Crypt Epithelium Research Articles

Histological features of Clostridioides difficile colitis in patients with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) are at increased risk for Clostridioides difficile infection, although clinically important infections can be difficult to recognise. C. difficile infection does not produce pseudomembranes when it occurs in IBD patients. These individuals may also be colonised by the organism, in which case diarrhoeal symptoms are not necessarily attributed to C. difficile. We performed this study to determine whether any features distinguished C. difficile-associated colitis from an IBD flare. We reviewed the clinical, endoscopic and biopsy findings from 50 patients with established IBD and worsening diarrhoea, including 22 with concurrent positive C. difficile stool toxin polymerase chain reaction (PCR) assays and 28 with negative C. difficile assay results. We found that C. difficile-infected patients had symptoms and endoscopic findings that were indistinguishable from active IBD. Although most biopsy samples from patients with C. difficile infection showed chronic active colitis indistinguishable from IBD, some displayed neutrophilic infiltrates unaccompanied by plasma cell-rich inflammation involving superficial (41%) and crypt (18%) epithelium as well as neutrophilic infiltrates within lamina propria distant from foci of cryptitis (32%). All three of these features were significantly more common among infected than uninfected patients (4, 0 and 4%; P = 0.002, P = 0.03 and P = 0.02, respectively). Although colonic biopsies from IBD patients with C. difficile infection usually lack features that aid distinction from colitic flares, some cases show an acute colitis pattern not seen in IBD alone. When identified in biopsies from symptomatic IBD patients, these changes should alert pathologists to the possibility of this clinically important infection.

Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/β-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

Histological diversity of anti-PD1-induced colitis.

Histological data on anti-PD1-associated colitis are limited, while the colitis subtypes are still not clearly defined and different terms are being used. The aim of the study was to explore the histopathology of anti-PD1-induced colitis. Colonic biopsies from 9 patients under anti-PD1 agents presenting diarrhea were examined. Histological evaluation revealed colitis of mild to moderate severity in almost all cases. Four distinct dominant histological patterns were identified with nearly the same incidence: Ulcerative colitis (UC)-like (n=2), GVHD-like (n=2), collagenous-like (n=3) and a mixed colitis pattern combining features of microscopic and UC-like colitis (n=2). The latter was additionally characterized by high crypt epithelium apoptosis and cryptitis with mixed inflammatory infiltrate. Thickening of the subepithelial band of collagen, detachment of the surface epithelium and increased apoptosis of the crypt epithelium were commonly encountered features, irrespective of colitis subtype. CD4/CD8 ratio was lower in the "combined" and higher in the GVHD-like subtype. Anti-PD1-induced colitis is expressed by different patterns of injury which share distinct histological hallmarks harboring diagnostic value, while a "combined" colitis subtype is being established. The histological alterations are indicative of mucosa barrier damage after antΙ-PD1 treatment and its participation in the pathogenetic process.

Histological and histochemical study of the intestine in invasive species Acridotheres tristis in Iraq

The current study was conducted to find out the most important histological and histochemistry differences in the bird A. trisris intestine. The bird samples were dissected; to remove and fixed in 10% formalin, preparing them for histological and histochemical study. The results showed there was a histological differences in the bird intestine. Histological sections showed that the mucosa of A. tristis small intestine was presented numerous long villi which lined by simple columnar epithelium. In the bird large intestine the mucosa was presented numerous villi which lined by simple columnar epithelium. The histochemical sections of A. tristis small and large intestine showed that with Alcian blue (pH2.5) stain the epithelium of villi and intestinal crypts showed that the goblet cells contained acidic mucopolysaccharide. While the enterocytes showed negative reaction. With PAS stain, the sections of villi showed strong neutral mucopolysaccharid within goblet cells.

Comparative histological and Histochemical study of the esophagus in two invasive species Acridotheres tristis and Tilapia zillii in Iraq

The current study conducted on two invasive species, the common Myna Acridotheres tristis and Tilapia Tilapia zillii in Iraq. The study aims to identify the histological and histochemical aspects of the esophagus in both species. Histological sections showed that the esophagus composed of four tunica: mucosa, submucosa, muscularis and adventia. The lumen of these parts displayed numerous long folds, these folds were lined by non-keratinized stratifies squamous epithelium in A. tristis, while in Tilapia zillii these folds lined by pseudo stratified columnar epithelium. The tunica mucosa showed absence of muscularis mucosa in both species, so the lamina propria was continued with tunica submucosa. In both species, the histochemical sections showed that the submucosal glands with Alcian blue stain contained acidic mucopolysacharrids, meanwhile the sections with PAS stain showed strong neutral secretions within submucosal glands. The middle part of Tilapia esophagus with alcian blue (pH 2.5) stain appeared that the epithelial crypts contained acidic mucopolysacharrids but no reaction within the epithelium, meanwhile with PAS stain showed strong neutral mucopolysacharrids within crypts and weak neutral mucopolysacharrids within epithelium.

Morphological features of the intestinal mucosa in ulcerative colitis in various forms of the disease

Background. Chronic inflammatory bowel disease (CIBD) is one of the most complex nosological groups in gastroenterology, due to the constant increase in their prevalence, shifting the onset to an earlier age, increasing the frequency of severe complicated forms. The most common representative of CIBD is nonspecific ulcerative colitis (UC). At the heart of UC is a diffuse ulcerative-inflammatory lesion of the colon. The leading role in the diagnosis of UC belongs to the comparison of the results of endoscopic and morphological methods of research. The morphological method of colonobiopsy examination in UC is the gold standard of diagnosis and makes it possible to determine the signs of disease activity even in endoscopic remission. In contrast to descriptive histology, pathological anatomy, quantitative morphology complements and clarifies the data of studies conducted by conventional morphological methods from the standpoint of generalized approaches. The purpose of quantitative morphology is the maximum objectification of the study of qualitative and quantitative manifestations of pathological processes, nosological units, their pathogenesis, morphogenesis, as well as the exclusion of measurement errors and, as far as possible, the subjectivity of the researcher. The purpose of the study: to conduct a comparative histological examination of biopsies of the mucous membrane of the colon in patients with UC of different severity of the disease. Materials and methods. Histological examination of intestinal biopsy specimens was performed in 55 patients with ulcerative colitis. Patients were divided according to the severity of the disease. Group I (n=11)— mild form, group II (n=39)— moderate form and group III (n=5)— patients with severe UC. Results. In 63.6% with a mild severity of UC edema was not observed, the condition of the epithelium was unchanged, the architecture is not disturbed. Crypt abscesses were detected in 54.6% of cases, atrophic changes— in 90.9%. Inflammatory infiltrate consisted mainly of neutrophils ((176.0±8.22) per 1 mm2 stroma) and mononuclear cells ((232.4±22.83) per 1 mm2 stroma), which indicates the activation of regenerative functions of the mucous membrane. From morphometric measurements the highest indicator was the height of the crypt epithelium (38.3±1.49) μm. With an moderate degree of UC in 33.3% of cases there was a violation of the integrity of the epithelium, atrophic changes were diagnosed in 23.1% of cases, violations of crypt architecture— in 17.9%, the presence of crypt abscesses— 48.8%, edema— 64.1%. Morphometric measurement of intestinal mucosal biopsies with moderate severity of UC showed the highest crypt depth (452.6±8.12) μm, crypt epithelial height (42.3±0.33) μm and the number of goblet cells (26.6±1.43). The height of the crypt epithelium was positively correlated with the number of goblet cells (r=0.30; p <0.05). Erosions, general epithelial disorders, crypt abscesses, and atrophic changes were observed in the majority of patients with severe UC in 40%. In severe UC, the smallest crypt depth (426.1±22.37) μm, crypt epithelial height (36.0±2.57)μm, the number of goblet cells (10.5±1.04) and the largest epithelial thickness were observed (51.5±2.81) μm. Conclusions. The presence of crypt abscesses was mainly observed in the group of mild and moderate forms of the disease. Mucosal edema was more common in the group with moderate severity disease. Increased numbers of eosinophils and neutrophils were observed in the group with severety disease.

The Efficacy of Encapsulated Gamma-Cyclodextrin/Tributyrin on Growth and Inflammation in a Dextran Sodium Sulfate-Challenge Piglet Model

ObjectivesInflammatory bowel diseases are common in adults, and their incidence is increasing in children. Butyrate, a short chain fatty acid, can improve gut health, but its bitterness limits incorporation into foods. Herein, the efficacy of tributyrin (TB), a butyrate analog, encapsulated with ɣ-cyclodextrin (ɣ-CD) on growth and intestinal inflammation in a piglet model of dextran-sodium sulfate (DSS)-induced colitis was investigated. MethodsTwo-day-old piglets were randomized to three diets: control (formula alone, CON, N = 41), CD (CON + 9.0 mM ɣ-CD, N = 41), or CDTB (CON + 9.0 mM ɣ-CD/TB, N = 43). Half of the piglets in each group received DSS (1.25 g/kg body weight) from postnatal day (PND) 14 to 18. Blood and intestinal samples were collected on PND 19 and 27. IL-6 and TNF-α concentration in serum and colon mucosa were assessed by ELISA and IL-6, TNF-α, IL-10 and IFN-γ gene expression in colonic tissue were measured by qPCR. A quantitative histological grading system was used to assess 4 independent parameters: epithelial lining, cell infiltration, crypt damage and extent of the intestinal section affected. ResultsAverage daily weight gain (WG) between PND2-10 and 10–19 were affected by diet (P < 0.05), while colitis led to lower WG from PND19-27. A diet and colitis interaction indicated that after PND22 CDTB-COL piglets weighed less than CON and CD (P = 0.051). Diet had no impact on the descending colon (DC) morphology in the colitis animals, but the severity of the DC inflammation increased from PND19 and 27 (P < 0.0001). TNF-α and IL-6 concentrations were elevated in the ascending colon (AC) at PND19, but by PND27 concentrations were similar to non-colitis levels. A similar pattern was found in the DC, except that IL-6 remained elevated on PND27. IL-6, TNF-α and IFN-γ gene expression were significantly higher in the DC of colitis vs. non-colitis animals on PND27. Diet and colitis had no effect on serum TNF-α and IL-6. ConclusionsThe piglet as a model of DSS colitis is a feasible alternative to rodents. Dietary ɣ-CD/TB reduced growth rate and did not ameliorate the inflammatory process of DSS-induced colitis in neonatal pigs. Herein, oral administration of encapsulated TB did not reduce DSS-induced tissue damage or inflammation, however, additional studies are needed to confirm these observations. Funding SourcesNIFA, ISDA 2017–67,017-26,519.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

Hypoxic Cell-Derived Extracellular Vesicles Aggravate Rectal Injury Following Radiotherapy via MiR-122-5p.

Radiation-induced rectal injury is a common side effect of radiotherapy. Hypoxia often occurs after radiotherapy. This study aimed to explore the bystander effect of hypoxia on radiation-induced rectal injury. In vivo, apoptosis increased nearby the highly hypoxic area in the rectal tissues in the mouse models of radiation-induced rectal injury, indicating the potential involvement of hypoxia. In vitro, flow cytometry and Western blotting showed that both hypoxia and hypoxic human intestinal epithelial crypt (HIEC) cell supernatant promoted apoptosis in normoxic HIEC cells. The pro-apoptotic effect of extracellular vesicles (EVs) derived from hypoxic HIEC cell to normoxic HIEC cells was then determined. MiR-122-5p was chosen for further studies through a microRNA (miRNA) microarray assay and apoptosis was alleviated in cells receiving miR-122-5p inhibiting hypoxic EVs. Together, our study demonstrated that the miR-122-5p containing-EVs derived from hypoxic HIEC cells promoted apoptosis in normoxic HIEC cells. Hypoxic EV-derived miR-122-5p plays a critical pathologic role in radiation-induced rectal injury and may be a potential therapeutic target.

Moniezia benedeni infection enhances neuromedin U (NMU) expression in sheep (Ovis aries) small intestine

BackgroundNeuromedin U (NMU) plays an important role in activating the group 2 innate lymphoid cells (ILC2s) and initiating the host’s anti-parasitic immune responses. It is aimed to explore the distribution characteristics of NMU in the sheep small intestine and the influence of Moniezia benedeni infection on them. In the present study, the pET-28a-NMU recombinant plasmids were constructed, and Escherichia coli. BL21 (DE3) were induced to express the recombinant protein. And then, the rabbit anti-sheep NMU polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of NMU in the intestine of normal and Moniezia benedeni-infected sheep were detected by ELISA.ResultsThe results showed that the molecular weight of the obtained NMU recombinant protein was consistent with the expected molecular (13 kDa) and it was expressed in the form of inclusion body. The titer and specificity of obtained rabbit anti-sheep NMU polyclonal antibody were good. The results of immunofluorescence analysis showed that the nerve fibers which specifically expressed NMU mainly extended from the ganglion in the submucosal to lamina propria (LP) in the sheep small intestine, and the expression level was relatively high; especially on the nerve fibers of LP around the intestinal glands. The expression levels were gradually increased from the duodenum to the ileum, and the levels in the jejunum and ileum were significantly higher than that in the duodenum (P < 0.05). In addition, scattered NMU positive cells were distributed in the epithelium of the jejunal crypts. Moniezia benedeni infection increased the expression of NMU in each intestinal segment, especially in the jejunum and ileum there were significant increase (P < 0.05).ConclusionsIt was suggested that Moniezia benedeni infection could be detected by the high expression of NMU in sheep enteric nervous, and which laid the foundation for further studies on whether NMU exerts anti-parasitic immunity by activating ILC2s. In addition, NMU was expressed in some intestinal gland epitheliums, which also provided a basis for studying its roles in regulation of the immune homeostasis. The present study laid the foundation for further revealing the molecular mechanism of sheep’s neural-immune interaction network perceiving the colacobiosis of parasites.

Ketone body \u03b2-hydroxybutyrate ameliorates colitis by promoting M2 macrophage polarization through the STAT6-dependent signaling pathway

BackgroundKetone body β-hydroxybutyrate (BHB) has received more and more attentions, because it possesses a lot of beneficial, life-preserving effects in the fields of clinical science and medicine. However, the role of BHB in intestinal inflammation has not yet been investigated.MethodsColonic mucosa of inflammatory bowel disease (IBD) patients and healthy controls were collected for evaluation of BHB level. Besides, the therapeutic effect of exogenous BHB in a murine model of acute dextran sulfate sodium (DSS)-induced colitis were assessed by body weight change, colon length, disease activity index, and histopathological sections. The regulatory effectors of BHB were analyzed by RT-qPCR, immunofluorescence, and microbe analysis in vivo. Moreover, the molecular mechanism of BHB was further verified in bone marrow-derived macrophages (BMDMs).ResultsIn this study, significantly reduced BHB levels were found in the colonic mucosa from IBD patients and correlated with IBD activity index. In addition, we demonstrated that the administration of exogenous BHB alleviated the severity of acute experimental colitis, which was characterized by less weight loss, disease activity index, colon shortening, and histology scores, as well as decreased crypt loss and epithelium damage. Furthermore, BHB resulted in significantly increased colonic expression of M2 macrophage-associated genes, including IL-4Ra, IL-10, arginase 1 (Arg-1), and chitinase-like protein 3, following DSS exposure, suggesting an increased M2 macrophage skewing in vivo. Moreover, an in vitro experiment revealed that the addition of BHB directly promoted STAT6 phosphorylation and M2 macrophage-specific gene expression in IL-4-stimulated macrophages. Besides, we found that BHB obviously increased M2 macrophage-induced mucosal repair through promoting intestinal epithelial proliferation. However, the enhancement effect of BHB on M2 macrophage-induced mucosal repair and anti-inflammation was completely inhibited by the STAT6 inhibitor AS1517499.ConclusionsIn summary, we show that BHB promotes M2 macrophage polarization through the STAT6-dependent signaling pathway, which contributes to the resolution of intestinal inflammation and the repair of damaged intestinal tissues. Our finding suggests that exogenous BHB supplement may be a useful therapeutic approach for IBD treatment.

A Proposal to Perform High Contrast Imaging of Human Palatine Tonsil with Cross Polarized Optical Coherence Tomography

The palatine tonsils provide the first line of immune defense against foreign pathogens inhaled or ingested. However, a disruption in the epithelial layer within the tonsil crypts can lead to recurrent acute tonsillitis (RAT). Current imaging techniques suffer from poor resolution and contrast and do not allow a classification of the severity of RAT. We have developed a cross-polarized optical coherence tomography system. The system can detect a change in the polarization of the light after the light-tissue interaction. We demonstrate improved resolution and contrast in tonsil imaging with the developed method. Intensity, as well as retardance images of the excised tonsil tissue, were acquired. Features such as crypt epithelium, lymphoid follicles, and dense connective tissue were observed with improved contrast. Cross polarized optical coherence tomography can be a valuable tool in the clinic to evaluate palatine tonsils as it would allow visualizing common tonsil features without the need for any external contrast agent.

Modeling and Computer Simulation of Viscoelastic Crypt Deformation

Colorectal cancer morphogenesis begins at the cellular level from cell mutations in the intestinal epithelium cavities called crypts. These mutations lead to a pressure difference in the epithelium crypt walls, which causes deformation and generates visible abnormalities in the epithelium. The geometrical modeling of these crypts and the mathematical modeling of the physical process that cause the deformations can be simulated by using a Finite Element Method. The method solves numerically the system of PDE equations that governs this phenomenon and permits to estimate the deformations of the crypt walls. We simulate in this work the crypt deformation when the cell mutations appear in several regions of the crypt epithelium.

Histoarchitecture of Large Intestine and Lympho Glandular Complex of Pig

Background: To have a better understanding of the lympho glandular complexes (LGC) along with the histoarchitecture of large intestine and associated M cells, the proposed study have been undertaken. Methods: Present study was conducted on 12 (twelve) numbers of apparently healthy weaned crossbred piglets of 3 to 4 months irrespective of sex. After humane sacrifice of the animals with the approval of institutional animal ethics committee, the representative tissue samples were collected preserved, processed and stained as per standard methods of Luna (1968). Result: Columnar cells predominated the follicle associated epithelium (FAE) and the crypts. M cells were recorded in the follicle associated epithelium as well as in lower crypt epithelium in toluidine blue stained ultra thin sections. M cells were observed with intraepithelial lymphocyte and basally placed euchromatic nucleus. In the colon and rectum of crossbred pigs the lympho glandular complex (LGC) typically consisted of a pore like depression on surface epithelium from which extensions of mucosal glands passed through the circular gap into lamina muscularis mucosae. The lymphatic aggregation of LGC in the lamina propria and tunica submucosa showed three distinct zone viz. follicle, interfollicular area and dome area and distinct FAE could be appreciated. The epithelium lining the solitary lymphatic nodule (SLN) showed (Periodic acid-Schiff) PAS positive reaction. The mucous substance showed strong reaction in the gland in LGC and the FAE epithelium the number of goblet was less. The LGC was formed by aggregation of lymphoid follicles with invagination of crypt gland inside the follicle in the tunica sub mucosa. The follicle associated epithelium devoid of goblet cells were observed on the LGC with interspersed of diffuse migrating lymphocytes in the lamina propria. In the LGC, secondary follicle with interfollicular area was appreciated. In mesenteric lymph node the secondary follicles with inner germinal centre and outer corona could be seen in cortico medullary area. Meshwork of reticular fibers was abundantly present around the crypts in the lamina propria and in the pore like depression of LGC and around the secondary follicle. Collagen fibers could be seen surrounding the LGC and glands. Scanty collagen fibres were observed in the inter-follicular glands. Innervations of nerve fibre was observed around the lymphoid follicle and penetrating the LGC. The mucosal glands were PAS positive.

Extracellular histones cause intestinal epithelium injury and disrupt its barrier function in vitro and in vivo

Extracellular histones are cytotoxic to various cells and have been extensively proven a vital mediator of multiple organ injuries. However, the effect of extracellular histones on the intestine remains largely unknown. This study aimed to clarify the effect of extracellular histones on the intestine. IEC-6, a cell line of rat small intestinal epithelial crypt, and C57BL/6 or ICR mice were treated with histones. The IEC-6 cells treated with histones from 20 μg/mL to 200 μg/mL for 0−24 h displayed a decline of cell viability and an increase of cell death in a concentration- and time-dependent manner. Moreover, histones (100 μg/mL) induced IEC-6 apoptosis through activating caspase 3 and necroptosis through up-regulation of receptor-interacting serine/threonine protein kinase 1 and 3 (RIPK1 and RIPK3), phosphorylated mixed-lineage kinase domain-like protein (p-MLKL) along with the decrease of caspase-8. Histones treatment disturbed zonular occludens 1 (ZO-1) expression and increased permeability of IEC-6 cell monolayer. In vivo, histones 50 mg/kg injection caused mice intestinal edema, loss apex of villus, epithelial lifting down the sides of the villi, and increased neutrophil infiltration. Elevation of serum intestinal fatty acid binding protein (I-FABP), d-lactate, or Diamine oxidase (DAO) and loss of tight junction protein, ZO-1, at 3 h and 6 h after histones injection strongly indicated severe intestinal epithelium injury, which led to increased permeability of the intestine. In conclusion, extracellular histones cause intestinal epithelial damage via direct cytotoxicity. Consequently, intestinal epithelial tight junction and barrier integrity are disrupted, which may play pivotal roles in diverse diseases.

TNFAIP3 DRIVES COLITIS THROUGH DEATH CELL AND TNFα PRODUCTION

Abstract IBD is characterized by inflammation involving a complex interplay between innate and adaptive immunity, non-immune cells, and intestinal microbes. To understand the contributions of innate immunity to inflammation during colitis we have developed a chronic model of innate immune-mediated colitis wherein RAG1-/- mice are crossed with mice expressing TNFAIP3 in intestinal epithelial cells (villin-TNFAIP3 mice). These villin-TNFAIP3 x RAG1-/- (TRAG) mice develop early onset, 100% penetrant, chronic colitis driven by gut microbes. To understand the inflammatory signals that lead to innate colitis in this model, we crossed TRAG mice with TNFα-/- mice (FRAG mice) and compared the severity of colitis in TRAG vs. FRAG mice. In addition, we characterized TNFα-producing myeloid populations from the lamina propria of TRAG mice. Finally, we assessed the expression and localization of proteins involved in cell death pathways, to unravel the mechanisms driving innate-immune mediated colitis. Our results show that, compared to RAG1-/- mice, TRAG mice have significantly increased numbers of lamina propria leukocytes, including increased numbers of neutrophils, inflammatory monocytes, macrophages, dendritic cells, and innate lymphoid cells. TRAG mice lacking TNFα (FRAG mice) had significantly lower numbers of all these leukocyte subsets. Consistent with this, FRAG mice had significantly lower histological colitis scores, compared to TRAG mice. Inflammatory monocytes, monocytes, macrophages, and neutrophils were found to be the main sources of TNFα production, while TNFα production was null or minimal in dendritic cells, natural killer cells, ILC1, ILC3 and IC2. Macrophages and inflammatory monocytes in TRAG mice were characterized by the expression of iNOS, whereas macrophages and monocytes of FRAG and RAG1-/- mice were notably marked by Arginase1 expression. Localization of active caspase3, phosphor-MLKL and TUNEL positive cells indicated extensive epithelial crypt cell death in TRAG mice that was markedly suppressed in FRAG mice. Together our results show that TNFα drives innate immune mediated colitis, potentially through induction of IEC necroptosis. TNFAIP3 is well known as a negative regulator of receptor-mediated NFκB and MAPK activation and therefore attempts to increase TNFAIP3 expression are considered promising avenues for prevention of inflammation. The present study, indicating that intestinal epithelial cell expression of TNFAIP3 drives TNFα-induced IEC death and innate colitis, highlights potential tissue-specific roles for TNFAIP3 that should be considered in the paradigm of TNFAIP3 as a target for prevention of inflammation.

P012 The effect of Oncostatin M on human intestinal organoids

Abstract Background We have previously shown that human subepithelial myofibroblasts (SEMFs), which are of mesenchymal origin, express the receptor of Oncostatin M (OSM), which is further induced when SEMFs are previously exposed to IL-1α and TNF-α. Human Intestinal Organoids (HIOs), derived from embryonic stem cells (ESC), form epithelial crypts consisting of several subtypes of epithelial cells and are surrounded by cells of mesenchymal origin. Recently, we reported that the mesenchymal component of HIOs is gradually reduced, as culture passages increase. The aim of our study was to examine the effect of exogenous OSM on fibrotic and pro-inflammatory marker expression of HIOs, with and without the combined presence of IL-1α and TNF-α. Methods The human ESC line (H1)-derived HIOs were developed using a commercially available kit and characterized by immunofluorescence in all differentiation stages. HIOs from passage 2 were stimulated with either 100ng/ml OSM for 12 hours or 5ng/ml IL-1α and 50ng/ml TNF-α for 24 hours and then with 100ng/ml OSM for 12 hours. mRNA expression of fibrotic markers, Collagen Type I, III, and Fibronectin, and pro-inflammatory markers, CCL2, CXCL10 and CXCL11 were examined by reverse transcription quantitative PCR. Results OSM alone significantly downregulated both Collagen Type I and III mRNA expression, and had no effect on Fibronectin expression. When HIOs were previously exposed to IL-1α and TNF-α, however, OSM stimulation resulted in significant upregulation of Collagen Type I and Fibronectin, and had no effect on Collagen Type III. Regarding the pro-inflammatory marker expression, OSM alone induced the expression of CCL2, CXCL10 and CXCL11, with a greater effect observed when HIOs were previously exposed to IL-1α and TNF-α. Conclusion Our findings indicate that OSM induces the expression of both fibrotic and pro-inflammatory factors in early passages of HIOs, previously exposed to IL-1a and TNF-a, as has been previously observed on SEMFs. Since the mesenchymal cell component of HIOs is strongly present in early passages and OSM’s receptor is mainly found to be expressed in these cells, our results suggest OSM affects HIOs via the cells that are of mesenchymal origin.

1001. Chronic Colonization with Toxigenic Clostridioides difficile Strains Drives Colonic Tumorigenesis in Mice

BackgroundLong-term effects of chronic and/or recurrent C. difficile infections (CDI) are not well understood, and any potential role of CDI in colorectal cancer (CRC) risk is presently unknown. While pursuing efforts to identify novel procarcinogenic microbes, we identified two mucosal slurries from CRC patients (3728T and 3752T) that were tumorigenic in germ-free (GF) ApcMin/+ mice. Surprisingly, both of these CRC patient slurries were positive for C. difficile by 16S rRNA amplicon sequencing. Given the ability of other chronic infections to promote tumorigenesis (e.g., H. pylori), we hypothesized that chronic colonization with C. difficile could promote tumorigenesis in the colon. MethodsA consortium of 30 bacterial isolates including a toxigenic tcdA+ tcdB+ C. difficile strain (CIm_3728T) was cultured from GF ApcMin/+ mice gavaged with the 3728T slurry. This consortium was gavaged into additional GF ApcMin/+ mice with or without C. difficile strains CIm_3728T, CIm_3752T (isolated from mice gavaged with the 3752T slurry), or isogenic tcdA/tcdB mutants of the M7404 R027 strain. Single cell RNA sequencing (scRNAseq), high dimensional (HD) flow cytometry, and fluorescence in situ hybridization (FISH) with EUB338 and Cd198 probes were performed on distal colons from mice gavaged with either complex CRC slurries or the 3728T isolates with CIm_3728T.Results C. difficile strains drove tumorigenesis of the 3728T isolate mixture (Fig. 1A,B). Tumorigenesis was associated with early procarcinogenic signaling and spatial changes including induction of Wnt signaling in colonic epithelial progenitor cells by scRNAseq, IL-17 induction in immune cells by HD flow cytometry, and bacterial biofilm invasion deep into epithelial crypts by FISH. Tumorigenesis correlated with chronic colonization with toxigenic strains of C. difficile and was toxin-dependent, as toxin mutant strains (M7404 tcdA-tcdB-) did not induce tumors.Figure 1. C. difficile strains from CRC patients induce distal colonic tumorigenesis in germ-free (GF) ApcMin/+ mice. A consortium of 30 bacteria, including C. difficile, were isolated from mice gavaged with the 3728T human CRC mucosal slurry. These isolates were then gavaged into additional GF ApcMin/+ mice, with or without C. difficile isolates from mice gavaged with the 3728T slurry or 3752T slurry. (A) Colonic tumor numbers in GF ApcMin/+ mice at 10 wk p.i. demonstrate that C. difficile (Cd) drives the tumorigenesis of this 30-member bacterial consortium. (B) Gross tumors can be observed in the colon of a representative mouse gavaged with the 3728T isolates with the CIm_3728T (top) or CIm_3752T (middle) strain of C. difficile but not in a mouse gavaged with the isolates lacking C. difficile (bottom).ConclusionToxigenic C. difficile strains isolated from human CRC mucosal slurries were pro-carcinogenic in mice, suggesting that C. difficile is a potential driver of CRC. Given the public health burden of C. difficile, further studies are warranted to determine whether C. difficile infections (initial, recurrent, and chronic asymptomatic) increase CRC risk in patients.Disclosures Jada Domingue, PhD, AstraZeneca (Employee) James White, PhD, Personal Genome Diagnostics (Consultant) Patricia J. Simner, PhD, Accelerate Diagnostics (Grant/Research Support)Affinity Biosensors (Grant/Research Support)BD Diagnostics (Consultant, Grant/Research Support)GeneCapture (Consultant)OpGen, Inc (Consultant, Grant/Research Support)Shionogi, Inc (Consultant) Karen C. Carroll, MD, MeMed (Scientific Research Study Investigator)Meridian Diagnostics, Inc. (Grant/Research Support)Pattern Diagnostics (Advisor or Review Panel member)Scanogen, Inc. (Advisor or Review Panel member) Karen C. Carroll, MD, Pattern Diagnostics, Inc. (Individual(s) Involved: Self): Grant/Research Support; Scanogen, Inc. (Individual(s) Involved: Self): Consultant Cynthia L. Sears, MD, Bristol Myers Squibb (Grant/Research Support)Ferring (Advisor or Review Panel member)Janssen (Grant/Research Support)

Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis

Calcium (Ca2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact invitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Single-cell sequencing of rotavirus-infected intestinal epithelium reveals cell-type specific epithelial repair and tuft cell infection

Intestinal epithelial damage is associated with most digestive diseases and results in detrimental effects on nutrient absorption and production of hormones and antimicrobial defense molecules. Thus, understanding epithelial repair and regeneration following damage is essential in developing therapeutics that assist in rapid healing and restoration of normal intestinal function. Here we used a well-characterized enteric virus (rotavirus) that damages the epithelium at the villus tip but does not directly damage the intestinal stem cell, to explore the regenerative transcriptional response of the intestinal epithelium at the single-cell level. We found that there are specific Lgr5+ cell subsets that exhibit increased cycling frequency associated with significant expansion of the epithelial crypt. This was accompanied by an increase in the number of immature enterocytes. Unexpectedly, we found rotavirus infects tuft cells. Transcriptional profiling indicates tuft cells respond to viral infection through interferon-related pathways. Together these data provide insights as to how the intestinal epithelium responds to insults by providing evidence of stimulation of a repair program driven by stem cells with involvement of tuft cells that results in the production of immature enterocytes that repair the damaged epithelium.