Introduction After birth, body surfaces transit from complete sterility to the densest microbial ecosystem known on Earth. The lower intestine alone is home to nearly 100 trillion microbes, forming a highly complex microbiota with over 1,000 operational taxonomic units (Hooper et al., 2012). Each individual harbors an idiosyncratic microbial consortia (Turnbaugh et al., 2010) that is thought to be shaped by host immunity, environment, and diet (De Filippo et al., 2010; Elinav et al., 2011; Faith et al., 2011; Garrett et al., 2007; Muegge et al., 2011). A sophisticated microbial-host crosstalk shapes immune adaptation and bacterial communities, which underlies mutualism. Intestinal microbes shape not only the neighboring intestinal epithelial cells (Cash et al., 2006; Chassin et al., 2010) but also the majority of sterile body compartments at mucosal and systemic sites (Cahenzli et al., 2013; Ganal et al., 2012; Hooper et al., 2012). The pervasive effect of commensal microbes is reflected by its contribution to health and disease, including inflammatory bowel disease, obesity, malnutrition, autoimmunity, and allergic asthma (Herbst et al., 2011; Markle et al., 2013; Olszak et al., 2012; Smith et al., 2013; Turnbaugh et al., 2009). Commensal microbial communities are thought to be a causal link between westernization and increasing immune disorders (Noverr and Huffnagle, 2005; Okada et al., 2010). Over the past few decades, westernized countries have experienced drastic changes in dietary habits and sanitation, including water decontamination, food pasteurization, sterilization, and uninterrupted cold chain delivery, vaccination, and widespread antibiotic use. All these factors have contributed to decreased infectious diseases (Bach, 2002). Over this period of improved hygiene, autoimmune and atopic allergic disorders have increased in incidence; an epidemiologic effect postulated to result from decreased acute infections during early childhood (Holt, 1998; Martinez and Holt, 1999; Strachan, 1989) or from a shift in commensal microbial communities (Braun-Fahrländer et al., 1999; Noverr and Huffnagle, 2005; Okada et al., 2010). Certainly, the western lifestyle shapes the composition of commensal bacterial consortia and their development over time (De Filippo et al., 2010; Koenig et al., 2011; Palmer et al., 2007; Walter and Ley, 2011). Although some mechanistic links between microbiota-induced immune regulation have been experimentally elucidated (Markle et al., 2013; Olszak et al., 2012), most models depend on particular microbes triggering disease (Elinav et al., 2011; Garrett et al., 2007). IgE antibodies play a central role in atopic allergic disease and immunity to parasites (Allen and Maizels, 2011; Gould and Sutton, 2008; Paul and Zhu, 2010). Healthy individuals maintain serum IgE concentrations at basal levels ( 90%) and L. murinus ASF361 (4%) are the two most abundant species present (high-throughput 16S amplicon sequencing) (data not shown). This suggests that increasing early-life diversity by adding less abundant additional species may already be sufficient to mediate protection from hyper-IgE. Elevated IgE Levels in Germ-free Mice Lead to Increased Amounts of Surface-Bound IgE on Mast Cells and Exaggerated Oral-Induced Systemic Anaphylaxis Given that IgE is known to regulate mast cell homeostasis (Kalesnikoff et al., 2001; Kitaura et al., 2003), we investigated whether the elevated levels of IgE in germ-free mice could alter mast-cell-mediated pathologies. We found that the amount of surface-bound IgE was significantly increased in peritoneal mast cells from germ-free and bicolonized mice in comparison to SPF mice (Figure 7A). Interestingly, cell-surface levels of CD117 (c-kit) were also increased on mast cells from germ-free and bicolonized mice in comparison to SPF mice, which may hint to a more immature phenotype (Boyce, 2004). Next, we evaluated whether tissue mast cell numbers were increased because of the elevated IgE levels in germ-free mice. The number of cutaneous mast cells, as identified by chloroacetate esterase (CAE) staining in the ear tissue of germ-free mice, was slightly, although not significantly, elevated under steady-state germ-free conditions in comparison to SPF conditions (Figure 7B). In addition, the reduced number of mast cells in the ear skin of SPF animals showed weaker CAE staining (Figure 7B). We investigated whether elevated IgE levels in germ-free mice have an impact on the severity of active antigen-induced anaphylaxis. It has been reported that gastrointestinal, cutaneous, and cardiovascular symptoms in oral antigen-induced anaphylaxis are dependent on IgE and mast cells (Ahrens et al., 2012), whereas, in systemic antigen-induced anaphylaxis, these adverse symptoms are mediated by both IgE and IgG pathways (Osterfeld et al., 2010). Germ-free and SPF BALB/c mice were systemically primed with OVA and aluminum potassium sulfate (AIK(SO4)2-12H2O) (Alum) or Alum alone. Mice were challenged 2 weeks later by intravenous injection of ovalbumin (OVA; active systemic anaphylaxis) or by oral gavage of OVA (active oral anaphylaxis). In response to systemic antigen challenge, germ-free mice developed hypothermia to the same level as SPF animals (Figure 7C). In contrast, germ-free mice showed significantly increased susceptibility to oral antigen-induced anaphylaxis (Figure 7C). Bicolonized C57BL/6 mice also displayed increased susceptibility to active oral anaphylaxis in comparison to SPF C57BL/6 mice (Figure S3). These data suggest that increased serum IgE levels in germ-free mice impact on mast cell homeostasis, which, in turn, leads to greatly increased sensitivity to oral-induced anaphylaxis. Discussion The hygiene hypothesis postulated that the quality and/or quantity of microbial exposure early in life might have an important impact on how the immune system behaves later in life (Strachan, 1989). Although the original hygiene hypothesis did not suggest a role for the intestinal commensal bacteria, in the past years, it has become clear that our intestinal inhabitants have a powerful influence on immune maturation at both mucosal and systemic sites. Systematic characterization of intestinal microbial communities in human babies has indicated that incidental environmental exposure plays a major role in determining the microbial community in each baby (Koenig et al., 2011; Palmer et al., 2007). Along with westernization, major changes in the environment have occurred that are likely to heavily influence the composition of the intestinal microbiota. Recent studies have also shown that children living in developing countries harbor greater intestinal bacterial diversity than children in developed countries (De Filippo et al., 2010; Lin et al., 2013). Decreased bacterial diversity and/or altered composition of the intestinal microbiota early in life are likely to contribute to the increased susceptibility to immune-mediated diseases in westernized countries. Using gnotobiotic mouse models, we provide experimental evidence that limited microbial association below a certain threshold of complexity or devoid of putative bacterial species or bacterial consortia with immunoregulatory properties during early life has profound negative and lasting effects on the induction of immune regulation, even when microbial diversity is increased above this threshold later in life. These data demonstrate the presence of a critical time window early in life during which appropriate microbial exposure has to occur in order to induce functional, life-long immune regulation that maintains serum IgE levels at baseline. In addition to allergic disorders and certain helminth infections, elevated IgE has been suggested to be a biomarker for primary immunodeficiencies (Liston et al., 2008). Data from numerous mouse models of genetic immunodeficiency suggest that reduced functional T cell populations may lead to immune dysregulation and elevated IgE levels as a consequence. There are most likely many pathways involved in the immune regulatory network that function to maintain IgE at low levels. The development of hyper-IgE in genetically immunocompetent germ-free mice indicates that exposure to commensal bacteria is a key factor in the development of a functional immune regulatory network. Germ-free mice also show elevated invariant natural killer T cells at mucosal sites (Olszak et al., 2012), which trigger heightened pathology in models of inflammatory bowel disease and allergic asthma. In this study, we show that elevated serum IgE titers leads to increased levels of surface-bound IgE on mast cells, and germ-free mice displayed exacerbated antigen-induced oral anaphylaxis in comparison to SPF mice, implying that dysregulated immune reactions such as food allergies may be highly dependent on adequate acquisition of bacterial consortia at early age. The fact that hygiene-mediated IgE does not require an underlying genetic immunodeficiency suggests that exposure to a diverse microbial population during a critical time window early in life is absolutely required in order to set the baseline immune regulatory state for life. It is interesting that isotype switch to IgE has been recently shown to be favored in immature B cells (Wesemann et al., 2011). Although the maturation state of the B cells undergoing IgE class switch was not investigated in the current study, B cells were found to switch to IgE in mucosal tissues, especially PP, and not in B cell developmental sites, such as bone marrow or spleen. Additional studies are required to carefully investigate the impact of microbial exposure on lymphocyte development and function. Although IgE alone does not constitute allergy and the simplicity of a bicolonization does not necessarily represent the restricted microbiota resulting from improved human hygiene, our results do show that diverse microbial colonization during a critical time window in neonatal life is required in order to limit default immune pathways, resulting in excessive IgE levels, mirroring one possible immune component for rationalizing the hygiene hypothesis. Experimental Procedures Mouse Strains, Hygiene Status, and Colonization Germ-free Mice C57BL/6, TCRβδ−/−, JH −/−, Rag1 −/− , TSLPR−/− (a kind gift from N. Harris [École Polytechnique Fédérale de Lausanne]) (all on a C57BL/6 background), NIH-Swiss, BALB/c, Swiss Webster, and NMRI mice were rederived to germ-free status via two-cell embryo transfer and bred and maintained in flexible-film isolators as described previously (Smith et al., 2007). Antigen-free Mice Germ-free mice were fed an irradiated (5M Rad Co-60 for 20 hr) elemental antigen-free diet consisting of extensively hydrolyzed proteins supplemented with fats, vitamins, and minerals (Pregestimil, Enfamil). Bedding consisted of endotoxin-free sand (baked at 250°C for 30 min). Gnotobiotic Mice Gnotobiotic mice associated with altered Schaedler flora (ASF) (Dewhirst et al., 1999) were originally obtained from germ-free mice cohoused with an ASF colonizer. For the generation of a bicolonized colony, germ-free C57BL/6 breeding pairs were orally gavaged with pure cultures of P. distasonis and L. murinus. Serum samples from low-complexity microbiota mice (Stecher et al., 2010; Endt et al., 2010) were kindly provided by W.-D. Hardt (Eidgenössische Technische Hochschule Zürich). E.coli HA107 was cultured and prepared for gavage of germ-free mice as described previously (Hapfelmeier et al., 2010), and 2 × 109−1010 CFU HA107 was administered by oral gavage. Maintenance of gnotobiotic mice is described in the Supplemental Information. SPF Mice C57BL/6, BALB/c, and TCRβδ−/− mice were purchased from Taconic or maintained in the central animal facilities at McMaster University or the University of Bern. MHC II−/− and IL-4Rα−/− mice were a kind gift from N. Harris. For SPF colonization experiments, germ-free mice were cohoused with an SPF mouse in individually ventilated cages. All animal experiments were carried out in accordance with the McMaster University animal utilization protocols and the Canadian Council on Animal Care guidelines or in accordance with Swiss federal regulations. Isotype-Specific ELISA for the Detection of Serum Antibodies Blood was collected in serum-separating tubes and total serum IgE, IgA, IgM, IgG1, IgG2a, IgG2c, and IgG3 concentrations determined by sandwich ELISA as described in the Supplemental Information. Cell Isolation and Quantitative Real Time PCR In order to obtain single-cell suspensions, lymph nodes (pLNs, PPs, and MLNs) were digested with 0.14 Wünsch U/ml Liberase C1 or Liberase TL (Roche Applied Science). Then, lymphoid tissues (spleen, pLNs, MLNs, and PPs) were homogenized and filtered through a 40 μm cell strainer. Lymphocytes from cLP were isolated as described previously (Geuking et al., 2011). Cells were resuspended in TRIzol reagent (Invitrogen) and RNA isolated according to the manufacturer’s instructions. qPCR was performed with SYBR Green Supermix or SsoFast EvaGreen Supermix (Bio-Rad) with specific primer pairs (see Table S1 and the Supplemental Information). Genomic DNA was isolated from cecal contents or fecal pellets with the QIAamp DNA Stool Mini Kit (Qiagen) and analyzed with SsoFast EvaGreen Supermix (Bio-Rad) with the use of primers specific for the 16S rRNA genes of the individual ASF species (see Table S2). PCR and analysis were performed on an iQ5 or CFX384 (Bio-Rad) platform and software. Flow Cytometry and Flow Cytometry Sorting Antibodies and corresponding clones used for flow cytometry are listed in the Supplemental Information. Dead cells were excluded with live/dead fixable dye eFluor 506 (eBioscience) and Fc receptors blocked with CD16/CD32 Fc blocking antibody. Data were acquired on a FACSCalibur (BD Biosciences) or LSRII (BD Biosciences) and analyzed with FlowJo (Tree Star). For FACS sorting, splenocytes were enriched for CD4+ T cells or CD19+ B cells (>80% of lymphocytes) with CD19 or CD4 magnetic beads (Miltenyi Biotec) and sorted for IgM+CD19+B220+CD3− (naive B cells) or CD3+CD25−CD45RBhigh (naive CD4+ T cells) populations on a FACSAria (BD Biosciences). Axenic In Vivo Administration of mAb Twice a week, germ-free mice (28 days old) were administered (200 μg intraperitoneal injected) one of the following sterile mAbs: anti-CD4 (clone YTS191.1.2), anti-iL-4 (clone 11B11), anti-TSLP (clone 28F12, a kind gift from A. Farr [University of Washington]), or isotype control (35.61). In order to abrogate PP ontogeny in vivo, anti-iL7Rα (3 mg, clone A7R34, Bio X Cell) was administered (intravenous [i.v.] injection) to germ-free pregnant mice at E14.5–E15.5. The absence of PP and germ-free status was confirmed at the end of each experiment. Axenic Adoptive Cell Transfer For cotransfer of purified naive B and T cells, 1 × 106 FACS-sorted splenic B and T cells were i.v. injected into germ-free Rag1 −/− recipients. B cells were purified from WT C57BL/6, IL-4Rα−/−, or MHC II-deficient mice. Mast Cell Quantification Samples were fixed in 10% formalin and processed by standard histological techniques prior to paraffin embedding. Then, 6 μm sections were deparaffinized and stained with an α-Naphthyl Chloroacetate Esterase Kit (Sigma-Aldrich) according to the manufacturer’s instructions. The sections were counterstained with Gill’s No.3 Hematoxylin Solution for 30 s. Antigen-Induced Oral and Systemic Anaphylaxis Germ-free, bicolonized, or SPF mice were subcutaneously injected with 50 μg of ovalbumin (Sigma-Aldrich) in the presence of 2 mg of Alum adjuvant (Sigma- Aldrich) in sterile PBS or with Alum alone. Two weeks later, mice received either an oral (50 mg/250 μl PBS) or systemic (100 μg ovalbumin in 500 μl PBS) ovalbumin challenge. For the oral challenge, mice were deprived of food for 4 hr prior to gavage. Rectal temperatures were monitored every 5–10 min for 90 min with a TCAT-2LV animal temperature controller (Physitemp Instruments).
