B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing.

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.

Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-ß1/FAK/ERK pathway.

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-ß1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC.

Mucin-interacting proteins: from function to therapeutics.

Mucins are high molecular weight glycoproteins that are involved in regulating diverse cellular activities both in normal and pathological conditions. Mucin activity and localization is mediated by several molecular mechanisms, including discrete interactions with other proteins. An understanding of the biochemistry behind the known interactions between mucins and other proteins, coupled with an appreciation of their pathophysiological significance, can lend insight into the development of novel therapeutic agents. Indeed, a recent study demonstrated that a cell permeable inhibitor, PMIP, that disrupts the MUC1-EGFR interaction, is effective in killing breast cancer cells in vitro and in tumor models.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Golgi organization is a determinant of stem cell function in the small intestine.

Cell-to-cell signalling between niche and stem cells regulates tissue regeneration. While the identity of many mediating factors is known, it is largely unknown whether stem cells optimize their receptiveness to niche signals according to the niche organization. Here, we show that Lgr5+ small intestinal stem cells (ISCs) regulate the morphology and orientation of their secretory apparatus to match the niche architecture, and to increase transport efficiency of niche signal receptors. Unlike the progenitor cells lacking lateral niche contacts, ISCs orient Golgi apparatus laterally towards Paneth cells of the epithelial niche, and divide Golgi into multiple stacks reflecting the number of Paneth cell contacts. Stem cells with a higher number of lateral Golgi transported Epidermal growth factor receptor (Egfr) with a higher efficiency than cells with one Golgi. The lateral Golgi orientation and enhanced Egfr transport required A-kinase anchor protein 9 (Akap9), and was necessary for normal regenerative capacity in vitro . Moreover, reduced Akap9 in aged ISCs renders ISCs insensitive to niche-dependent modulation of Golgi stack number and transport efficiency. Our results reveal stem cell-specific Golgi complex configuration that facilitates efficient niche signal reception and tissue regeneration, which is compromised in the aged epithelium.

Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine.

Cytokines are immunomodulatory proteins that orchestrate cellular networks in health and disease. Among these, interleukin (IL)-10 is critical for the establishment of intestinal homeostasis, as mutations in components of the IL-10 signaling pathway result in spontaneous colitis. Whether IL-10 plays other than immunomodulatory roles in the intestines is poorly understood. Here, we report that il10, il10ra, and il10rb are expressed in the zebrafish developing intestine as early as 3 days post fertilization. CRISPR/Cas9-generated il10-deficient zebrafish larvae showed an increased expression of pro-inflammatory genes and an increased number of intestinal goblet cells compared to WT larvae. Mechanistically, Il10 promotes Notch signaling in zebrafish intestinal epithelial cells, which in turn restricts goblet cell expansion. Using murine organoids, we showed that IL-10 modulates goblet cell frequencies in mammals, suggesting conservation across species. This study demonstrates a previously unappreciated IL-10-Notch axis regulating goblet cell homeostasis in the developing zebrafish intestine and may help explain the disease severity of IL-10 deficiency in the intestines of mammals.

The spatial transcriptomic landscape of the healing mouse intestine following damage.

The intestinal barrier is composed of a complex cell network defining highly compartmentalized and specialized structures. Here, we use spatial transcriptomics to define how the transcriptomic landscape is spatially organized in the steady state and healing murine colon. At steady state conditions, we demonstrate a previously unappreciated molecular regionalization of the colon, which dramatically changes during mucosal healing. Here, we identified spatially-organized transcriptional programs defining compartmentalized mucosal healing, and regions with dominant wired pathways. Furthermore, we showed that decreased p53 activation defined areas with increased presence of proliferating epithelial stem cells. Finally, we mapped transcriptomics modules associated with human diseases demonstrating the translational potential of our dataset. Overall, we provide a publicly available resource defining principles of transcriptomic regionalization of the colon during mucosal healing and a framework to develop and progress further hypotheses.

Muc17 protects intestinal epithelial cells from enteroinvasive E. coli infection by promoting epithelial barrier integrity.

The membrane-bound mucin MUC17 (mouse homolog Muc3) is highly expressed on the apical surface of intestinal epithelia and is thought to play a role in epithelial restitution and protection. Therefore, we hypothesized that MUC17 has a role in protection of the intestinal mucosa against luminal pathogens. Human intestinal cell lines were transfected by electroporation (Caco-2 and HT 29/19A) and by retroviral expression vector (LS174T, a cell line with high levels of MUC17 expression) using MUC17 siRNA. Transepithelial electrical resistance, permeability, tight-junction protein expression, adhesion, and invasion in response to enteroinvasive Escherichia coli (EIEC) were measured in all cell lines. In some experiments, the effect of the addition of exogenous purified crude mucin or recombinant Muc3 cysteine-rich domain protein (Muc3 CRD1-L-CRD2) as preventative or protective treatment was tested. Reduction of endogenous MUC17 is associated with increased permeability, inducible nitric oxide synthase and cyclooxygenase 2 induction, and enhanced bacterial invasion in response to EIEC exposure. Bacterial adhesion is not affected. Exogenous mucin (Muc3) and recombinant Muc3CRD treatment had a small but significant effect in attenuating the effects of EIEC infection. In conclusion, these data suggest that both native and exogenous MUC17 play a role in attachment and invasion of EIEC in colonic cell lines and in maintaining epithelial barrier function.

Perfluorooctanesulfonic acid modulates barrier function and systemic T-cell homeostasis during intestinal inflammation.

The intestinal epithelium is continuously exposed to deleterious environmental factors that might cause aberrant immune responses leading to inflammatory disorders. However, what environmental factors might contribute to disease are poorly understood. Here, to overcome the lack of in vivo models suitable for screening of environmental factors, we used zebrafish reporters of intestinal inflammation. Using zebrafish, we interrogated the immunomodulatory effects of polyfluoroalkyl substances, which have been positively associated with ulcerative colitis incidence. Exposure to perfluorooctanesulfonic acid (PFOS) during 2,4,6-trinitro-benzene sulfonic acid (TNBS)-induced inflammation enhanced the expression of proinflammatory cytokines as well as neutrophil recruitment to the intestine of zebrafish larvae, which was validated in the TNBS-induced colitis mouse model. Moreover, PFOS exposure in mice undergoing colitis resulted in neutrophil-dependent increased intestinal permeability and enhanced PFOS translocation into the circulation. This was associated with a neutrophil-dependent expansion of systemic CD4+ T cells. Thus, our results indicate that PFOS worsens inflammation-induced intestinal damage with disruption of T-cell homeostasis beyond the gut and provides a novel in vivo toolbox to screen for pollutants affecting intestinal homeostasis.

Liver X receptor regulates Th17 and RORγt+ Treg cells by distinct mechanisms.

The gastrointestinal microenvironment, dominated by dietary compounds and the commensal bacteria, is a major driver of intestinal CD4+ T helper (Th) cell differentiation. Dietary compounds can be sensed by nuclear receptors (NRs) that consequently exert pleiotropic effects including immune modulation. Here, we found that under homeostatic conditions the NR Liver X receptor (LXR), a sensor of cholesterol metabolites, regulates RORγt+ CD4 T cells in the intestine draining mesenteric lymph node (MLN). While LXR activation led to a decrease, LXR-deficiency resulted in an increase in MLN Th17 and RORγt+ Tregs. Mechanistically, LXR signaling in CD11c+ myeloid cells was required to control RORγt+ Treg. By contrast, modulation of MLN Th17 was independent of LXR signaling in either immune or epithelial cells. Of note, horizontal transfer of microbiota between LXRα-/- and WT mice was sufficient to only partially increase MLN Th17 in WT mice. Despite LXRα deficiency resulted in an increased abundance of Ruminococcaceae and Lachnospiraceae bacterial families compared to littermate controls, microbiota ablation (including SFB) was not sufficient to dampen LXRα-mediated expansion of MLN Th17. Altogether, our results suggest that LXR modulates RORγt+ Treg and Th17 cells in the MLN through distinct mechanisms.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Retinoic acid induced cytokines are selectively modulated by liver X receptor activation in zebrafish.

Nuclear receptors (NRs) rapidly activate/repress gene expression to detour immune responses and allow tissue adaptation to constant environmental changes. However, the effect of combined NRs in the immune system is often unclear due to the lack of reliable experimental models that recapitulate the complex interaction between NRs in vivo. Here, we used the zebrafish to investigate the immunological outcome of combining the activation of retinoic acid receptor (RAR), liver X receptor (LXR) and the cytoplasmic sensor aryl hydrocarbon receptor (AHR). Although simultaneous activation did not affect the expression of respective bona-fide target genes, RAR-induced il17a/f3 was antagonized by LXR and AHR, whereas il22 was antagonized by AHR but not LXR. In addition, RA decreased il10 expression, which was further decreased by LXR activation. Thus, using combinatorial NR activation in zebrafish larvae, we show that LXR antagonizes the expression of selected RA-induced cytokines and provide a strategy to tailor the cytokine milieu.

Lysophosphatidic Acid-Mediated GPR35 Signaling in CX3CR1+ Macrophages Regulates Intestinal Homeostasis.

Single-nucleotide polymorphisms in the gene encoding G protein-coupled receptor 35 (GPR35) are associated with increased risk of inflammatory bowel disease. However, the mechanisms by which GPR35 modulates intestinal immune homeostasis remain undefined. Here, integrating zebrafish and mouse experimental models, we demonstrate that intestinal Gpr35 expression is microbiota dependent and enhanced upon inflammation. Moreover, murine GPR35+ colonic macrophages are characterized by enhanced production of pro-inflammatory cytokines. We identify lysophosphatidic acid (LPA) as a potential endogenous ligand produced during intestinal inflammation, acting through GPR35 to induce tumor necrosis factor (Tnf) expression in macrophages. Mice lacking Gpr35 in CX3CR1+ macrophages aggravate colitis when exposed to dextran sodium sulfate, which is associated with decreased transcript levels of the corticosterone-generating gene Cyp11b1 and macrophage-derived Tnf. Administration of TNF in these mice restores Cyp11b1 expression and intestinal corticosterone production and ameliorates DSS-induced colitis. Our findings indicate that LPA signals through GPR35 in CX3CR1+ macrophages to maintain TNF-mediated intestinal homeostasis.

Conserved transcriptomic profile between mouse and human colitis allows unsupervised patient stratification.

Clinical manifestations and response to therapies in ulcerative colitis (UC) are heterogeneous, yet patient classification criteria for tailored therapies are currently lacking. Here, we present an unsupervised molecular classification of UC patients, concordant with response to therapy in independent retrospective cohorts. We show that classical clustering of UC patient tissue transcriptomic data sets does not identify clinically relevant profiles, likely due to associated covariates. To overcome this, we compare cross-sectional human data sets with a newly generated longitudinal transcriptome profile of murine DSS-induced colitis. We show that the majority of colitis risk-associated gene expression peaks during the inflammatory rather than the recovery phase. Moreover, we achieve UC patient clustering into two distinct transcriptomic profiles, differing in neutrophil-related gene activation. Notably, 87% of patients in UC1 cluster are unresponsive to two most widely used biological therapies. These results demonstrate that cross-species comparison enables stratification of patients undistinguishable by other molecular approaches.

Cytochrome P4501-inhibiting chemicals amplify aryl hydrocarbon receptor activation and IL-22 production in T helper 17 cells.

The aryl hydrocarbon receptor (AHR) controls interleukin 22 production by T helper 17 cells (Th17). IL-22 contributes to intestinal homeostasis but has also been implicated in chronic inflammatory disorders and colorectal cancer, highlighting the need for appropriate regulation of IL-22 production. Upon activation, the AHR induces expression of cytochrome P4501 (CYP1) enzymes which in turn play an important feedback role that curtails the duration of AHR signaling by metabolizing AHR ligands. Recently we described how agents that inhibit CYP1 function potentiate AHR signaling by disrupting metabolic clearance of the endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). In the present study, we investigated the immune-modulating effects of environmental pollutants such as polycyclic aromatic hydrocarbons on Th17 differentiation and IL-22 production. Using Th17 cells deficient in CYP1 enzymes (Cyp1a1/1a2/1b1-/-) we show that these chemicals potentiate AHR activation through inhibition of CYP1 enzymes which leads to increases in intracellular AHR agonists. Our findings demonstrate that IL-22 production by Th17 cells is profoundly enhanced by impaired CYP1-function and strongly suggest that chemicals able to modify CYP1 function or expression may disrupt AHR-mediated immune regulation by altering the levels of endogenous AHR agonist(s).

ß7 integrins contribute to intestinal tumor growth in mice.

The gut homing receptor integrin α4ß7 is essential for the migration of pro-inflammatory T cells into the gut mucosa. Since intestinal neoplasia has been associated with chronic inflammation, we investigated whether interfering with gut-homing affects intestinal tumorigenesis. Using chemically induced and spontaneous intestinal tumor models we showed that lack of ß7 integrin significantly impairs tumor growth without affecting tumor frequencies, with a mild translatable effect on overall survival. This correlates with human data showing lower MAdCAM-1 expression and disease-free survival in colorectal cancer patients. Thus, paradoxically in contrast to extra-intestinal tumors, blocking migration of immune cells into the gut might have a positive therapeutic effect on intestinal neoplasia.

Flt3 ligand expands bona fide innate lymphoid cell precursors in vivo.

A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (ILC) lineage has been recently characterized. While specific requirements of transcription factors for CHILPs development has been partially described, their ability to sense cytokines and react to peripheral inflammation remains unaddressed. Here, we found that systemic increase in Flt3L levels correlated with the expansion of Lineage (Lin)negα4ß7+ precursors in the adult murine bone marrow. Expanded Linnegα4ß7+ precursors were bona fide CHILPs as seen by their ability to differentiate into all helper ILCs subsets but cNK in vivo. Interestingly, Flt3L-expanded CHILPs transferred into lymphopenic mice preferentially reconstituted the small intestine. While we did not observe changes in serum Flt3L during DSS-induced colitis in mice or plasma from inflammatory bowel disease (IBD) patients, elevated Flt3L levels were detected in acute malaria patients. Interestingly, while CHILP numbers were stable during the course of DSS-induced colitis, they expanded following increased serum Flt3L levels in malaria-infected mice, hence suggesting a role of the Flt3L-ILC axis in malaria. Collectively, our results indicate that Flt3L expands CHILPs in the bone marrow, which might be associated with specific inflammatory conditions.

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer.

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn's disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in our understanding of IBD. Of the chemical induced colitis models, dextran sodium sulfate (DSS) induced colitis model is a relatively simple and very widely used model of experimental colitis.