to large scRNA-seq datasets consisting of ∼ millions of cells.

Metalloendopeptidase ADAM-like Decysin 1 (ADAMDEC1) in Colonic Subepithelial PDGFRα+ Cells Is a New Marker for Inflammatory Bowel Disease.

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα+ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα+ cells. ADAMDEC1 protein was mainly released from PDGFRα+ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα+ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45- PDGFRα+ cells in DSS-induced colitis mice, with only minimal expression in CD45+ CD64+ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα+ cells, but not in CD64+ macrophages found in human colonic mucosal tissue affected by Crohn's disease. In summary, PDGFRα+ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn's disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn's disease.

Presence of Natural Killer B Cells in Simian Immunodeficiency Virus-Infected Colon That Have Properties and Functions Similar to Those of Natural Killer Cells and B Cells but Are a Distinct Cell Population.

Here, we report the appearance of natural killer B (NKB) cells within the colon during simian immunodeficiency virus (SIV) infection of susceptible monkeys. Using RNA sequencing (RNAseq) and flow cytometry, we show that NKB cells are unique cells with features and functions of both NK and B cells. NKB cells express receptors and ligands found on B cells that are important for (i) antigen presentation; (ii) activities associated with class switching, affinity maturation, and B-cell memory formation in secondary lymphoid follicles; and (iii) antigen recognition. The predominant immunoglobulins (Igs) expressed on NKB cells are IgA, although NKB cells can express surface IgM and IgG. There is dominant lambda expression over the kappa light chain characteristic of mucosal B cells. In addition to B-cell aspects, NKB cells express NK cell activation receptors and Fas ligand. We show in this study that NKB cells express perforin and granzymes and lyse cells in a lytic assay. In addition to NK cell cytolytic function, NKB cells also produce the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin-18 (IL-18). Finally, we noted the increased capacity of NKB cells to proliferate compared to NK cells and CD8+ T cells from the SIV-infected colon. The increased proliferation and inflammatory cytokine production may be related to the relatively high expression levels of IL-15 receptor beta, IL-7 receptor, IL-18 receptor, and 41BB relative to the same receptors on CD8 and NK cells. The properties of NKB cells may point to their role in the enhanced inflammation observed in the SIV-infected gut. IMPORTANCE There is low-level but significant mucosal inflammation in the gastrointestinal tract secondary to human immunodeficiency virus (HIV) infection that has long-term consequences for the infected host. This inflammation most likely originates from the immune response that appears as a consequence of HIV. Here, we show in an animal model of HIV that the chronically SIV-infected gut contains cytotoxic natural killer B cells that produce inflammatory cytokines and proliferate during infection.

Matrine protects colon mucosal epithelial cells against inflammation and apoptosis via the Janus kinase 2 /signal transducer and activator of transcription 3 pathway.

Ulcerative colitis (UC) is a type of chronic disease of inflammation, and matrine has anti-inflammatory activity. However, it is unclear that whether matrine can alleviate UC. This study aimed to evaluate the effect of matrine on DSS-induced intestinal epithelial cell injury. Cell viability was performed by MTT assay. Then cell apoptosis was analyzed using the TUNEL assay and flow cytometry. The levels of interleukin (IL)-2, IL-6, TNF-α, and IL-1ß were evaluated using qRT-PCR. Myeloperoxidase (MPO) activity was detected using ELISA assay. Nitric oxide (NO) production was detected by the Griess reagent. Bax, cleaved caspase-3, Bcl-2, JAK2, p-JAK2, STAT3, p-STAT3, STAT5, p-STAT5 levels were measured by Western blot. Bax (6A7) was asses using immunoprecipitation and immunofluorescence assays. The results illustrated that cell viability was inhibited as the concentration of DSS increased. Matrine did not affect cell viability at the concentration of 0-2 mg/ml but inhibited cell viability in a time-independent manner. Matrine suppressed the levels of pro-inflammatory factors, MPO activity, NO production, and apoptosis of DSS-stimulated cells. Furthermore, we found that matrine inhibited the levels of p-JAK2/JAK2 and p-STAT3/STAT3 but did not affect p-STAT5/STAT5. AG490 treatment further enhanced the effect of matrine on the apoptosis and pro-inflammatory factor levels in DSS-induced cells. In summary, matrine protected NCM460 cell against injury by inactivating the JAK2/STAT3 pathway. These data suggested for the first time that matrine may effective in treating UC.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

AIM: The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. METHODS: Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. RESULTS: pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. CONCLUSIONS: The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage.

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

Deoxycholic acid delays the wound healing of colonic epithelial cells via transmembrane G-protein-coupled receptor 5.

BACKGROUND AND AIM: Efficient intestinal wound healing is essential for good prognoses of ulcerative colitis (UC). Although bile acids and the transmembrane G-protein-coupled receptor (TGR) 5 have been reported to affect wound healing in intestinal epithelial cells, the detailed underlying mechanisms are unclear. Here, we investigated the role of TGR5 in wound healing in the context of colonic epithelial cells in the presence of bile acids. METHODS: The expression of TGR5 in the colonic epithelium of both a dextran sulfate sodium (DSS)-induced colitis mouse model (recovery phase), and UC patients in clinical remission, was evaluated. Young adult mouse colonic epithelial (YAMC) cells were then used to evaluate wound healing after treatment with deoxycholic acid (DCA); TGR5 was silenced in YAMC cells via shRNA-transfection, and a wound-healing assay in the presence of DCA was performed. Furthermore, we investigated the role of the activation of AKT in the context of wound healing. RESULTS: The expression of TGR5 was decreased in the colonic epithelium of both mice with DSS-induced colitis and UC patients. Additionally, DCA significantly delayed wound healing in YAMC cells but not in TGR5 silenced ones. Of note, the DCA-induced activation of AKT signaling in YAMC cells was inhibited by TGR5 silencing, and AKT inhibitors prevented the wound healing delay induced by DCA. CONCLUSIONS: Overall, we show that DCA delays wound healing in the context of colonic epithelial cells through AKT activation. These results may support the development of new therapeutic approaches for epithelial regeneration in UC.

Colonic healing requires Wnt produced by epithelium as well as Tagln+ and Acta2+ stromal cells.

Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes.

Regulatory T lymphocyte (Treg) homing reactions mediated by G protein-coupled receptor (GPCR)-ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15-C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short-chain fatty acid-C10ORF99/GPR15-induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15-C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR-ligand pairing leads to physiological adaptation during vertebrate evolution.

Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro-inflammatory host-microbe interactions in the colon.

The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host-microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy-to-handle in vitro triple coculture model that combines chemically-induced macrophage-like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human-derived gut microbiota samples for 16 h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human-derived gut microbiota samples, respond via pro-inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro-inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host-microbe research questions in a stable in vitro environment.

Detection of DNA damage by alkaline comet assay in mouse colonic mucosa.

We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).