Paneth Cells Research Articles

Absence of Paneth Cell Metaplasia to Predict Clinical Relapse in Ulcerative Colitis with Endoscopically Quiescent Mucosa.

Paneth cells play multiple roles in maintaining intestinal homeostasis. However, the clinical role of Paneth cell metaplasia (PCM) in ulcerative colitis (UC) remains unclear. We aimed to investigate the relationship between PCM and relapse in patients with UC and compare the usefulness of PCM with other histological indexes, including mucin depletion (MD) and basal plasmacytosis (BP). Patients with UC in clinical remission (CR) who underwent colonoscopy to confirm a Mayo endoscopic subscore (MES) ≦1 with biopsies from the distal colon were enrolled into this retrospective cohort study. Biopsy samples were evaluated for histological findings of PCM, MD, and BP. Clinical relapse was defined as partial Mayo score ≧3 or medication escalation. Multivariate analysis was performed to determine independent predictors of relapse among the three histological findings, MES, and patient background, and relapse prediction models were generated. Eighty-three patients were enrolled in this study (MES 0, n = 47; MES 1, n = 36). The number of PCM cases was significantly higher in patients with prolonged CR than that in those with relapse (p = 0.01). Multivariate analysis showed that the absence of PCM and MD were related to relapse in all the patients. In patients with MES 1, the absence of PCM was the only risk factor significantly and independently associated with relapse (hazard ratio, 4.51 [1.15-17.7]; p = 0.03). The absence of PCM was a histological risk factor for relapse in patients with MES 1, implying a protective role for PCM in remission and a new index for mucosal healing.

Interaction between microRNA-195 and HuR regulates Paneth cell function in the intestinal epithelium by altering SOX9 translation.

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here, we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.NEW & NOTEWORTHY Our results indicate that intestinal epithelial tissue-specific transgenic miR-195 expression decreases the levels of SOX9 expression, along with reduced numbers of Paneth cells. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restores Paneth cell development ex vivo. miR-195 inhibits SOX9 translation by preventing binding of HuR to Sox9 mRNA. These findings suggest that interaction between miR-195 and HuR controls Paneth cell function via SOX9 in the intestinal epithelium.

Changes at small intestine induced by food-fish contaminated with ciguatoxins

Ciguateric syndrome is a food poisoning associated with the consumption of some species of fish that have accumulated ciguatoxins (CTXs) in their tissues. The effects of the syndrome occur with nervous imbalances which have been described for quite some time, and mentioned in sailing literature for centuries. In the last decade, research has been focused on the implementation of analytical methods for toxin identification and the study of action modes of CTXs to design effective treatments. However, an important aspect is to determine the damage that CTXs caused in the organs of affected individuals. In this work, the damages observed in tissues of mice, mainly in the small intestine, were analyzed. The animals were fed with CTX-contaminated fish muscle at concentrations 10-times below the median lethal dose (LD50) for 10 weeks. The analysis of tissues derived from the oral treatment resulted in an increased occurrence of Paneth cells, presence of lymphoid tissue infiltrating the mucosa and fibrous lesions in the mucosal layer of the small intestine. A decreasing weight in animals fed with toxic muscle was observed.

AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection.

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.IMPORTANCEWe observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.

Endogenous Hyaluronan Promotes Intestinal Homeostasis and Protects against Murine Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a complex, multifactorial gastrointestinal disorder predominantly affecting preterm infants. The pathogenesis of this condition involves a complex interplay between intestinal barrier dysfunction, microbial dysbiosis, and an altered immune response. This study investigates the potential role of endogenous hyaluronan (HA) in both the early phases of intestinal development and in the context of NEC-like intestinal injury. We treated neonatal CD-1 mouse pups with PEP1, a peptide inhibiting HA receptor interactions, from postnatal days 8 to 12. We evaluated postnatal intestinal developmental indicators, such as villi length, crypt depth, epithelial cell proliferation, crypt fission, and differentiation of goblet and Paneth cells, in PEP1-treated animals compared with those treated with scrambled peptide. PEP1 treatment significantly impaired intestinal development, as evidenced by reductions in villi length, crypt depth, and epithelial cell proliferation, along with a decrease in crypt fission activity. These deficits in PEP1-treated animals correlated with increased susceptibility to NEC-like injuries, including higher mortality rates, and worsened histological intestinal injury. These findings highlight the role of endogenous HA in supporting intestinal development and protecting against NEC.

The fate of secretory cells during intestinal homeostasis, regeneration, and tumor formation is regulated by Tcf4.

The single-layer epithelium of the gastrointestinal tract is a dynamically renewing tissue that ensures nutrient absorption, secretory and barrier functions and is involved in immune responses. The basis for this homeostatic renewal is the Wnt signaling pathway. Blocking this pathway can lead to epithelial damage, while its abnormal activation can result in the development of intestinal tumors. In this study, we investigated the dynamics of intestinal epithelial cells and tumorigenesis using a conditional mouse model. Using single-cell and bulk RNA sequencing and histological analysis, we elucidated the cellular responses following the loss of specific cell types. We focused on the fate of cells in the lower parts of the intestinal crypts and the development of colon adenomas. By partially inactivating the transcription factor Tcf4, a key effector of the Wnt signaling pathway, we analyzed the regeneration of isolated hyperproliferative foci (crypts). Our results suggest that the damaged epithelium is not restored by a specific regeneration program associated with oncofetal gene production, but rather by a standard homeostatic renewal pathway. Moreover, disruption of Tcf4 in secretory progenitors resulted in a significant shift in the cell lineage from Paneth cells to goblet cells, characterized by morphological changes and loss of Paneth cell-specific genes. We also found that hyperactivation of the Wnt signaling pathway in colonic adenomas correlated with the upregulation of genes typical of Paneth cells in the intestine, followed by the emergence of secretory tumor cells producing the Wnt3 ligand. The absence of Tcf4 led to a phenotypic shift of the tumor cells towards goblet cells. Our study presents a new model of epithelial regeneration based on the genetically driven partial elimination of intestinal crypts. We highlight the critical role of Tcf4 in the control of cell lineage decisions in the intestinal epithelium and colon tumors.

Ribonuclease 4 functions as an intestinal antimicrobial protein to maintain gut microbiota and metabolite homeostasis

Antimicrobial proteins contribute to host-microbiota interactions and are associated with inflammatory bowel disease (IBD), but our understanding on antimicrobial protein diversity and functions remains incomplete. Ribonuclease 4 (Rnase4) is a potential antimicrobial protein with no known function in the intestines. Here we find that RNASE4 is expressed in intestinal epithelial cells (IEC) including Paneth and goblet cells, and is detectable in human and mouse stool. Results from Rnase4-deficient mice and recombinant protein suggest that Rnase4 kills Parasutterella to modulate intestinal microbiome, thereby enhancing indoleamine-2,3-dioxygenase 1 (IDO1) expression and subsequently kynurenic and xanthurenic acid production in IECs to reduce colitis susceptibility. Furthermore, deceased RNASE4 levels are observed in the intestinal tissues and stool from patients with IBD, correlating with increased stool Parasutterella. Our results thus implicate Rnase4 as an intestinal antimicrobial protein regulating gut microbiota and metabolite homeostasis, and as a potential diagnostic biomarker and therapeutic target for IBD.

Western diet reduces small intestinal intraepithelial lymphocytes via FXR-Interferon pathway

The prevalence of obesity in the United States has continued to increase over the past several decades. Understanding how diet-induced obesity modulates mucosal immunity is of clinical relevance. We previously showed that consumption of a high fat, high sugar “Western” diet (WD) reduces the density and function of small intestinal Paneth cells, a small intestinal epithelial cell type with innate immune function. We hypothesized that obesity could also result in repressed gut adaptive immunity. Using small intestinal intraepithelial lymphocytes (IEL) as a readout, we found that in non-inflammatory bowel disease (IBD) subjects, high body mass index correlated with reduced IEL density. We recapitulated this in wild type (WT) mice fed with WD. A 4-week WD consumption was able to reduce IEL but not splenic, blood, or bone marrow lymphocytes, and the effect was reversible after another 2 weeks of standard diet (SD) washout. Importantly, WD-associated IEL reduction was not dependent on the presence of gut microbiota, as WD-fed germ-free mice also showed IEL reduction. We further found that WD-mediated Farnesoid X Receptor (FXR) activation in the gut triggered IEL reduction, and this was partially mediated by intestinal phagocytes. Activated FXR signaling stimulated phagocytes to secrete type I IFN, and inhibition of either FXR or type I IFN signaling within the phagocytes prevented WD-mediated IEL loss. Therefore, WD consumption represses both innate and adaptive immunity in the gut. These findings have significant clinical implications in the understanding of how diet modulates mucosal immunity.

Ileal Paneth Cell Phenotype is a Cellular Biomarker for Pouch Complications in Ulcerative Colitis.

Biomarkers that integrate genetic and environmental factors and predict outcome in complex immune diseases such as inflammatory bowel disease (IBD; including Crohn's disease [CD] and ulcerative colitis [UC]) are needed. We showed that morphologic patterns of ileal Paneth cells (Paneth cell phenotype [PCP]; a surrogate for PC function) is one such cellular biomarker for CD. Given the shared features between CD and UC, we hypothesized that PCP is also associated with molecular/genetic features and outcome in UC. Because PC density is highest in the ileum, we further hypothesized that PCP predicts outcome in UC subjects who underwent total colectomy and ileal pouch-anal anastomosis (IPAA). Uninflamed ileal resection margins from UC subjects with colectomy and IPAA were used for PCP and transcriptomic analyses. PCP was defined using defensin 5 immunofluorescence. Genotyping was performed using Immunochip. UC transcriptomic and genotype associations of PCP were incorporated with data from CD subjects to identify common IBD-related pathways and genes that regulate PCP. The prevalence of abnormal ileal PCP was 27%, comparable to that seen in CD. Combined analysis of UC and CD subjects showed that abnormal PCP was associated with transcriptomic pathways of secretory granule maturation and polymorphisms in innate immunity genes. Abnormal ileal PCP at the time of colectomy was also associated with pouch complications including de novo CD in the pouch and time to first episode of pouchitis. Ileal PCP is biologically and clinically relevant in UC and can be used as a biomarker in IBD.

Effect of acetylcholinesterase inhibition on immune cells in the murine intestinal mucosa

The gastrointestinal tract (GI) is the largest immune organ whose function is controlled by a complex network of neurons from the enteric nervous system (ENS) as well as the sympathetic and parasympathetic system. Evolving evidence indicates that cross-communication between gut-innervating neurons and immune cells regulates many essential physiological functions including protection against mucosal infections. We previously demonstrated that following paraoxon treatment, 70 % of the mice were able to survive an oral infection with S. typhimurium, a virulent strain of Salmonella enterica serovar Typhimurium. The present study aims to investigate the effect that rivastigmine, a reversible AChE inhibitor used for the treatment of neurodegenerative diseases, has on the murine immune defenses of the intestinal mucosa. Our findings show that, similar to what is observed with paraoxon, administration of rivastigmine promoted the release of secretory granules from goblet and Paneth cells, resulting in increased mucin layer. Surprisingly, however, and unlike paraoxon, rivastigmine treatment did not affect overall mortality of infected mice. In order to investigate the mechanistic basis for the differential effects observed between paraoxon and rivastigmine, we used multi-color flowcytometric analysis to characterize the immune cell landscape in the intraepithelial (IE) and lamina propria (LP) compartments of intestinal mucosa. Our data indicate that treatment with paraoxon, but not rivastigmine, led to an increase of resident CD3+CD8+ T lymphocytes in the ileal mucosa (epithelium and lamina propria) and CD11b− CD11c+ dendritic cells in the LP. Our findings indicate the requirement for persistent cholinergic pathway engagement to effect a change in the cellular landscape of the mucosal tissue that is necessary for protection against lethal bacterial infections. Moreover, optimal protection requires a collaboration between innate and adaptive mucosal immune responses in the intestine.

Dysplasia Detected in Patients With Serrated Epithelial Change Is Frequently Associated With an Invisible or Flat Endoscopic Appearance, Nonconventional Dysplastic Features, and Advanced Neoplasia.

The significance of serrated epithelial change (SEC), defined as endoscopically invisible hyperplastic polyp (HP)-like mucosal change identified in patients with inflammatory bowel disease (IBD), remains unclear. Although some studies reported an increased risk of synchronous and/or metachronous colorectal neoplasia in patients with SEC, including advanced neoplasia (high-grade dysplasia or colorectal cancer), the development of SEC is not significantly associated with increased colonic inflammation. This contrasts with the reported positive correlation between increased colonic inflammation and the risk of colorectal neoplasia in ulcerative colitis, arguing against the notion that SEC may represent a form of dysplasia. As such, this study aimed to characterize the features of synchronous and metachronous dysplasia detected in patients with SEC to identify factors contributing to the increased risk of colorectal neoplasia, including advanced neoplasia, observed in a subset of these patients. Clinicopathologic features of 46 IBD patients with SEC (n=109) and synchronous and/or metachronous dysplasia (n=153) were analyzed. All dysplastic lesions were subtyped as either conventional or nonconventional dysplasia. As controls, 45 IBD patients with endoscopically visible or polypoid HP (n=75) and synchronous and/or metachronous dysplasia (n=87) were analyzed. The SEC group included 28 (61%) men and 18 (39%) women with a mean age of 58 years and a long history of IBD (mean duration: 23 years). The majority of patients (n=34; 74%) had ulcerative colitis, and 12 (26%) had Crohn's disease. Thirty-nine (85%) patients had a history of pancolitis, and 2 (4%) had concomitant primary sclerosing cholangitis. Twenty-seven (59%) patients had multifocal SEC. SEC was predominantly found in the left colon (n=52; 48%) and rectum (n=34; 31%). Dysplasia in the SEC group was often endoscopically invisible or flat (n=42; 27%) and demonstrated nonconventional dysplastic features (n=49; 32%). Six nonconventional subtypes were identified in the SEC group, including 17 (11%) dysplasia with increased Paneth cell differentiation, 12 (8%) hypermucinous dysplasia, 8 (5%) crypt cell dysplasia, 7 (5%) goblet cell deficient dysplasia, 3 (2%) sessile serrated lesion-like dysplasia, and 2 (1%) traditional serrated adenoma-like dysplasia. Advanced neoplasia was detected in 11 (24%) patients. The SEC group was more likely to have nonconventional dysplasia (32%, P <0.001), invisible/flat dysplasia (27%, P <0.001), and advanced neoplasia (24%, P <0.001) than the control group (7%, 2%, and 0%, respectively). High-risk nonconventional subtypes (ie, hypermucinous, crypt cell, and goblet cell deficient dysplasias) accounted for 18% of all dysplastic lesions in the SEC group, which were not seen in the control group ( P <0.001). The SEC group (n=35; 76%) also had a higher rate of concordance between the location of SEC and the area of synchronous/metachronous dysplasia than the control group (n=22; 49%) ( P =0.007). In conclusion, dysplasia detected in patients with SEC is often endoscopically invisible/flat (27%), nonconventional (32%, including the high-risk subtypes), and found in the same colonic segment as SEC (76%), which may in part explain why some patients with SEC are associated with an increased risk of colorectal neoplasia, including advanced neoplasia. The finding of SEC may warrant a careful follow-up colonoscopy with increased random biopsy sampling, especially in the segment of colon with SEC.

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn’s disease severity

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn’s disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

IFN-γ stimulates Paneth cell secretion through necroptosis mTORC1 dependent.

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

Nur77 as a novel regulator of Paneth cell differentiation and function

Serving as a part of intestinal innate immunity, Paneth cells play an important role in intestinal homeostasis maintenance via their multiple functions. However, the regulation of Paneth cells has been proven to be complex and diverse. Here, we identified nuclear receptor Nur77 as a novel regulator of Paneth cell differentiation and function. Nur77 deficiency led to the loss of Paneth cells in murine ileal crypts. Intestinal tissues or organoids with Nur77 deficiency exhibited the impaired intestinal stem cell niche and failed to enhance antimicrobial peptide expression after Paneth cell degranulation. The defects in Paneth cells and antimicrobial peptides in Nur7-/- mice led to intestinal microbiota disorders. Nur77 deficiency rendered postnatal mice susceptible to necrotizing enterocolitis. Mechanistically, Nur77 transcriptionally inhibited Dact1 expression to activate Wnt signaling activity, thus promoting Paneth cell differentiation and function. Taken together, our data suggest the regulatory role of Nur77 in Paneth cell differentiation and function and reveal a novel Dact1-mediated Wnt inhibition mechanism in Paneth cell development.

Intestinal lysozyme engagement of Salmonella Typhimurium stimulates the release of barrier-impairing InvE and Lpp1

Lysozyme is a β-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella invitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.

Clinical manifestations, diagnosis and long-term prognosis of adult autoimmune enteropathy: Experience from Peking Union Medical College Hospital.

Autoimmune enteropathy (AIE) is a rare disease whose diagnosis and long-term prognosis remain challenging, especially for adult AIE patients. To improve overall understanding of this disease's diagnosis and prognosis. We retrospectively analyzed the clinical, endoscopic and histopathological characteristics and prognoses of 16 adult AIE patients in our tertiary medical center between 2011 and 2023, whose diagnosis was based on the 2007 diagnostic criteria. Diarrhea in AIE patients was characterized by secretory diarrhea. The common endoscopic manifestations were edema, villous blunting and mucosal hyperemia in the duodenum and ileum. Villous blunting (100%), deep crypt lymphocytic infiltration (67%), apoptotic bodies (50%), and mild intraepithelial lymphocytosis (69%) were observed in the duodenal biopsies. Moreover, there were other remarkable abnormalities, including reduced or absent goblet cells (duodenum 94%, ileum 62%), reduced or absent Paneth cells (duodenum 94%, ileum 69%) and neutrophil infiltration (duodenum 100%, ileum 69%). Our patients also fulfilled the 2018 diagnostic criteria but did not match the 2022 diagnostic criteria due to undetectable anti-enterocyte antibodies. All patients received glucocorticoid therapy as the initial medication, of which 14/16 patients achieved a clinical response in 5 (IQR: 3-20) days. Immunosuppressants were administered to 9 patients with indications of steroid dependence (6/9), steroid refractory status (2/9), or intensified maintenance medication (1/9). During the median of 20.5 months of follow-up, 2 patients died from multiple organ failure, and 1 was diagnosed with non-Hodgkin's lymphoma. The cumulative relapse-free survival rates were 62.5%, 55.6% and 37.0% at 6 months, 12 months and 48 months, respectively. Certain histopathological findings, including a decrease or disappearance of goblet and Paneth cells in intestinal biopsies, might be potential diagnostic criteria for adult AIE. The long-term prognosis is still unsatisfactory despite corticosteroid and immunosuppressant medications, which highlights the need for early diagnosis and novel medications.

Intestinal Stem Cells Remodel in Response to Acute Notch Inhibition and Paneth Cell Loss

INTRODUCTION: Intestinal epithelial cells are rapidly renewed throughout life by intestinal stem cells (ISCs). ISC function is regulated by niche factors that orchestrate cellular proliferation and differentiation. Of these, Notch signaling is essential for ISC self-renewal and regulates the cell fate choice between absorptive cells (enterocyte) vs secretory (Paneth, goblet, enteroendocrine, and tuft) cells. Yet, the Notch niche is not fully defined. In the mature small intestine, Paneth cells neighbor each ISC and act as Notch niche cells, providing the Notch signal by expressing the Notch ligands DLL1 and DLL4 on their surface. However, studies in mice have shown that ISCs can function in the immature intestine before Paneth cells are formed, and when Paneth cells are depleted in the adult intestine. We previously observed that acute Notch inhibition via the inhibitor dibenzazepine (DBZ) resulted in Paneth cell apoptosis with intact ISCs displaying transient dysfunction. Here, we aimed to define the Notch niche after Paneth cell apoptosis in adult mice and in the developing intestine. We hypothesized that during acute Notch inhibition, ISCs remodel to compensate for Paneth cell loss by activating Notch ligand expression, and that this remodeling mimics the state of immature ISCs prior to Paneth cell emergence. METHODS: We isolated Lgr5+ ISCs by FACS from Lgr5-GFP-CreERT2 mice 24 hours after DBZ or vehicle treatment and performed bulk RNAseq analysis. To define the Notch niche during development, we analyzed postnatal intestines from Notch ligand reporter mice (Dll1-mCherry and Dll4-mCherry) crossed with the ISC reporter mice (Lgr5-GFP-CreERT2). RESULTS: ISCs isolated from Paneth cell-depleted intestines upregulated expression of Notch ligands Dll4 and Dll4 and various secretory cell markers, with expected downregulation of Notch target genes, but no alteration of other stem cell genes. This suggests that after acute Notch inhibition, Lgr5+ ISCs maintain partial stem cell identity while acquiring a signature resembling that of secretory progenitors. In the immature intestine, we showed by immunohistology that sporadic cells expressing secretory cell markers (LYZ, MUC2, and CHGA) localized to the intervillus region and co-stained with Dll1- or Dll4-mCherry+. This suggests that multiple differentiated cell types could provide the Notch signal to immature ISCs. We also observed occasional Lgr5-GFP+ ISCs that were co-positive for Dll1-mCherry, suggesting that immature ISCs can express Notch ligands at homeostasis. Further experimentation will determine whether these Dll1+ ISCs are similar to the remodeled adult ISCs after Paneth cell depletion. CONCLUSIONS: Acute Notch inhibition directly remodels ISCs to express the Notch ligands, Dll1 and Dll4 in the adult intestine. In the immature intestine, multiple secretory cell types appear to comprise the immature Notch niche. This project was funded by NIH R01 DK118023 awarded to Dr. Linda Samuelson. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Erbb3 regulation of secretory differentiation in the intestinal epithelium

Background: Previous work by our lab showed that intestinal-specific deletion of the ErbB3 receptor tyrosine kinase results in increased ileal Paneth cell numbers. These cells produce host defense peptides and support stem cells with growth factors. The mechanisms by which ErbB3 regulates Paneth cells are not well understood, but a candidate mediator is the polycomb repressor complex 1 component BMI1, which marks secretory progenitor cells. Furthermore, while there are no Paneth cells in the mouse colon, deep crypt secretory (DCS) cells are a distinct Reg4+ population that have some functional overlap with both Paneth and goblet cells; we will also test whether ErbB3 regulates DCS differentiation. Overall we are testing the hypothesis that ErbB3 regulates secretory cell numbers through BMI1. Methods: We bred Villin-Cre;ErbB3flox/flox mice (ErbB3ΔIE) to delete ErbB3 from the intestinal epithelium. Colonic sections from ErbB3ΔIE and ErbB3flox/flox littermates were subjected to RNAscope in situ hybridization for Reg4. RNA from mucosal scrapings (both ileum and colon) was assessed by qPCR for Reg4, Bmi1, and Lgr5. To test signaling mechanisms, we treated HT-29 cells with NRG-1β to activate ErbB3, +/- inhibitors to PI3K (LY294002), MEK/MAPK (U0126), or BMI1 (PTC-209). Colonoids and enteroids from ErbB3ΔIE and ErbB3flox/flox mice were used to confirm responses in a non-transformed system. Results: In ErbB3ΔIE mice, the colonic epithelium showed increased Reg4 levels and more Reg4+ cells compared to littermate controls, similar to our published data for the Paneth cell marker Lyz1 in ileum. BMI1 RNA and protein levels were also increased in both small and large intestinal mucosal scrapings from ErbB3ΔIE mice vs. littermates. ErbB3ΔIE colonoids had increased Reg4 versus control colonoids, and NRG-1β reduced levels in control but not ErbB3ΔIE cultures. Ileal enteroids showed a decrease in Lyz1 expression with Bmi1 inhibition and an increase in Bmi1 expression with PI3K and MAPK blockade. In HT-29 cells, NRG-1β treatment reduced Reg4 and Bmi1 expression. Conversely, Reg4 and Bmi1 were induced by PI3K inhibition with LY294002 (p&lt;0.03) (p&lt;0.0005) or MEK/MAPK inhibition with U0126 (p&lt;0.05) (p&lt;0.0001). Conclusions: NRG1-ErbB3 signaling restricts both ileal Paneth ( Lyz1+) and colonic DCS ( Reg4+) cell numbers. Furthermore, ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling and BMI1 activity promotes secretory cell development. Since loss of DCS or Paneth cells has been shown to disrupt the intestinal stem cell niche and secretory cell markers are altered in several intestinal disease states, these results may point to potential future therapeutic avenues targeting this regulatory mechanism. NIH/NIDDK award R01DK095004. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Paneth Cell TCF4 Deficiency Alters Gut Microbiota and Host Tissue Gene Expression

Introduction: The mucosal immune system is crucial in regulating gut microbiota composition. Paneth cells, the specialized secretory cells in the small intestine, produce α-defensins that regulate gut microbiota. The Wnt signaling pathway with T-cell factor-4 (TCF4) as the terminal signaling molecule regulates Paneth cell differentiation and function. Wnt signaling plays a role in Paneth cell α-defensin expression. TCF4 binds to the α-defensin promoter and increases the transcriptional activity. In this study, we investigated the role of Paneth cell TCF4 in regulating gut microbiota and the host intestinal gene expression. Materials and methods: We generated the Paneth cell-specific TCF4 knockout mice by crossing Tcf4flox/flox mice with the Defa6Cre mice. Tcf4flox/flox-Defa6Cre (KO) were compared with Tcf4flox/flox and Defa6Cre mice (WT). Colonic flushing from these mice was subjected to metatranscriptomics, metagenomics, and proteomics. Colonic tissue was analyzed for transcriptomics. Plasma lipopolysaccharide (LPS) was measured to evaluate endotoxemia. Intestinal mucosal inflammatory response and Paneth cell function were assessed by RT-PCR for cytokines, defensins, and lysozyme. Bioinformatics was applied to assess the taxonomic diversity and functional activity of the gut microbiome, colonic luminal peptides, and colonic mucosal gene expression. Results: The metatranscriptome profile of colonic contents showed an altered microbial diversity in KO mice compared to WT mice. PCoA analyses indicated that the microbial composition in the three strains was distinctly different from each other. At the phylum level, Proteobacteria and viruses such as Pisuviricota, Cressdnaviricota, and Artiverviricota were higher in KO mice than in WT mice. LEfSe analyses showed that the abundance of numerous bacterial and viral populations, such as Firmicutes, Clostridia, and Geminiviridae, was high in KO mice. Functional profiling further confirmed the alteration of microbial diversity in the KO mice. The increased abundance of bacteria in KO mice was related to human diseases, infectious diseases, and bacterial toxin production. Proteomic analyses indicated that nearly 36% of proteins identified in the KO mice were significantly altered compared to WT mice. The Reactome analyses indicated that the altered proteins in the KO mice were related to the immune system, cell cycle, cell signaling, and protein metabolism. RT-PCR of intestinal tissue showed that the expression of Defa6, lysozyme, and Reg3γ was low in KO mice compared to WT mice. On the other hand, the expression of IL-1β, IL-6, and MCP1 in the intestine was higher in KO mice compared to WT mice. These changes in microbiota and mucosa in the KO mice were associated with increased plasma LPS. Finally, there was a significant sex-dependent differences in the Paneth TCF4 activity on gut microbiota, intestinal gene expression and endotoxemia. Conclusion: This study indicates that the Paneth cell TCF4 is essential in regulating the gut microbial composition and host intestinal gene expression. NIH/NIAID, UO1-AI170019 and U01-AI172991. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.