Steven E. Domino

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.

Polarity Proteins Control Ciliogenesis via Kinesin Motor Interactions

BACKGROUND Cilia are specialized organelles that play a fundamental role in several mammalian processes including left-right axis determination, sperm motility, and photoreceptor maintenance. Mutations in cilia-localized proteins have been linked to human diseases including cystic kidney disease and retinitis pigmentosa. Retinitis pigmentosa can be caused by loss-of-function mutations in the polarity protein Crumbs1 (CRB1), but the exact role of CRB1 in retinal function is unclear. RESULTS Here we show that CRB3, a CRB1-related protein found in epithelia, is localized to cilia and required for proper cilia formation. We also find that the Crumbs-associated Par3/Par6/aPKC polarity cassette localizes to cilia and regulates ciliogenesis. In addition, there appears to be an important role for the polarity-regulating 14-3-3 proteins in this process. Finally, we can demonstrate association of these polarity proteins with microtubules and the microtubular motor KIF3/Kinesin-II. CONCLUSIONS Our findings point to a heretofore unappreciated role for polarity proteins in cilia formation and provide a potentially unique insight into the pathogenesis of human kidney and retinal disease.

Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota

Significance Our data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner. We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1–2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2− mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2+ mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2+ and Fut2− mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2− mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241–4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2− mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241–4247 operon was also seen in humanized Fut2− mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

Deficiency of Reproductive Tract α(1,2)Fucosylated Glycans and Normal Fertility in Mice with Targeted Deletions of the FUT1 or FUT2 α(1,2)Fucosyltransferase Locus

ABSTRACT The fucose α(1→2) galactose β structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation. Fucα(1→2)Galβ moieties and cognate fucosyltransferases are also expressed by epithelial cells of the male reproductive tract and have been implicated in sperm maturation events that may contribute to fertilization. To determine directly if Fucα(1→2)Galβ moieties are required for fertility, we have generated strains of mice that are deficient in genes encoding FUT1 and FUT2, a pair of GDP-l-fucose:β(1→4)-d-galactosyl-R2-α-l-fucosyltransferase enzymes (EC 2.4.1.69 ) responsible for Fucα(1→2)Galβ synthesis and expression. FUT1 null mice and FUT2 null mice develop normally and exhibit no gross phenotypic abnormalities. The Fucα(1→2)Galβ epitope is absent from the uterine epithelia of FUT2 null mice and from the epithelia of the epididymis of FUT1 null mice. Fully normal fertility is observed in FUT1 null intercrosses and in FUT2 null intercrosses. These observations indicate that Fucα(1→2)Galβ moieties are not essential to blastocyst-uterine epithelial cell interactions required for implantation and are not required for sperm maturation events that permit fertilization and that neither the FUT loci nor their cognate fucosylated glycans are essential to normal development.

Ketamine kinetics in unmedicated and diazepam-premedicated subjects

Plasma ketamine concentrations after diazepam and placebo pretreatment were examined in a double‐blind, randomized, cross‐over study. Eight healthy male subjects received either diazepam or a 0.9% NaCl placebo before ketamine and received the alternate combination 5 to 24 days later. Ten minutes before ketamine dosing, diazepam, 0.3 mg/kg, or placebo in equal volume was injected intravenously at a rate not exceeding 5 mg/min. Ketamine, 2.2 mg/kg iv, was injected over 1 min. For the clinically relevant period for anesthesia (1 to 30 min), diazepam‐ketamine treatment resulted in higher plasma levels at most time points, but diazepam pretreatment did not alter plasma levels of metabolite KI and pseudometabolite KII nor the 24‐hr urinary excretion of ketamine, KI, and KII. Ketamine kinetics followed a three‐term exponential decline under both treatment conditions. After placebo‐ketamine dosing, plasma t½s were as follows: distribution (πt½) = 24.1 sec, redistribution (αt½) = 4.68 min, and elimination (βt½) = 2.17 hr. After diazepam‐ketamine dosing, t½s were: πt½ = 25.0 sec, αt½ = 6.37 min, and βt½ = 2.32 hr.

Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues.

Increased Susceptibility of Secretor Factor Gene Fut2-Null Mice to Experimental Vaginal Candidiasis

ABSTRACT Fut2-LacZ-null mice, which are a model of the human ABO and Lewis nonsecretor group, display increased susceptibility to experimental yeast vaginitis, indicating a role for α(1,2)fucosylated cervical glycans in mucosal defense. However, the lack of significant effect of competitive inhibition by exogenous neoglycoproteins in this study emphasizes the complexity of Candida-epithelial cell adhesion events.

Gender-Specific Effects on Gestational Length and Birth Weight by Early Pregnancy BPA Exposure

CONTEXT AND OBJECTIVE Effects of prenatal exposure to bisphenol A (BPA) on gestational and birth outcomes are controversial. The aim of the study was to evaluate the relationship between prenatal exposure to BPA and birth and gestational outcomes. design, setting, participants, and outcome: Levels of unconjugated (uBPA) and BPA glucuronide in 80 matching samples of pregnant women during the first trimester of pregnancy and at delivery and matching term cord blood obtained from a prospective study conducted at the University of Michigan Hospitals were determined using a methodology validated in the National Institutes of Environmental Health Sciences funded Round Robin study and related to pregnancy outcomes. RESULTS Highest levels of uBPA were found in maternal term samples followed by first trimester maternal (M1) samples and cord blood. A 2-fold increase in M1 uBPA was associated with 55-g less birth weight when male and female pregnancies were combined and 183-g less birth weight with only female pregnancies. A 2-fold increase in maternal term uBPA was associated with an increased gestational length of 0.7 days for all pregnancies and 1.1 days for only female pregnancies. CONCLUSION Higher uBPA exposure levels during first trimester and term are associated with sex-specific reduction in birth weight and increase in gestational length, respectively. Race, parity, and employment have an effect on BPA exposure. Because low birth weight is associated with adverse health outcomes, effect of early pregnancy BPA levels on reducing birth weight highlights the risk posed by developmental exposure to BPA.

Molecular Cloning, Genomic Mapping, and Expression of TwoSecretor Blood Group α(1,2)Fucosyltransferase Genes Differentially Regulated in Mouse Uterine Epithelium and Gastrointestinal Tract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal α(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three α(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an α(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fucα1→2Galβ epitopes proposed to mediate blastocyst adhesion.

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyers patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.