Scattered crypt intestinal epithelial cell apoptosis induces necrotizing enterocolitis via intricate mechanisms.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear. METHODS: A novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IECs-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The three days old neonatal 3xTg-iAPcIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms. RESULTS: Treatment of 3xTg-iAPcIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8+ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8+ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in NEC pathogenesis. CONCLUSIONS: Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8+ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.

Severe gut mucosal injury induces profound systemic inflammation and spleen-associated lymphoid organ response.

Clinical evidence indicates a connection between gut injuries, infections, inflammation, and an increased susceptibility to systemic inflammation. Nevertheless, the animal models designed to replicate this progression are inadequate, and the fundamental mechanisms are still largely unknown. This research explores the relationship between gut injuries and systemic inflammation using a Dextran Sulfate Sodium (DSS)-induced colonic mucosal injury mouse model. Continuous treatment of adult mice with 4% DSS drinking water yielded a remarkable mortality rate by day 7, alongside intensified gut injury and detectable peripheral inflammation. Moreover, RNAscope in situ hybridization with 16S rRNA probe noted bacterial penetration into deeper colon compartments of the mice following treatment with DSS for 7 days. Histological analysis revealed inflammation in the liver and lung tissues of DSS-treated mice. In addition, we found that DSS-treated mice exhibited elevation of Alanine transaminase (ALT) and Aspartate transaminase (AST) in peripheral blood and pro-inflammatory cytokine levels in the liver. Notably, the DSS-treated mice displayed a dampened metabolic profile, reduced CD45 marker expression, and an increase in apoptosis within the lymphoid organ such as spleen. These findings suggest that high-dose DSS-induced gut injury gives rise to sepsis-like systemic inflammation characterized by multiple organ injury and profound splenocyte apoptosis and dysfunction of CD45+ cells in the spleen, indicating the role of the spleen in the pathogenesis of gut-derived systemic inflammation. Together, the severe colonic mucosal injury model facilitates research into gut damage and associated peripheral immune responses, providing a vital framework for investigating mechanisms related to clinically relevant, gut-derived systemic inflammation.

Feeding mode influences dynamic gut microbiota signatures and affects susceptibility to anti-CD3 mAb-induced intestinal injury in neonatal mice.

Feeding modes influence the gut microbiome, immune system, and intestinal barrier homeostasis in neonates; how feeding modes impact susceptibility to neonatal gastrointestinal (GI) diseases is still uncertain. Here, we investigated the impact of dam feeding (DF) and formula feeding (FF) on features of the gut microbiome and physiological inflammation during the first 2 days of postnatal development and on the susceptibility to intestinal injury related to the inflammatory state in neonatal mouse pups. 16S rRNA sequencing data revealed microbiome changes, lower α-diversity, and a distinct pattern of ß-diversity including expansion of f_Enterobacteriaceae and f_Enterococcaceae in the ileum of FF pups compared with DF pups by postnatal day (P)2. Together with gut dysbiosis, the FF cohort also had greater ileal mucosa physiological inflammatory activity compared with DF pups by P2 but maintained normal histological features. Interestingly, FF but not DF mouse pups developed necrotizing enterocolitis (NEC)-like intestinal injury within 24 h after anti-CD3 mAb treatment, suggesting that FF influences the susceptibility to intestinal injury in neonates. We further found that NEC-like incidence in anti-CD3 mAb-treated FF neonatal pups was attenuated by antibiotic treatment. Collectively, our data suggest that FF predisposes mouse pups to anti-CD3 mAb-induced intestinal injury due to abnormal f_Enterobacteriaceae and f_Enterococcaceae colonization. These findings advance our understanding of FF-associated microbial colonization and intestinal inflammation, which may help inform the development of new therapeutic approaches to GI diseases like NEC in infants.NEW & NOTEWORTHY This report shows that a feeding mode profoundly affects gut colonization in neonatal mice. Furthermore, our results demonstrate that formula feeding predisposes mouse pups to anti-CD3 mAb-induced necrotizing enterocolitis (NEC)-like intestinal injury upon inadequate microbial colonization. The study suggests the role of the combined presence of formula feeding-associated dysbiosis and mucosal inflammation in the pathogenesis of NEC and provides a new mouse model to study this disease.

SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8SARS-CoV-2) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNß in lung epithelial cells. Taken together, ORF8SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future.

MFG-E8 Plays an Important Role in Attenuating Cerulein-Induced Acute Pancreatitis in Mice.

Milk fat globule-EGF factor 8 (MFG-E8) is a secreted glycoprotein that regulates tissue homeostasis, possesses potent anti-inflammatory properties, and protects against tissue injury. The human pancreas expresses MFG-E8; however, the role of MFG-E8 in the pancreas remains unclear. We examined the expression of MFG-E8 in the pancreas at baseline and during cerulein-induced acute pancreatitis in mice and determined whether MFG-E8 attenuates the progression of pancreatitis, a serious inflammatory condition that can be life-threatening. We administered cerulein to wild-type (WT) and Mfge8 knockout (KO) mice to induce pancreatitis. Immunoblot analysis showed that MFG-E8 is constitutively expressed in the murine pancreas and is increased in mice with cerulein-induced acute pancreatitis. In situ hybridization revealed that ductal epithelial cells in the mouse pancreas express Mfge8 transcripts at baseline. During pancreatitis, Mfge8 transcripts were abundantly expressed in acinar cells and endothelial cells in addition to ductal epithelial cells. Knocking out Mfge8 in mice exacerbated the severity of cerulein-induced acute pancreatitis and delayed its resolution. In contrast, administration of recombinant MFG-E8 attenuated cerulein-induced acute pancreatitis and promoted repair of pancreatic injury in Mfge8 KO mice. Taken together, our study suggests that MFG-E8 protects the pancreas against inflammatory injury and promotes pancreatic tissue repair. MFG-E8 may represent a novel therapeutic target in acute pancreatitis.

Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism.

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3'-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3'-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3'-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration.

BACKGROUND & AIMS: Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation. METHODS: We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs. RESULTS: In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, mice with LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19ΔEx1/+ mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19ΔEx1/+ mice vs control mice. Crypt epithelial cells from H19ΔEx1/+ mice proliferated more slowly than those from control mice after exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium. CONCLUSIONS: The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration.

Sirtuin-6 preserves R-spondin-1 expression and increases resistance of intestinal epithelium to injury in mice.

Sirtuin-6 (Sirt6) is a critical epigenetic regulator, but its function in the gut is unknown. Here, we studied the role of intestinal epithelial Sirt6 in colitis-associated intestinal epithelial injury. We found that Sirt6, which is predominantly expressed in epithelial cells in intestinal crypts, is decreased in colitis in both mice and humans. Colitis-derived inflammatory mediators including interferon-γ and reactive oxygen species strongly inhibited Sirt6 protein expression in young adult mouse colonocyte (YAMC) cells. The susceptibility of the cells to injurious insults was increased after knockdown of Sirt6 expression. In contrast, YAMC cells with Sirt6 overexpression exhibited more resistance to injurious insult. Furthermore, intestinal epithelial-specific Sirt6 (Sirt6IEC-KO) knockout mice exhibited greater susceptibility to dextran sulfate sodium (DSS)-induced colitis. RNA sequencing transcriptome analysis revealed that inflammatory mediators such as tumor necrosis factor (TNF)-α suppressed expression of R-spondin-1 (Rspo1, a critical growth factor for intestinal epithelial cells) in Sirt6-silenced YAMC cells in vitro. In addition, lipopolysaccharide was found to inhibit colonic Rspo1 expression in Sirt6IEC-KO mice but not their control littermates. Furthermore, Sirt6IEC-KO mice with DSS-induced colitis also exhibited in a significant decrease in Rspo1 expression in colons. In vitro, knockdown of Rspo1 attenuated the effect of ectopic expression of Sirt6 on protection of YAMC cells against cell death challenges. In conclusion, Sirt6 plays an important role in protecting intestinal epithelial cells against inflammatory injury in a mechanism associated with preserving Rspo1 levels in the cells.

Spherical nucleic acid targeting microRNA-99b enhances intestinal MFG-E8 gene expression and restores enterocyte migration in lipopolysaccharide-induced septic mice.

Milk fat globule-EGF factor 8 (MFG-E8) maintains the intestinal homeostasis by enhancing enterocyte migration and attenuating inflammation. We previously reported that sepsis is associated with down-regulation of intestinal MFG-E8 and impairment of enterocyte migration. Here, we showed that impairment of intestinal epithelial cell migration occurred in lipopolysaccharide (LPS)-induced septic mice. Treatment of RAW264.7 cells (a murine macrophage-like cell line) with LPS increased expression of miR-99b, a microRNA that is predicted to target mouse MFG-E8 3'UTR. Using a luciferase assay, we showed that miR-99b mimic suppressed the activity of a reporter containing MFG-E8 3'UTR. This suggests the role of miR-99b in inhibition of MFG-E8 gene expression. In addition, we developed an anti-miR99b spherical nucleic acid nanoparticle conjugate (SNA-NC(anti-miR99b)). Treatment of both naïve and LPS-challenged cells with SNA-NC(anti-miR99b) enhanced MFG-E8 expression in the cells. Administration of SNA-NC(anti-miR99b) rescued intestinal MFG-E8 expression in LPS-induced septic mice and attenuated LPS inhibitory effects on intestinal epithelial cell migration along the crypt-villus axis. Collectively, our study suggests that LPS represses MFG-E8 expression and disrupts enterocyte migration via a miR-99b dependent mechanism. Furthermore, this work shows that SNA-NC(anti-miR99b) is a novel nanoparticle-conjugate capable of rescuing MFG-E8 gene expression and maintaining intestinal epithelial homeostasis in sepsis.

Molecular Mechanisms Underlying the Regulation of the MFG-E8 Gene Promoter Activity in Physiological and Inflammatory Conditions.

Milk fat globule-EGF factor 8 (MFG-E8) is expressed by macrophages and plays an important role in attenuating inflammation and maintaining tissue homeostasis. Previously, we and others found that lipopolysaccharide (LPS) inhibits MFG-E8 gene expression in macrophages. Here, we characterized the 5'-flanking region of the mouse MFG-E8 gene. To functionally analyze the upstream regulatory region of the MFG-E8 gene, a series of luciferase reporter gene constructs containing deleted or mutated regulatory elements were prepared. Using the luciferase assay, we revealed that Sp1 binding motifs within the proximal promoter region were necessary for full activity of the MFG-E8 promoter, whereas AP-1 like binding sequence at -372 played a role in governing the promoter activity at a homeostatic level. With chromatin immunoprecipitation assay, we showed that Sp1 and c-Jun physically interact with the MFG-E8 promoter region in vivo. In addition, Sp1 was found to regulate the MFG-E8 promoter activity positively and c-Jun negatively. Furthermore, we demonstrated that LPS inhibited MFG-E8 promoter activity via targeting Sp1 and AP-1-like motifs in the 5'-flanking region. Collectively, our data indicate that Sp1 and AP-1-related factors are involved in the regulation of MFG-E8 gene transcription by targeting their binding sites in the 5'-flanking region under physiological and inflammatory states.

Dissociation of hepatic insulin resistance from susceptibility of nonalcoholic fatty liver disease induced by a high-fat and high-carbohydrate diet in mice.

Liver steatosis in nonalcoholic fatty liver disease is affected by genetics and diet. It is associated with insulin resistance (IR) in hepatic and peripheral tissues. Here, we aimed to characterize the severity of diet-induced steatosis, obesity, and IR in two phylogenetically distant mouse strains, C57BL/6J and DBA/2J. To this end, mice (male, 8 wk old) were fed a high-fat and high-carbohydrate (HFHC) or control diet for 16 wk followed by the application of a combination of classic physiological, biochemical, and pathological studies to determine obesity and hepatic steatosis. Peripheral IR was characterized by measuring blood glucose level, serum insulin level, homeostasis model assessment of IR, glucose intolerance, insulin intolerance, and AKT phosphorylation in adipose tissues, whereas the level of hepatic IR was determined by measuring insulin-triggered hepatic AKT phosphorylation. We discovered that both C57BL/6J and DBA/2J mice developed obesity to a similar degree without the feature of liver inflammation after being fed an HFHC diet for 16 wk. C57BL/6J mice in the HFHC diet group exhibited severe pan-lobular steatosis, a marked increase in hepatic triglyceride levels, and profound peripheral IR. In contrast, DBA/2J mice in the HFHC diet group developed only a mild degree of pericentrilobular hepatic steatosis that was associated with moderate changes in peripheral IR. Interestingly, both C57BL/6J and DBA/2J developed severe hepatic IR after HFHC diet treatment. Collectively, these data suggest that the severity of diet-induced hepatic steatosis is correlated to the level of peripheral IR, not with the severity of obesity and hepatic IR. Peripheral rather than hepatic IR is a dominant factor of pathophysiology in nonalcoholic fatty liver disease.

MFG-E8 and HMGB1 are involved in the mechanism underlying alcohol-induced impairment of macrophage efferocytosis.

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.

Luminal-applied flagellin is internalized by polarized intestinal epithelial cells and elicits immune responses via the TLR5 dependent mechanism.

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn's lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

Milk fat globule-EGF factor 8 is a critical protein for healing of dextran sodium sulfate-induced acute colitis in mice.

Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8(+/+)) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8(+/+) and MFG-E8(-/-) mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8(+/+) mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8(+/+) mice. MFG-E8(-/-)mice developed more severe crypt-epithelial injury than MFG-E8(+/+) mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8(-/-) and MFG-E8(+/+) mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.

Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis.

Peptidoglycan (PGN) is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb PGN from the lumen. Nonetheless, how PGN is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized PGN transport in vitro using IEC-18 and HT29-CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual-labeled PGN by enterocytes. Engulfed PGN was found to form a complex with PGN recognition protein-3, which may facilitate delivering PGN in vivo. Utilizing electronic microscopy, we revealed that uptake of apical PGN across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of PGN using the transwell system. Our data indicated that apically loaded PGN was exocytosed to the basolateral compartment with exosomes by HT29-CL19A cells. The PGN-contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled PGN, we found that luminal PGN was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre-confluent immature but not post-confluent mature C2BBe1 cells engulfed PGN via a toll-like receptor 2-dependent manner. Together, our findings suggest that (1) crypt-based immature intestinal epithelial cells play an important role in transport of luminal PGN over the intestinal epithelium; and (2) luminal PGN is transcytosed across intestinal epithelia via a toll-like receptor 2-mediated phagocytosis-multivesicular body-exosome pathway. The absorbed PGN and its derivatives may facilitate maintenance of intestinal immune homeostasis.

Probiotic preparation VSL#3 alters the distribution and phenotypes of dendritic cells within the intestinal mucosa in C57BL/10J mice.

Probiotic nutrients have shown promise in therapy for the treatment of gastrointestinal inflammation, infection, and atopic disease. Intestinal dendritic cells (DC) play a critical role in shaping the intestinal immune response. In this study, we tested the effect of a probiotic preparation (VSL#3) on DC distribution and phenotypes within the intestinal mucosa using a lineage depletion-based flow cytometric analysis. In naïve C57BL/10J mice, intestinal mucosal DC were composed of plasmacytoid DC (pDC) and myeloid DC (mDC). The pDC were the dominant form in lamina propria and Peyer's patches, whereas mDC were the prevailing type in the mesenteric lymph nodes. Additional characterization of pDC and mDC with flow cytometry revealed that they expressed heterogeneous phenotypes in the intestinal mucosa. In mice gavaged with the probiotic VSL#3 for 7 d, the proportion of pDC within the lamina propria was >60% lower, whereas the pDC subset in the mesenteric lymph nodes was more than 200% greater than in sham-treated controls (P < 0.01). Within pDC, the proportion of functionally unique CX3CR1(+) DC was greater than in controls in both the lamina propria and the Peyer's patches (P < 0.01). In contrast to pDC, the mDC number was greater than in controls in all intestinal lymphoid tissue compartments in VSL#3-treated mice (P < 0.01). In conclusion, this study suggests that phenotypically and functionally distinct DC subsets are localized to specific lymphoid tissues within the intestinal mucosa and that the VSL#3 probiotic nutritional supplement alters the distribution of the DC subsets within the intestinal mucosa. These changes may be important in the alteration of mucosal immunity following probiotic VSL#3 therapy.

Milk fat globule-EGF factor 8 mRNA expression in rat splanchnic tissues during postnatal development.

Milk fat globule-EGF factor (MFG-E8) is a protein that binds to alphavbeta3/5 integrin and phosphatidylserine. It plays a role in apoptotic cell removal, tissue remodeling and intestinal epithelial wound-healing process. In the present study, we examined the expression of MFG-E8 mRNA in the small intestine, liver and lungs during postnatal development. Sprague-Dawley rats were sacrificed at different ages (E20, P0, P5, P10 and P21). Total RNA was extracted from various tissues including the small intestine, liver and lungs. The MFG-E8 mRNA expression was quantified by real-time PCR. We found that the MFG-E8 mRNA was expressed at a low level on E20 in the liver. Within 24 hours after birth, its expression was markedly increased. It was then stably expressed at high level during the postnatal period. In contrast, in the small intestine, MFG-E8 mRNA significantly decreased by more than 60% (p<0.001) within 24 hours after birth compared to E20, then it gradually regained E20 values by P10. It was persistently expressed until P21. In the lungs, MFG-E8 is constitutively expressed prenatally at E20. Its expression did not change during the postnatal period. In summary, our study indicated that MFG-E8 is extensively expressed in the small intestine, liver, and lungs. As opposed to other organs, the expression of intestinal MFG-E8 is decreased during the first week of life. It is possible that this could contribute to the predisposition of the neonatal intestine to inflammation.

Milk fat globule-EGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium.

Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.

Lysozyme-modified probiotic components protect rats against polymicrobial sepsis: role of macrophages and cathelicidin-related innate immunity.

Severe sepsis is associated with dysfunction of the macrophage/monocyte, an important cellular effector of the innate immune system. Previous investigations suggested that probiotic components effectively enhance effector cell functions of the immune system in vivo. In this study, we produced bacteria-free, lysozyme-modified probiotic components (LzMPC) by treating the probiotic bacteria, Lactobacillus sp., with lysozyme. We showed that oral delivery of LzMPC effectively protected rats against lethality from polymicrobial sepsis induced by cecal ligation and puncture. We found that orally administrated LzMPC was engulfed by cells such as macrophages in the liver after crossing the intestinal barrier. Moreover, LzMPC-induced protection was associated with an increase in bacterial clearance in the liver. In vitro, LzMPC up-regulated the expression of cathelicidin-related antimicrobial peptide (CRAMP) in macrophages and enhanced bactericidal activity of these cells. Furthermore, we demonstrated that surgical stress or cecal ligation and puncture caused a decrease in CRAMP expression in the liver, whereas enteral administration of LzMPC restored CRAMP gene expression in these animals. Using a neutralizing Ab, we showed that protection against sepsis by LzMPC treatment required endogenous CRAMP. In addition, macrophages from LzMPC-treated rats had an enhanced capacity of cytokine production in response to LPS or LzMPC stimulation. Together, our data suggest that the protective effect of LzMPC in sepsis is related to an enhanced cathelicidin-related innate immunity in macrophages. Therefore, LzMPC, a novel probiotic product, is a potent immunomodulator for macrophages and may be beneficial for the treatment of sepsis.