Enterocyte Apoptosis Research Articles

Preventive effects of indole-3-carbinol against alcohol-induced liver injury in mice via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms: Role of gut-liver-adipose tissue axis

Indole-3-carbinol (I3C), found in Brassica family vegetables, exhibits antioxidant, anti-inflammatory, and anti-cancerous properties. Here, we aimed to evaluate the preventive effects of I3C against ethanol (EtOH)-induced liver injury and study the protective mechanism(s) by using the well-established chronic-plus-binge alcohol exposure model. The preventive effects of I3C were evaluated by conducting various histological, biochemical, and real-time PCR analyses in mouse liver, adipose tissue, and colon, since functional alterations of adipose tissue and intestine can also participate in promoting EtOH-induced liver damage. Daily treatment with I3C alleviated EtOH-induced liver injury and hepatocyte apoptosis, but not steatosis, by attenuating elevated oxidative stress, as evidenced by the decreased levels of hepatic lipid peroxidation, hydrogen peroxide, CYP2E1, NADPH-oxidase, and protein acetylation with maintenance of mitochondrial complex I, II, and III protein levels and activities. I3C also restored the hepatic antioxidant capacity by preventing EtOH-induced suppression of glutathione contents and mitochondrial aldehyde dehydrogenase-2 activity. I3C preventive effects were also achieved by attenuating the increased levels of hepatic proinflammatory cytokines, including IL1β, and neutrophil infiltration. I3C also attenuated EtOH-induced gut leakiness with decreased serum endotoxin levels through preventing EtOH-induced oxidative stress, apoptosis of enterocytes, and alteration of tight junction protein claudin-1. Furthermore, I3C alleviated adipose tissue inflammation and decreased free fatty acid release. Collectively, I3C prevented EtOH-induced liver injury via attenuating the damaging effect of ethanol on the gut-liver-adipose tissue axis. Therefore, I3C may also have a high potential for translational research in treating or preventing other types of hepatic injury associated with oxidative stress and inflammation.

Colonization with Escherichia coli EC 25 protects neonatal rats from necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a significant cause of morbidity and mortality in premature infants; yet its pathogenesis remains poorly understood. To evaluate the role of intestinal bacteria in protection against NEC, we assessed the ability of naturally occurring intestinal colonizer E. coli EC25 to influence composition of intestinal microbiota and NEC pathology in the neonatal rat model. Experimental NEC was induced in neonatal rats by formula feeding/hypoxia, and graded histologically. Bacterial populations were characterized by plating on blood agar, scoring colony classes, and identifying each class by sequencing 16S rDNA. Binding of bacteria to, and induction of apoptosis in IEC-6 enterocytes were examined by plating on blood agar and fluorescent staining for fragmented DNA. E. coli EC 25, which was originally isolated from healthy rats, efficiently colonized the intestine and protected from NEC following introduction to newborn rats with formula at 106 or 108 cfu. Protection did not depend significantly on EC25 inoculum size or load in the intestine, but positively correlated with the fraction of EC25 in the microbiome. Introduction of EC25 did not prevent colonization with other bacteria and did not significantly alter bacterial diversity. EC25 neither induced cultured enterocyte apoptosis, nor protected from apoptosis induced by an enteropathogenic strain of Cronobacter muytjensii. Our results show that E. coli EC25 is a commensal strain that efficiently colonizes the neonatal intestine and protects from NEC.

Supplementary choline attenuates olive oil lipid emulsion-induced enterocyte apoptosis through suppression of CELF1/AIF pathway.

Enterocyte apoptosis induced by lipid emulsions is a key cause of intestinal atrophy under total parenteral nutrition (TPN) support, and our previous work demonstrated that olive oil lipid emulsion (OOLE) could induce enterocyte apoptosis via CUGBP, Elav‐like family member 1 (CELF1)/ apoptosis‐inducing factor (AIF) pathway. As TPN‐associated complications are partially related to choline deficiency, we aimed to address whether choline supplementation could attenuate OOLE‐induced enterocyte apoptosis. Herein we present evidence that supplementary choline exhibits protective effect against OOLE‐induced enterocyte apoptosis both in vivo and in vitro. In a rat model of TPN, substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE. In cultured Caco‐2 cells, supplementary choline attenuated OOLE‐induced apoptosis and mitochondria dysfunction by suppressing CELF1/AIF pathway. Compared to OOLE alone, the expression of CELF1 and AIF was significantly decreased by supplementary choline, whereas the expression of Bcl‐2 was evidently increased. No obvious alterations were observed in Bax expression and caspase‐3 activation. Mechanistically, supplementary choline repressed the expression of CELF1 by increasing the recruitment of CELF1 mRNA to processing bodies, thus resulting in suppression of its protein translation. Taken together, our data suggest that supplementary choline exhibits effective protection against OOLE‐induced enterocyte apoptosis, and thus, it has the potential to be used for the prevention and treatment of TPN‐induced intestinal atrophy.

Protective effects of oral glutathione on fasting-induced intestinal atrophy through oxidative stress

AIMTo determine whether oral glutathione (GSH) administration can alleviate the effects of fasting-induced intestinal atrophy in the small intestinal mucosa.METHODSRats were divided into eight groups. One group was fed ad libitum, another was fed ad libitum and received oral GSH, and six groups were administrated saline (SA) or GSH orally during fasting. Mucosal height, apoptosis, and cell proliferation in the jejunum were histologically evaluated. iNOS protein expression (by immunohistochemistry), nitrite levels (by high performance liquid chromatography, as a measure of NO production), 8-hydroxydeoxyguanosine formation (by ELISA, indicating ROS levels), glutathione/oxidized glutathione (GSH/GSSG) ratio (by enzymatic colorimetric detection), and γ-glutamyl transpeptidase (Ggt1) mRNA levels in the jejunum (by semi-quantitative RT-PCR) were also estimated.RESULTSOral GSH administration was demonstrated to drastically reduce fasting-induced intestinal atrophy in the jejunum. In particular, jejunal mucosal height was enhanced in GSH-treated animals compared to SA-treated animals [527.2 ± 6.9 for 50 mg/kg GSH, 567.6 ± 5.4 for 500 mg/kg GSH vs 483.1 ± 4.9 (μm), P < 0.01 at 72 h]. This effect was consistent with decreasing changes in GSH-treated animals compared to SA-treated animals for iNOS protein staining [0.337 ± 0.016 for 50 mg/kg GSH, 0.317 ± 0.017 for 500 mg/kg GSH vs 0.430 ± 0.023 (area of staining part/area of tissue), P < 0.01 at 72 h] and NO [2.99 ± 0.29 for 50 mg/kg GSH, 2.88 ± 0.19 for 500 mg/kg GSH vs 5.34 ± 0.35 (nmol/g tissue), P < 0.01 at 72 h] and ROS [3.92 ± 0.46 for 50 mg/kg GSH, 4.58 ± 0.29 for 500 mg/kg GSH vs 6.42 ± 0.52 (8-OHdG pg/μg DNA), P < 0.01, P < 0.05 at 72 h, respectively] levels as apoptosis mediators in the jejunum. Furthermore, oral GSH administration attenuated cell proliferation decreases in the fasting jejunum [182.5 ± 1.9 for 500 mg/kg GSH vs 155.8 ± 3.4 (5-BrdU positive cells/10 crypts), P < 0.01 at 72 h]. Notably, both GSH concentration and Ggt1 mRNA expression in the jejunum were also attenuated in rats following oral administration of GSH during fasting as compared with fasting alone [0.45 ± 0.12 vs 0.97 ± 0.06 (nmol/mg tissue), P < 0.01; 1.01 ± 0.11 vs 2.79 ± 0.39 (Ggt1 mRNA/Gapdh mRNA), P < 0.01 for 500 mg/kg GSH at 48 h, respectively].CONCLUSIONOral GSH administration during fasting enhances jejunal regenerative potential to minimize intestinal mucosal atrophy by diminishing fasting-mediated ROS generation and enterocyte apoptosis and enhancing cell proliferation.

Rack1 function in intestinal epithelia: regulating crypt cell proliferation and regeneration and promoting differentiation and apoptosis.

Previously, we showed that receptor for activated C kinase 1 (Rack1) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages, and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. NEW & NOTEWORTHY Our findings reveal novel functions for receptor for activated C kinase 1 (Rack1) in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia.

Feedback regulation of steady-state epithelial turnover and organ size.

Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss. However, the mechanistic basis of this equilibrium is unknown. Here we show that robustly precise turnover of the adult Drosophila intestine arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell division. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic epidermal growth factors (EGFs) by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated β-catenin (Armadillo in Drosophila) and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers activation of the EGF receptor (Egfr) in stem cells within a discrete radius. When we blocked apoptosis, E-cadherin-controlled feedback suppressed divisions, and the organ retained the same number of cells. When we disrupted feedback, apoptosis and divisions were uncoupled, and the organ developed either hyperplasia or atrophy. Together, our results show that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place at which a replacement cell is needed. In this way, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.

Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in Drosophila.

Introduction of exogenous biomacromolecules into living systems is of great interest in genome editing, cancer immunotherapy, and stem cell reprogramming. Whereas current strategies generally depend on nucleic acids transfection, direct delivery of functional proteins that provides enhanced specificity, increased safety, and fast and temporal regulation is highly desirable. Nevertheless, intracellular delivery of intact and bioactive proteins, especially in vivo, remains poorly explored. In this study, we developed a nanodiamonds (NDs)-based protein delivery system in cultured cells and in Drosophila that showed high adsorption, high efficiency, and effective cytosolic release of fully functional proteins. Through live-cell imaging, we observed a novel phenomenon wherein a substantial amount of internalized NDs-protein complex rejected fusion with the early endosome, thereby evading protein degradation in the lysosome. More significantly, we demonstrated that dietary NDs-RNase induced apoptosis in enterocytes, stimulating regenerative divisions in intestinal stem cells and increasing the number of stem cells and precursor cells in Drosophila intestine. As stem cells are poorly accessible by exogenous agents in vivo, NDs-mediated oral delivery of proteins provides a new approach to modulate the stem cell microenvironment for intestinal remodeling, which has important implications for colorectal cancer therapy and regenerative medicine.

Lawsonia intracellularis in Pigs: Progression of Lesions and Involvement of Apoptosis.

The purpose of this study was to follow the progression of gross and histologic lesions and apoptosis events in Lawsonia intracellularis-infected enterocytes through the course of the disease, proliferative enteropathy (PE). Thirty 5-week-old pigs were divided into 2 groups: 20 challenged and 10 control animals. Groups of 3 pigs, 2 challenged and 1 control, were euthanized at 1, 3, 5, 8, 11, 15, 19, 24, 29, and 35 days after inoculation. Complete necropsies were performed with gross evaluation. Tissue samples from different sites of the gastrointestinal tract and other visceral organs were collected for routine histologic staining and for immunohistochemistry (IHC) for L. intracellularis. In addition, caspase-3, terminal deoxyuridine nick-end labeling assay, and electron microscopy were performed in ileum samples. Macroscopic and histologic lesions suggestive of PE were first detected 11 days after infection and continued through day 24. L. intracellularis antigen was first detected in the intestine by IHC on day 5 after inoculation, and the bacterium was first detected by transmission electron microscopy on day 15. Positive IHC staining for [L. intracellularis] and enterocyte proliferation, but no gross lesion, were detected on day 29. All 3 pigs euthanized on day 35 were grossly and histologically normal and IHC negative. Hyperplastic crypts in challenge pigs had more apoptotic cells on days 15, 19, and 24 postinfection ( P < .05) compared to control pigs. Our results demonstrated the progression of lesions and infection by L. intracellularis and that inhibition of enterocyte apoptosis is not involved in the pathogenesis of proliferative enteropathy.

C-Jun N-terminal kinase 2 promotes enterocyte survival and goblet cell differentiation in the inflamed intestine.

c-Jun N-terminal kinases (JNKs) contribute to immune signaling but their functional role during intestinal mucosal inflammation has remained ill defined. Using genetic mouse models, we characterized the role of JNK1 and JNK2 during homeostasis and acute colitis. Epithelial apoptosis, regeneration, differentiation, and barrier function were analyzed in intestinal epithelium-specific (ΔIEC) or complete JNK1 and bone marrow chimeric or complete JNK2 deficient mice as well as double-knockout animals (JNK1ΔIECJNK2-/-) during homeostasis and acute dextran sulfate sodium (DSS)-induced colitis. Results were confirmed using human HT-29 cells and wild-type or JNK2-deficient mouse intestinal organoid cultures. We show that nonhematopoietic JNK2 but not JNK1 expression confers protection from DSS-induced intestinal inflammation reducing epithelial barrier dysfunction and enterocyte apoptosis. JNK2 additionally enhanced Atonal homolog 1 expression, goblet cell and enteroendocrine cell differentiation, and mucus production under inflammatory conditions. Our results identify a protective role of epithelial JNK2 signaling to maintain mucosal barrier function, epithelial cell integrity, and mucus layer production in the event of inflammatory tissue damage.

Inflammation and Apoptosis: Dual Mediator Role for Toll-like Receptor 4 in the Development of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of neonatal gastrointestinal mortality; effective interventions are lacking with limited understanding of the pathogenesis of NEC. The importance of Toll-like receptor 4 (TLR4) signaling in NEC is well documented; however, the potential mechanisms that regulate enterocyte inflammation and apoptosis remain unclear. The aim of this study was to characterize the role of TLR4-mediated inflammation and apoptosis in the development of NEC and to determine the major apoptotic pathways and regulators in the process. TLR4-deficient C57BL/10ScNJ mice and lentivirus-mediated stable TLR4-silent cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide in vivo or lipopolysaccharide stimulation in vitro. Enterocyte apoptosis was evaluated by TUNEL or Annexin analysis. The expression of TLR4, caspase3, caspase8, caspase9, Bip, Bax, Bcl-2, and RIP was detected by Western blot and immunofluorescence. Inflammatory factors such as tumor necrosis factor-α and interleukin-2 were examined by Luminex. Defect of TLR4 led to suppressed enterocytes apoptosis both in vitro and in vivo; the expression of caspase3, caspase8, Bip, and Bax was decreased; and caspase9 and Bcl-2 were increased. NEC severity was attenuated in TLR4-deficient mice compared with wild-type counterparts, and enterocytes apoptosis was correlated with NEC severity. RIP and cytokine level of tumor necrosis factor-α and interleukin-2 were also decreased. TLR4-induced inflammation and apoptosis play a critical role in the pathogenesis of NEC. TLR4 inhibition, combined with extrinsic (caspase8) and/or endoplasmic reticulum stress (Bip) apoptosis signaling blockade could serve as a potential effective treating strategy for NEC.

Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells

Background and Aims: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. Methods: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. Results: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. Conclusion: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.

Asparagine improves intestinal integrity, inhibits TLR4 and NOD signaling, and differently regulates p38 and ERK1/2 signaling in weanling piglets after LPS challenge.

Asparagine (Asn), an activator of ornithine decarboxylase (ODC), stimulates cell proliferation in intestinal epithelial cells. We hypothesized that Asn can mitigate LPS-induced injury of intestinal structure and barrier function by regulating inflammatory signaling pathways. We executed the following experiment using weanling pigs for each of the groups: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 0.5% Asn; (4) LPS + 1.0% Asn. After 21-d feeding, pigs received an i.p. injection of either saline or LPS. Four h after injection, the mid-jejunum and mid-ileum samples were collected. We found that Asn restored ODC expression that was decreased by LPS treatment. Asn also restored intestinal morphology and barrier function that were impaired by LPS treatment. In addition, Asn down-regulated intestinal caspase-3 protein expression and TNF-α concentration, and decreased the mRNA expression of intestinal TLR4, TLR4 downstream signals (myeloid differentiation factor 88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6 and NOD1, NOD2 and their adaptor molecule (receptor-interacting serine/threonine-protein kinase 2). Moreover, Asn decreased p38 phosphorylation but increased ERK1/2 phosphorylation. Our results suggest that Asn improves intestinal integrity during an inflammatory insult, which appears to be related to the decrease of intestinal pro-inflammatory cytokine (via TLR4, NODs and p38) and of enterocyte apoptosis (via p38 and ERK1/2).

Fenofibrate reduces intestinal damage and improves intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

Fenofibrate (FEN) is known as a nuclear receptor activator which regulates many pathophysiological processes, such as oxidative stress, inflammation, and leukocyte endothelium interactions. Recent studies have demonstrated an anti-oxidant, anti-inflammatory, and anti-ischemic role of FEN in the attenuation of ischemia-reperfusion (IR) injury in the kidney, liver, brain, and heart. The purpose of the present study was to examine the effect of FEN on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) sham-FEN rats underwent laparotomy and were treated with intraperitoneal (IP) FEN (20mg/kg); (3) IR rats underwent occlusion of both the superior mesenteric artery and the portal vein for 30min followed by 24h of reperfusion, and (4) IR-FEN rats underwent IR and were treated with IP FEN immediately before abdominal closure. Intestinal structural changes, Park's injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24h following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real-time PCR, Western blot, and immunohistochemistry. Treatment with FEN resulted in a significant decrease in Park's injury score in jejunum (32%) and ileum (33%) compared to IR animals. IR-FEN rats also demonstrated a significant increase in mucosal weight in jejunum (23%) and ileum (22%), mucosal DNA (38%) and protein (65%) in jejunum, villus height in jejunum (17%) and ileum (21%), and crypt depth in ileum (14%) compared to IR animals. IR-FEN rats also experienced significant proliferation rates as well as lower apoptotic indices in jejunum and ileum which was accompanied with higher Bcl-2 levels compared to IR animals. Treatment with fenofibrate prevents intestinal mucosal damage and stimulates intestinal epithelial cell turnover following intestinal IR in a rat model.

Expression of CD68 and Fas Ligand in Colon Mucosa of Patients with Inflammatory Bowel Disease as Prognostic Markers of Cancerogenesis

As it is known, colon carcinogenesis is associated with the inflammatory bowel disease (IBD). Colorectal cancer (CRC) being observed in 5.5-13.5 % of patients with ulcerative colitis (UC) and in 0.4-0.8 % of patients with Crohn disease (CD). The gut mucosa of patients with CD, but not with UC, as well as the stroma of CRC have elevated numbers of CD68(+) macrophages. It is known also that the expression of Fas Ligand (FasL), representing another branch of immunoreactivity, in the lesions of CRC and UC but not of CD is upregulated. The fact that enterocyte apoptosis is increased in lesions of CD and lymphocytes in UC are resistant to Fas-mediated apoptosis can be the keypoint for understanding the hypothesis [17], suggesting that cells express FasL and are able to kill Fas-expressing activated lymphocytes and escape rejection by the immune system, which can be the basis for differences in association of UC and CD with CRC. The aim of this study was to evaluate the number of CD68(+) macrophages and to analyze FasL expression in colon mucosa of patients with UC, CD and CRC in order to assess its value for prognosis of CRC. Expression of CD68 and FasL was analyzed immunohistochemically in the samples of colon mucosa taken from the affected areas of 4 patients with UC, 6 patients with CD and 10 patients with CRC. In addition in 7 CRC patients the samples taken from unaffected areas were analyzed. We find average expression of FasL in both unaffected and affected areas of CRC patients was higher than in CD patients (p = 0.04, p = 0.00). Average expression of FasL in affected areas of CRC patients was higher (p = 0.02) than in UC patients and the same (p = 0.23) as in unaffected areas of CRC patients. The revealed associations support the possibility to use expression of CD68 and FasL as prognostic markers for transformation of IBD to CRC.

Irinotecan- and 5-fluorouracil-induced intestinal mucositis: insights into pathogenesis and therapeutic perspectives.

Intestinal mucositis and diarrhea are common manifestations of anticancer regimens that include irinotecan, 5-fluorouracil (5-FU), and other cytotoxic drugs. These side effects negatively impact therapeutic outcomes and delay subsequent cycles of chemotherapy, resulting in dose reductions and treatment discontinuation. Here, we aimed to review the experimental evidence regarding possible new targets for the management of irinotecan- and 5-FU-related intestinal mucositis. A literature search was performed using the PubMed and MEDLINE databases. No publication time limit was set for article inclusion. Here, we found that clinical management of intestinal mucositis and diarrhea is somewhat ineffective at reducing symptoms, possibly due to a lack of specific targets for modulation. We observed that IL-1β contributes to the apoptosis of enterocytes in mucositis induced by 5-FU. However, 5-FU-related mucositis is far less thoroughly investigated with regard to specific molecular targets when compared to irinotecan-related disease. Several studies have proposed that a correlation exists between the intestinal microbiota, the enterohepatic recirculation of active metabolites of irinotecan, and the establishment of mucositis. However, as reviewed here, this association seems to be controversial. In addition, the pathogenesis of irinotecan-induced mucositis appears to be orchestrated by interleukin-1/Toll-like receptor family members, leading to epithelial cell apoptosis. IL-1β, IL-18, and IL-33 and the receptors IL-1R, IL-18R, ST2, and TLR-2 are potential therapeutic targets that can be modulated to minimize anticancer agent-associated toxicity, optimize cancer treatment dosing, and improve clinical outcomes. In this context, the pathogenesis of mucositis caused by other anticancer agents should be further investigated.

Effect of Chelerythrine on Intestinal Cell Turnover following Intestinal Ischemia-Reperfusion Injury in a Rat Model.

Background Chelerythrine (CHE) is a benzophenanthridine alkaloid that is a potent, selective, and cell-permeable protein kinase C inhibitor. The purpose of the present study was to examine the effect of CHE on intestinal recovery and enterocyte turnover after intestinal ischemia-reperfusion (IR) injury in rats. Methods Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) sham-CHE rats underwent laparotomy and were treated with intraperitoneal CHE; (3) IR-rats underwent occlusion of both superior mesenteric artery and portal vein for 30 minutes followed by 48 hours of reperfusion, and (4) IR-CHE rats underwent IR and were treated with intraperitoneal CHE immediately before abdominal closure. Intestinal structural changes, Park injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real Western blot and immunohistochemistry. Results Treatment with CHE resulted in a significant decrease in Park injury score in jejunum (threefold decrease) and ileum (twofold decrease), and parallel increase in mucosal weight in jejunum and ileum, villus height in jejunum and ileum, and crypt depth in ileum compared with IR animals. IR-CHE rats also experienced a significantly lower apoptotic index in jejunum and ileum, which was accompanied by a lower Bax/Bcl2 ratio compared with IR animals. Conclusions Treatment with CHE inhibits programmed cell death and prevents intestinal mucosal damage following intestinal IR in a rat.

Antibody blockade of mucosal addressin cell adhesion molecule-1 attenuates proinflammatory activity of mesenteric lymph after hemorrhagic shock and resuscitation

The current study was designed to determine the effects of antibody blockade of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on the proinflammatory activity of mesenteric lymph after hemorrhagic shock and resuscitation (HS/R). Rats were subjected to HS/R with or without treatment with MAdCAM-1 polyclonal antibody. MAdCAM-1 expression and lymphocyte infiltration in rats were examined. Post-shock mesenteric lymph was collected, filtered to remove lymphocytes, and transfused into recipient mice to induce systemic inflammation and intestinal injury. The proinflammatory activity of post-shock lymph in mice was determined by examining intestinal permeability, enterocyte apoptosis, intestinal lactate levels, lung myeloperoxidase (MPO) activity, and serum cytokine levels. Survival of recipient mice was determined over a 1-week time period. Rats subjected to HS/R had increased MAdCAM-1 expression and lymphocyte infiltration in the intestine. Antibody blockade of MAdCAM-1 attenuated the increased lymphocyte infiltration after HS/R (P < .05). Post-shock mesenteric lymph transfusion significantly increased mortality accompanied by increases in gut permeability, enterocyte apoptosis, intestinal lactate levels, lung MPO activity, and serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor-β (all P < .05). Antibody blockade of MAdCAM-1 in rats subjected to HS/R attenuated the proinflammatory activity of post-shock mesenteric lymph, with abrogation of lymph transfusion-induced increases in mortality, gut permeability, epithelial cell apoptosis, intestinal lactate levels, lung MPO activity, and serum levels of IL-1β, IL-6, and TNF-α (all P < .05). These findings demonstrate that antibody blockade of MAdCAM-1 attenuates the proinflammatory activity of mesenteric lymph after HS/R.

Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis.

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

A Novel Function for P2Y2 in Myeloid Recipient-Derived Cells during Graft-versus-Host Disease.

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2(-/-) mice to test the role of this receptor in GvHD. P2y2(-/-) recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2(-/-) inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2(+) cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD.

Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo

We compared the mechanisms of porcine delatacoronavirus (PDCoV) induced death of infected enterocytes in vivo and infected LLC porcine kidney (LLC-PK) and swine testicular (ST) cells in vitro. We conducted histologic analysis and immunofluorescence (IF) staining for the detection of PDCoV antigens, and TUNEL assay in singly or serially cut tissue sections from the small and large intestines of four, 11- to 14-day-old gnotobiotic pigs, inoculated orally with 8.8–11.0log10 genomic equivalents (GE) of US PDCoV strains OH-FD22 or OH-FD100 (n=3), or mock (n=1). Similar comparative assays were done on LLC-PK and ST cells inoculated with the cell-adapted PDCoV strain OH-FD22-P44 (passage 44) in cell culture medium with 2.5–10μg/ml of trypsin and 1% pancreatin, respectively. At post-inoculation days 3–4, infected pigs showed severe watery diarrhea and/or vomiting and mainly, diffuse, severe atrophic enteritis, with mild to moderate cytoplasmic vacuolation of the enteroctyes lining the atrophied villous epithelium. By IF, PDCoV antigens were evident in villous or crypt epithelial cells. No PDCoV antigen-positive, small and large intestinal villous or crypt epithelial cells, of which cytoplasm was also either vacuolated or morphologically normal, showed positive TUNEL staining. In contrast, by double IF and TUNEL staining, most of the TUNEL-positive signals (apoptotic nuclear fragmentation) were found in PDCoV antigen-positive LLC-PK and ST cells that also showed cytopathic effects, such as cell rounding, detachment and clumping in clusters. Secondary annexin V/propidium iodide (PI) staining revealed increased numbers of annexin V- or PI-positive LLC-PK and ST cells at 21h after inoculation, compared to the negative controls. Thus, PDCoV does not induce apoptosis in the infected intestinal enterocytes in vivo, but in two infected cell lines of swine origin, LLC-PK and ST cells.