Di Meng

The Mechanism of Excessive Intestinal Inflammation in Necrotizing Enterocolitis: An Immature Innate Immune Response

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

Probiotics prevent necrotizing enterocolitis by modulating enterocyte genes that regulate innate immune-mediated inflammation.

Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1β-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.

Glucocorticoids and microbiota regulate ontogeny of intestinal fucosyltransferase 2 requisite for gut homeostasis

At weaning, the intestinal mucosa surface glycans change from predominantly sialylated to fucosylated. Intestinal adaptation from milk to solid food is regulated by intrinsic and extrinsic factors. The contribution by glucocorticoid, an intrinsic factor, and colonization by microbiota, an extrinsic factor, was measured as the induction of α1,2/3-fucosyltransferase and sucrase-isomaltase (SI) activity and gene expression in conventionally raised, germ-free, and bacteria-depleted mice. In conventionally raised mice, cortisone acetate (CA) precociously accelerated SI gene expression up to 3 weeks and fut2 to 4 weeks of age. In germ-free mice, CA treatment induces only SI expression but not fucosyltransferase. In post-weaning bacteria-deficient (germ-free and bacteria-depleted) mice, fut2 expression remains at low suckling levels. In microbiota deficient mice, intestinal fut2 (but not fut1, fut4 or fut7) was induced only by adult microbiota, but not immature microbiota or CA. Fut2 induction could also be restored by colonization by Bacteroides fragilis, but not by a B. fragilis mutant unable to utilize fucose. Restoration of fut2 expression (by either microbiota or B. fragilis) in bacteria-depleted mice is necessary for recovery from dextran sulfate sodium-induced mucosal injury. Thus, glucocorticoids and microbes regulate distinct aspects of gut ontogeny: CA precociously accelerates SI expression and, only in colonized mice, fut2 early expression. The adult microbiota is required for the fut2 induction responsible for the highly fucosylated adult gut phenotype and is necessary for recovery from intestinal injury. Fut2-dependent recovery from inflammation may explain the high incidence of inflammatory disease (Crohns and necrotizing enterocolitis) in populations with mutant FUT2 polymorphic alleles.

Mast cell-induced lung injury in mice infected with H5N1 influenza virus

ABSTRACT Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.

Exploring the interplay of barrier function and leukocyte recruitment in intestinal inflammation by targeting fucosyltransferase VII and trefoil factor 3

Intestinal mucosal integrity is dependent on epithelial function and a regulated immune response to injury. Fucosyltransferase VII (Fuc-TVII) is an essential enzyme required for the expression of the functional ligand for E- and P-selectin. Trefoil factor 3 (TFF3) is involved in both protecting the intestinal epithelium against injury as well as aiding in wound repair following injury. The aim of the present study was to assess the interplay between barrier function and leukocyte recruitment in intestinal inflammation. More specifically, we aimed to examine how targeted disruption of Fuc-TVII either in wild-type or TFF3(-/-) mice would alter their susceptibility to colonic injury. TFF3 and Fuc-TVII double-knockout mice (TFF3/Fuc-TVII(-/-) mice) were generated by mating TFF3(-/-) and Fuc-TVII(-/-) mice. Colitis was induced by administration of dextran sodium sulfate (DSS) (2.5% wt/vol) in the drinking water. Changes in baseline body weight, diarrhea, and fecal blood were assessed daily. Upon euthanasia, extents of colonic inflammation were assessed macroscopically, microscopically, and through quantification of myeloperoxidase (MPO) activity. Colonic lymphocyte subpopulations were assessed at 6 days after administration of DSS by flow cytometry and immunohistochemistry. No baseline intestinal inflammation was found in TFF3/Fuc-TVII(-/-), TFF3(-/-), Fuc-TVII(-/-), or wild-type mice. Loss of Fuc-TVII resulted in a reduction in disease severity whereas TFF3(-/-) mice were markedly more susceptible to DSS-induced colitis. Remarkably, the loss of Fuc-TVII in TFF3(-/-) mice markedly decreased the severity of DSS-induced colitis as evidenced by reduced weight loss, diarrhea, decreased colonic MPO levels and improved survival. Furthermore, the loss of TFF3 resulted in increased severity of spontaneous colitis in IL-2/beta-microglobulin-deficient mice. These studies highlight the importance of the interplay between factors involved in the innate immune response, mucosal barrier function, and genes involved in regulating leukocyte recruitment and other aspects of the immune response.

Toll-like receptor-4 in human and mouse colonic epithelium is developmentally regulated: a possible role in necrotizing enterocolitis

Background:Necrotizing enterocolitis (NEC) is an immature intestinal condition resulting in devastating intestinal inflammation due to unknown mechanisms. Evidence has suggested that intestinal maturation attenuates the severity of NEC and Toll-like receptor 4 (TLR4) has been suggested to play a critical role in its pathogenesis. We investigated whether maturational effects of TLR4 expression in immature colon might contribute to the development of NEC.Methods:TLR4 colonocyte expression was detected by immunofluorescence confocal microscopy. Interleukin-6 (IL-6) levels were assayed by an enzyme-linked immunosorbent assay (ELISA).Results:TLR4 expression was high in fetal colonic epithelium in human and mouse, with earlier gestation having a higher surface/cytoplasm distribution. TLR4 remained high in mouse postnatal day 1 but the surface/cytoplasm distribution was reduced. TLR4 decreased in amount and then was expressed in crypts in the mature human and mouse colon. Hydrocortisone (HC) reduced the surface/cytoplasm distribution of TLR4 in human fetal colon. Elevated IL-6 levels in immature colon after lipopolysaccharide were attenuated by HC in human and mouse.Conclusion:Expression, localization, and signaling of TLR4 in colonic epithelium may be developmentally regulated. HC may accelerate the TLR developmental pathway change to an adult type, which may account for its impact on TLR4 signaling.

Apoptosis and pro-inflammatory cytokine response of mast cells induced by influenza A viruses.

The pathogenesis of the influenza A virus has been investigated heavily, and both the inflammatory response and apoptosis have been found to have a definitive role in this process. The results of studies performed by the present and other groups have indicated that mast cells may play a role in the severity of the disease. To further investigate cellular responses to influenza A virus infection, apoptosis and inflammatory response were studied in mouse mastocytoma cell line P815. This is the first study to demonstrate that H1N1 (A/WSN/33), H5N1 (A/Chicken/Henan/1/04), and H7N2 (A/Chicken/Hebei/2/02) influenza viruses can induce mast cell apoptosis. They were found to do this mainly through the mitochondria/cytochrome c-mediated intrinsic pathway, and the activation of caspase 8-mediated extrinsic pathway was here found to be weak. Two pro-apoptotic Bcl-2 homology domain 3 (BH3) -only molecules Bim and Puma appeared to be involved in the apoptotic pathways. When virus-induced apoptosis was inhibited in P815 cells using pan-caspase (Z-VAD-fmk) and caspase-9 (Z-LEHD-fmk) inhibitors, the replication of these three subtypes of viruses was suppressed and the secretions of pro-inflammatory cytokines and chemokines, including IL-6, IL-18, TNF-α, and MCP-1, decreased. The results of this study may further understanding of the role of mast cells in host defense and pathogenesis of influenza virus. They may also facilitate the development of novel therapeutic aids against influenza virus infection.

Chronic Heat Stress Inhibits Immune Responses to H5N1 Vaccination through Regulating CD4 + CD25 + Foxp3 + Tregs

Chronic heat stress (CHS) is known to have negative impacts on the immune responses in animals and increases their susceptibility to infections including the highly pathogenic avian influenza virus H5N1. However, the role of regulatory T cells (Tregs) in CHS immunosuppression remains largely undefined. In this study, we demonstrated a novel mechanism by which CHS suppressed both Th1 and Th2 immune responses and dramatically decreased the protective efficacy of the formalin-inactivated H5N1 vaccine against H5N1 influenza virus infection. This suppression was found to be associated with the induced generation of CD4+CD25+FoxP3+ Tregs and the increased secretions of IL-10 and TGF-β in CD4+ T cells. Adoptive transfer of the induced Tregs also suppressed the protective efficacy of formalin-inactivated H5N1 virus immunization. Collectively, this study identifies a novel mechanism of CHS immunosuppression mediated by regulating CD4+CD25+Foxp3+ Tregs.

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking. Methodology/Principal Findings Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8+ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8+ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8+ T cells. Conclusions/Significance Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.

Secretions of Bifidobacterium infantis and Lactobacillus acidophilus Protect Intestinal Epithelial Barrier Function

Objectives: The secreted metabolites of probiotics are cytoprotective to intestinal epithelium and have been shown to attenuate inflammation and reduce gut permeability. The present study was designed to determine the protective effects of probiotic conditioned media (PCM) from Bifidobacterium infantis (BCM) and Lactobacillus acidophilus (LCM) on interleukin (IL)-1&bgr;–induced intestinal barrier compromise. Methods: The epithelial barrier was determined by measuring the transepithelial electrical resistance (TER) across a Caco-2 cell monolayer using a Transwell model. The paracellular permeability was determined by fluorescein isothiocyanate–labeled dextran flux. The expression of tight junction (TJ) proteins and nuclear factor-kappa B (NF-&kgr;B) p65 were determined using Western blot and the distribution of NF-&kgr;B p65 was determined by immunofluorescence staining. Results: BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation (P < 0.05). The maximal increase of Caco-2 TER occurred at 4 hours of treatment with a PCM concentration of 15%. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1&bgr;. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers (P < 0.05). IL-1&bgr; stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells (P < 0.05), which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1&bgr; stimulation; however, a similar response in occludin was not demonstrated. The IL-1&bgr;–induced nuclear translocation of NF-&kgr;B p65 in Caco-2 cells was prevented by pretreatment with both PCMs. Conclusions: BCM and LCM protected the intestinal barrier against IL-1&bgr; stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1&bgr;–induced NF-&kgr;B activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.