Natsumi Takeyama

Innate lymphoid cells regulate intestinal epithelial cell glycosylation

INTRODUCTION The combination of food intake and the resident gut microbiota exposes the gastrointestinal (GI) tract to numerous antigens. Intestinal epithelial cells (ECs) compose a physical barrier separating the internal organs from the gut microbiota and other pathogenic microorganisms entering the GI tract. Although anatomically contained, the gut microbiota is essential for developing appropriate host immunity. Thus, the mucosal immune system must simultaneously maintain homeostasis with the gut microbiota and protect against infection by pathogens. Maintenance of the gut microbiota requires epithelial cell-surface glycosylation, with fucose residues in particular. Epithelial fucosylation is mediated by the enzyme fucosyltransferase 2 (Fut2). Polymorphisms in the FUT2 gene are associated with the onset of multiple infectious and inflammatory diseases and metabolic syndrome in humans. ILC3s regulate epithelial glycosylation. Commensal bacteria, including segmented filamentous bactiera (SFB), induce IL-22 production by ILC3. LT is produced by ILC3 in a commensal bacteria–independent manner. ILC3-derived IL-22 and LT cooperatively induce the production of Fut2 and subsequent epithelial fucosylation, which protects the host against Salmonella typhimurium infection. RATIONALE Despite its importance, the mechanisms underlying epithelial fucosylation in the GI tract is not well understood. In particular, although commensals such as Bacteroides thetaiotaomicron induce epithelial fucosylation, how mucosal immune cells participate in this process is unknown. We used a combination of bacteriological, gnotobiological, and immunological techniques to elucidate the cellular and molecular basis of epithelial fucosylation by mucosal immune cells in mice, especially innate lymphoid cells (ILCs). To explore the role of ILCs in the induction and maintenance of epithelial fucosylation, we used genetically engineered mice lacking genes associated with the development and function of ILCs. To investigate the physiological functions of ILC-induced epithelial fucosylation, we used a Fut2-deficient mouse model of S. typhimurium infection. RESULTS The induction and maintenance of Fut2 expression and subsequent epithelial fucosylation in the GI tract required type 3 ILCs (ILC3s) that express the transcription factor RORγt and the cytokines interleukin-22 (IL-22) and lymphotoxin (LT). Commensal bacteria, including segmented filamentous bacteria (SFB), induced fucosylation of intestinal columnar ECs and goblet cells. Expression of IL-22 by ILC3 required commensal bacteria, whereas LT was expressed in a commensal-independent manner. Ablation of IL-22 or LT in ILC3 resulted in a marked reduction in epithelial fucosylation, demonstrating that both cytokines are critical for the induction and regulation of epithelial fucosylation. Fucosylation of ECs in response to the intestinal pathogen S. typhimurium was also mediated by ILC3. Compared with control mice, Fut2-deficient mice were more susceptible to pathogenic inflammation as a result of S. typhimurium infection, suggesting that epithelial fucosylation contributes to host defense against S. typhimurium infection. CONCLUSION We demonstrate the critical role of the cytokines IL-22– and/or LT-producing ILC3 in the induction and regulation of intestinal epithelial fucosylation. We also show that ILC3-mediated epithelial fucosylation protects the host from invasion of S. typhimurium into the intestine. Our results provide important details of the glycosylation system and homeostatic responses created by the trilateral ILC3–EC–commensal axis in the intestine. Modulation of mucosal immune cell–mediated epithelial glycosylation may provide novel targets for the treatment or prevention of infectious diseases in humans. Immune cells and bugs make a sugary coat Epithelial cells line the intestinal tract and help to keep the peace between our immune system and our trillions of gut microbes. Such peacekeeping requires glycosylated proteins (proteins with attached carbohydrate chains) present on the epithelial cell surface, but how glycosylation occurs is unclear. Goto et al. find that fucosylation (a type of glycosylation) of gut epithelial cells in mice requires gut microbes (see the Perspective by Hooper). This process also requires innate lymphoid cells there, which produce the cytokines interleukin-22 and lymphotoxin, presumably in response to microbial signals. These cytokines signal epithelial cells to add fucose to membrane proteins, which allows the détente between microbes and immune cells to continue. Science, this issue 10.1126/science.1254009; see also p. 1248 Glycosylation of gut epithelial cells requires gut microbes, innate lymphoid cells, and cytokines. [Also see Perspective by Hooper] Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host–microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria–dependent and –independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

Plant-based vaccines for animals and humans: recent advances in technology and clinical trials:

It has been about 30 years since the first plant engineering technology was established. Although the concept of plant-based pharmaceuticals or vaccines motivates us to develop practicable commercial products using plant engineering, there are some difficulties in reaching the final goal: to manufacture an approved product. At present, the only plant-made vaccine approved by the United States Department of Agriculture is a Newcastle disease vaccine for poultry that is produced in suspension-cultured tobacco cells. The progress toward commercialization of plant-based vaccines takes much effort and time, but several candidate vaccines for use in humans and animals are in clinical trials. This review discusses plant engineering technologies and regulations relevant to the development of plant-based vaccines and provides an overview of human and animal vaccines currently under clinical trials.

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies.

AIMS Insulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice. MAIN METHODS To localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2(+/+) mice. IA-2(-/-) mice served as a negative control. KEY FINDINGS Western blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells. SIGNIFICANCE The IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.

Species-specific anti-apoptotic activity of cellular prion protein in a mouse PrP-deficient neuronal cell line transfected with mouse, hamster, and bovine Prnp

The neuroprotective function of prion protein (PrP) was revealed first by the fact that reintroduction of the mouse prion protein gene (Prnp) into a mouse Prnp(-/-) neuronal cell line, HpL3-4, could prevent apoptosis induced by serum deprivation. In the present study, the anti-apoptotic activities of mouse, hamster, and bovine PrP were compared by expressing mouse PrP (MoPrP), hamster PrP (HaPrP), and bovine PrP (BoPrP) in HpL3-4 cells, respectively. Morphological analysis and DNA fragmentation assays demonstrated that HpL3-4 cells expressing HaPrP, BoPrP, and empty vector (EM) showed the typical features of apoptosis with DNA laddering and apoptotic bodies after serum deprivation, whereas HpL3-4 cells expressing MoPrP showed decreased levels of apoptosis in comparison. The levels of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytosol fractions of the cells correlated with the levels of DNA laddering. These results indicate a species-specific anti-apoptotic function of PrP exists, suggesting that the interaction of the mouse PrP with mouse host factors is required for its anti-apoptotic activity.

RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds

Key messageRNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2.AbstractA purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.

MucoRice-cholera Toxin B-subunit, a Rice-based Oral Cholera Vaccine, Down-regulates the Expression of α-Amylase/trypsin Inhibitor-like Protein Family as Major Rice Allergens

To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.

Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in both neonatal and weaned pigs. Because the cholera toxin B subunit (CTB) has a high level of amino acid identity to the ETEC heat-labile toxin (LT) B-subunit (LTB), we selected MucoRice-CTB as a vaccine candidate against ETEC-induced pig diarrhea. When pregnant sows were orally immunized with MucoRice-CTB, increased amounts of antigen-specific IgG and IgA were produced in their sera. CTB-specific IgG was secreted in the colostrum and transferred passively to the sera of suckling piglets. IgA antibodies in the colostrum and milk remained high with a booster dose after farrowing. Additionally, when weaned minipigs were orally immunized with MucoRice-CTB, production of CTB-specific intestinal SIgA, as well as systemic IgG and IgA, was induced. To evaluate the cross-protective effect of MucoRice-CTB against ETEC diarrhea, intestinal loop assay with ETEC was conducted. The fluid volume accumulated in the loops of minipigs immunized with MucoRice-CTB was significantly lower than that in control minipigs, indicating that MucoRice-CTB-induced cross-reactive immunity could protect weaned pigs from diarrhea caused by ETEC. MucoRice-CTB could be a candidate oral vaccine for inducing both passive and active immunity to protect both suckling and weaned piglets from ETEC diarrhea.

Enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice.

Porcine epidemic diarrhea virus (PEDV) is the major causative agent of fatal diarrhea in piglets. To study the pathogenic features of PEDV using a mouse model, PEDV with virulence in mice is required. In pursuit of this, we adapted a tissue-culture-passed PEDV MK strain to suckling mouse brains. PEDV obtained after ten passages through the brains (MK-p10) had increased virulence for mice, and its fusion activity in cultured cells exceeded that of the original strain. However, the replication kinetics of MK and MK-p10 did not differ from each other in the brain and in cultured cells. The spike (S) protein of MK-p10 had four amino acid substitutions relative to the original strain. One of these (an H-to-R substitution at residue 1,381) was first detected in PEDV isolated after eight passages, and both this virus (MK-p8) and MK-p10 showed enhanced syncytium formation relative to the original MK strain and viruses isolated after two, four, and six passages, suggesting the possibility that the H-to-R mutation was responsible for this activity. This mutation could be also involved in the increased virulence of PEDV observed for MK-p10.

Detection of highly pathogenic avian influenza virus infection in vaccinated chicken flocks by monitoring antibodies against non-structural protein 1 (NS1).

H5 and H7 highly pathogenic avian influenza virus (HPAIV) represent a major global concern in poultries and human health. Avian influenza (AI) vaccines are available but not preferred for field applications, primarily because vaccination interferes with sero-surveillances of AIV infection. To overcome the problem, ELISA systems using non-structural protein 1 (NS1) of AIV as antigens (NS1-ELISA) have been developed to measure anti-NS1 antibodies that are raised in AIV-infected but not in vaccinated chickens. However, some AI-vaccinated chickens having a weak anti-virus immune response may subsequently be infected with AIV and spread the virus. This raises a concern for the validity of NS1-ELISA to detect AIV infection in previously vaccinated chickens. In this study, we developed NS1-ELISA and assessed its feasibility to detect HPAIV infection in chickens previously immunized with H5 or H7 AI vaccines. The results indicated that the NS1-ELISA could identify HPAIV infection in both unvaccinated and vaccinated chickens at 1 week after infection in correlation with results from time-consuming virus isolation tests. Taken together, the NS1-ELISA system would be valuable tool to define HPAIV infection when AI vaccine program is in place.