Commensal DNA Limits Regulatory T Cell Conversion and Is a Natural Adjuvant of Intestinal Immune Responses

Immunity

Article Commensal DNA Limits Regulatory T Cell Conversion and Is a Natural Adjuvant of Intestinal Immune Responses Jason A. Hall,1,2,5 Nicolas Bouladoux,1,5 Cheng Ming Sun,1 Elizabeth A. Wohlfert,1 Rebecca B. Blank,1 Qing Zhu,3 Michael E. Grigg,4 Jay A. Berzofsky,3 and Yasmine Belkaid1,* 1Mucosal Immunology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA 2Immunology Graduate group, University of Pennsylvania, Philadelphia, PA 19104, USA 3Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 4Molecular Parasitology Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA 5These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.immuni.2008.08.009

SUMMARY

The intestinal tract is in intimate contact with the commensal microflora. Nevertheless, how commensals communicate with cells to ensure immune homeostasis is still unclear. In this study, we found that gut flora DNA (gfDNA) plays a major role in intestinal homeostasis through Toll-like receptor 9 (TLR9) engagement. Tlr9/ mice displayed increased frequencies of CD4+Foxp3+ regulatory T (Treg) cells within intestinal effector sites and reduced constitutive IL-17- and IFN-g-producing effector T (Teff) cells. Complementing this, gfDNA limited lamina propria dendritic cell-induced Treg cell conversion in vitro. Further, Treg/Teff cell disequilibrium in Tlr9/ mice led to impaired immune responses to oral infection and to oral vaccination. Impaired intestinal immune responses were recapitulated in mice treated with antibiotics and were reversible after reconstitution with gfDNA. Together, these data point to gfDNA as a natural adjuvant for priming intestinal responses via modulation of Treg/Teff cell equilibrium. INTRODUCTION Immune reactivity against intestinal flora or dietary antigen poses a substantial risk to the host and can lead to severe tissue damage (Izcue et al., 2006). To avoid this consequence, the host has a multitude of complementary regulatory mechanisms in place. Nevertheless, inability to overcome these regulatory mechanisms in the face of an invasive pathogen may compromise effective immunity. Lending weight to this paradox is the fact that commensal and pathogenic microbes interact with the host immune system through similar conserved ligands that are cardinal features of microorganisms (Sansonetti and Di Santo, 2007). Many of these ligands signal through the Toll-like family of receptors (TLRs) (Sansonetti and Di Santo, 2007). TLRs are widely expressed by cells of hematopoietic origin, as well as nonhematopoietic cells, including the epithelial cells lining the in-

testinal tract (Takeda et al., 2003). The initial identification of TLRs in the splenic and peripheral-blood-leukocyte compartments paved our understanding of how engagement of these molecules by invasive pathogens can activate inflammatory cascades that initiate adaptive immunity (Medzhitov et al., 1997). However, mucosal tissues via their interaction with commensals are the only environments in constant contact with TLR ligands. Yet, the purpose of TLR signaling by the commensal flora has only recently begun to be elucidated. For example, it is now clear that TLR signaling in the intestinal epithelial compartment is crucially involved in the maintenance of intestinal homeostasis and tissue repair (Rakoff-Nahoum et al., 2004). Further, these signals also positively regulate the sampling of lumenal contents by dendritic cells (DCs) from the underlying lamina propria compartment (Chieppa et al., 2006). Commensal floral interactions with TLRs have also been shown to mediate tolerance to food antigens (Bashir et al., 2004). CD4+ T cells of the Foxp3+ regulatory lineage (Treg cells) also mediate intestinal homeostasis. Their absence or failure to properly patrol the gut-associated lymphoid tissues (GALTs) leads to reactivity against the commensal flora and subsequent colitis (Izcue et al., 2006). We and others have shown that naive T cells can become Foxp3+ Treg cells after oral exposure to antigen (Coombes et al., 2007; Mucida et al., 2005; Sun et al., 2007). Although thymically derived Treg cells are essential for this protection, converted Treg cells formed in the GALTs after naive CD4+ T cell encounter with antigen may provide additional protection. This process was associated with the capacity of GALT antigen-presenting cells (APCs) to generate Treg cells via a mechanism that, in addition to TGF-b, is dependent on the vitamin A metabolite retinoic acid (Coombes et al., 2007; Denning et al., 2007; Sun et al., 2007). Previous work has demonstrated that TLR signaling can influence both the function and expansion of Treg cells (Sutmuller et al., 2006b). In some instances, this control is exerted via direct TLR engagement on Treg cells, whereas in others it occurs through T effectors (Teff) cells and/or APC stimulation. Although some interactions have been proposed to increase Treg cell activity, including TLR4 and TLR5 (Caramalho et al., 2003; Crellin et al., 2005), others involving TLR2, TLR8 and TLR9 have been shown to limit Treg cell function (Liu et al., 2006; Pasare and Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc. 637

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Medzhitov, 2003; Peng et al., 2005; Sutmuller et al., 2006a). TLR stimulation of Treg cells may also influence their proliferation (Liu et al., 2006; Sutmuller et al., 2006a). Putting these elements together, we find that the constant exposure of the intestinal immune system to the flora provides a rationale to consider the physiological impact of TLR signaling on the presence and function of Treg cells in the GALTs. TLR9 recognizes unmethylated cytosine phosphate guanosine (CpG) dinucleotides, which are abundant in prokaryotic DNA found in intestinal flora. With synthesized sequences containing CpG, previous studies have shown that engagement of TLR9 expressed on not just DCs but also on Treg and Teff cells can limit Treg cell’s suppressive function (Larosa et al., 2007; Pasare and Medzhitov, 2003). We have recently demonstrated that the presence of CpG at the time of Leishmania infection can limit accumulation of Treg cells in the infected dermis (Wu et al., 2006). Previous work identified an association between Crohn’s disease and a promoter polymorphism in the TLR9 gene in humans (Torok et al., 2004). Such an association supports the idea of a role for gut floral DNA (gfDNA) sensing in the pathophysiology of inflammatory bowel diseases (IBDs). However, whether host interaction between ligands expressed on gfDNA and TLR9 can influence Treg cells and subsequently local immune responses in the gut is unknown. In the present study, we investigated how constitutive interaction between gfDNA and TLR9 in the gut would influence intestinal immune responses. We addressed this by using a model of oral infection, microsporidia, and a model of oral vaccination, OVA, with the mutant form of Escherichia coli labile toxin (LT) as mucosal adjuvant (Chong et al., 1998). We found that in intestinal tissues, gfDNA acts as a natural immunological adjuvant and critically controls the balance between Treg and Teff cell frequency and function. This reveals a previously unidentified aspect of gut flora in the regulation of mucosal immunity. Such control appears to be essential for development of effective immune responses against invading microorganisms as well as mucosally administered vaccines and correlates with restricted peripheral Treg cell conversion. RESULTS TLR9 Signaling Regulates Treg/Teff Cell Ratio in Intestinal Tissues To address the role of TLR9 signaling in Treg cell homeostasis in the GALTs, we first compared Treg cell populations in the peripheral tissues and the GI tract of Tlr9/ and wild-type (WT) mice raised in the same animal facilities. With the exception of the mesenteric lymph nodes (MLNs), naive Tlr9/ mice had higher percentages and absolute numbers of Treg cells than their WT counterparts in most of the GALTs, including the intestinal epithelium lymphocyte (IEL), small intestinal lamina propria (LP), and Peyer’s patch (PP) compartments (Figures 1A and 1B and Figure S1A available online). These differences held in mice housed in the same cage. Moreover, differences were restricted to the small intestine compartment in light of the fact that Treg cell frequencies were similar in the spleen, MLN, and the large intestine LP of WT and Tlr9/ mice (Figures 1A and 1B). These elevated Treg cell frequencies did not appear to be caused by an ongoing reduction in Teff cells proliferation compared to Treg cells proliferation in Tlr9/ mice as assessed by staining for 638 Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc.

the nuclear-proliferation antigen, Ki-67 (Figure S1C). We did not observe this alteration in Foxp3+ expression patterns in either Tlr4/ or Tlr2/ mice (data not shown). The enhanced Treg cell frequencies found in the effector small intestine sites of Tlr9/ mice indicated that basal cytokine production by Teff cells might also be affected in these tissues. Because most conventional ab T cells are CD4+ in the LP and CD8+ in the IEL, we were specifically interested in cytokine production by these two subsets. Tlr9/ and WT mice have similar numbers of CD4+ and CD8+ T cells in these respective compartments (data not shown). Within the LP, we noted a significant reduction in the percentage and absolute number (Figures 1C and 1D) of CD4+ T cells producing IFN-g and/or IL-17 in the LP of Tlr9/ mice. IL-10 production was less affected (Figure 1C). Similarly, within the IEL, the proportion and absolute number of CD8+ T cells producing IFN-g were dramatically reduced in Tlr9/ mice (Figures 1C and 1D). Thus, in the absence of TLR9 signaling, a tissue specific disequilibrium of T cell subsets in the gut emerges, in which Treg cell frequencies are augmented and constitutive inflammatory cytokine production by Teff is reduced. TLR9 Signaling Is Requisite for Optimal Responses to Oral Infection and Vaccination We reasoned that the concomitant gain of Treg cell and loss of cytokine producing Teff in the gut tissues of Tlr9/ mice could influence their ability to mount protective immune responses during gastrointestinal infection. Microsporidia are obligate intracellular parasites capable of infecting a wide range of hosts through the alimentary tract (Didier, 2005). Infection with the species Encephalitozoon cuniculi (E. cuniculi) produces a robust IFN-g response that is required for protection against oral as well as intraperitoneal (i.p.) challenge in mice (Khan and Moretto, 1999; Moretto et al., 2004). To address this possibility, we took advantage of E. cuniculi’s ability to induce productive immune responses through both i.p. and oral routes of infection. Tlr9/ mice infected i.p. with E. cuniculi-mounted protective responses that were comparable to WT cohorts, on the basis of ELISA measurement of IFN-g secretion during antigen recall and parasite burden 11 days after infection (p.i.) (Figures 2A and 2B). Thus, recognition by TLR9 is not required for peripheral immunity to the parasite. Conversely, immune responses in orally infected Tlr9/ mice were severely impaired when compared to WT mice (Figures 2C and 2D). Recall responses revealed that antigen-specific IFN-g production was severely impaired in the IEL and LP of orally infected Tlr9/ mice (Figure 2C). Moreover, whereas restimulation of splenocytes from WT mice resulted in large secretion of IFN-g, the amount of IFN-g secreted by splenocytes from Tlr9/ mice was reduced by 75% (Figure 2C). IL-17 secretion was also significantly impaired in all tested tissues of infected Tlr9/ mice (Figure 2C). In accord with a defect in proinflammatory cytokine secretion, parasite burdens were significantly higher in orally infected Tlr9/ than in WT cohorts, both in the primary site (duodenum) and dissemination site (liver) (Figure 2D). This difference was associated with higher systemic concentration of IL-10 and IL-4 (Figures S1D and S1E), although we did not observe any bias toward a T helper 2 cell (Th2) response in the GALTs (Figure S1D). The increased Treg cell frequency within the IEL, LP, and PP persisted in orally infected Tlr9/ mice

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 1. TLR9 Signaling Regulates Treg Cell Frequency and Steady-State Teff Cell Cytokine Production in Gut-Associated Lymphoid Tissues of Uninfected Mice (A) Comparative assessment of CD4+Foxp3+ Treg cells in spleen, mesenteric lymph node (MLN), intestinal epithelium lymphocyte (IEL), intestinal lamina propria (LP), and Peyer’s patch (PP) compartments in age-matched naive WT and Tlr9/ mice. CD4+TCR-b+-gated cells were analyzed for expression of Foxp3 and CD25 by flow cytometry. Numbers in quadrants refer to the percentage of each subset. (B) Treg cell percentages in naive WT (closed circle) and Tlr9/ (open circle) mice. Each dot represents the results from one experiment (three mice pooled per group), and crossbars depict the mean of three independent experiments (*p < 0.05 compared with WT mice). (C) Loss of TLR9 reduces the basal frequency of IL-17- and IFN-g-producing CD4+ T cells in the LP and IFN-g-producing CD8a+ T cells in the IEL compartments. Numbers in quadrants refer to the percentage of each subset. (D) Absolute numbers of CD4+ and/or CD8a+ T lymphocytes producing IFN-g and IL-17 in naive WT (closed circle) and Tlr9/ (open circle) mice. Each dot represents one mouse and each bar represents the mean of three mice analyzed (*p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice). For (C) and (D), data shown are representative of two independent experiments with similar results.

when assessed on day 11 p.i. (Figure S1E). Thus, these data support the idea that augmented gut Treg/Teff cell ratios in Tlr9/ mice and/or defects in Teff cell priming may predispose them to impaired mucosal immune responses during oral challenges. To further probe the impaired intestinal immune responses in Tlr9/ mice, we employed an oral-vaccination model in which mice were gavaged with a mixture of OVA and the mucosal adjuvant, LT(R129G), a nontoxic mutant of the heat-labile enterotoxin of Escherichia coli (Chong et al., 1998). After immunization, single-cell suspensions of tissues were incubated with DCs

infected with vaccina virus expressing OVA or loaded with OVA peptide. As was the case with oral infection by E. cuniculi, markedly reduced amounts of both IFN-g and IL-17 were found in supernatants from the IEL and LP of Tlr9/ mice in comparison to WT animals (Figure 2E; Figures S2 and S1F). To determine the contribution of Treg cells to this phenotype, we treated mice with anti-CD25 (a-CD25) prior to immunization. As expected, a-CD25 treatment in WT mice enhanced IFN-g and IL-17 production (Figure 2E). Importantly, a-CD25 treatment in Tlr9/ mice restored IFN-g secretion to WT amounts, whereas treatment with Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc. 639

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 2. Tlr9/ Mice Mount Impaired Protective Responses to E. cuniculi after Oral Infection and Respond Poorly to Oral Vaccination WT and Tlr9/ mice were infected with E. cuniculi spores i.p. (A and B) or through an oral route (C and D). (A) and (C) show ELISA of IFN-g from i.p. infected mice and IFN-g and IL-17 from orally infected mice in supernatants of bulk leukocyte preparations from 11 days postinfected WT and Tlr9/ mice restimulated with uninfected (DC) or E. cuniculi-infected BMDC (inf. DC). As shown in (B) and (D), parasite loads are increased in orally infected Tlr9/ mice. Parasite loads were measured in the duodenum and liver of i.p. (B) or orally (D) infected mice 11 days postinfection by quantitative real-time PCR. Data shown are representative of three independent experiments with five mice per group (**p < 0.01). As shown in (E), WT and Tlr9/ mice were orally immunized with a mixture of OVA and the mutant E. coli LT(R129G) on day 0 and day 7 and treated with anti-CD25 or isotype control antibody. On day 14, IFN-g and IL-17 secretion by LP cells were evaluated by ELISA after in vitro restimulation with BMDC infected with recombinant vaccinia virus expressing OVA. For (A), (C), and (E), histograms represent the mean cytokine concentration of triplicate wells ± SD and are representative of at least two independent experiments with similar results (*p < 0.05; **p < 0.01; ***p < 0.001; n.s., non significant; ND, not detected).

isotype control yielded no increase. IL-17 secretion also rebounded to WT levels in the LP of a-CD25-treated Tlr9/ mice. Similar results were observed when Tlr9/ mice received a-CD25 prior to oral infection with E. cuniculi. Parasite control was also significantly increased in the duodenum and liver of Tlr9/ mice treated with a-CD25 compared to the isotype control (Figures S3A and S3B). Although a-CD25 treatment increased cytokine production to WT amounts, these amounts were still reduced relative to WT mice treated with a-CD25. This could suggest that in addition to increased Treg cell frequencies, impaired effector responses may account for the effect observed in absence of TLR9 signaling. However, because a large fraction of Foxp3 positive cells do not express CD25, antibody treatment is only partially efficient at reducing Foxp3 numbers and/or function in the GI tract (Figure S4). Taken together, these data suggest that the elevated proportion of Treg cells in Tlr9/ mice contributes to their impaired ability to respond to both oral infection with E. cuniculi and mucosal vaccination. TLR9 Signaling in the Hematopoietic Compartment Regulates Treg Cell Frequencies and Promotes Mucosal Effector Responses TLR9 is expressed in several radiosensitive hematopoietic lineages found within small intestinal tissue and heavily expressed 640 Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc.

on the surface of the intestinal epithelia derived from radioresistant stromal cells (Lee et al., 2006). Although our data demonstrate that TLR9 expression is required for intestinal mucosal responses, we wanted to better address the cellular compartment required for immune sensitization. To pursue this question, we generated bone marrow (BM) chimeric mice so that TLR9 expression in the intestinal tract was predominantly restricted to the epithelia or to hematopoietic lineages and that the origin of both compartments can be tracked on the basis of the expression of congenic markers. At 8 weeks after reconstitution, Treg cell frequencies were elevated in the Tlr9/ BM compartment relative to the WT BM compartment in reconstituted hosts (Figures 3A and 3B). Conversely, Treg cell frequencies were reduced in the WT BM compartment relative to the Tlr9/ BM compartment in reconstituted hosts (Figures 3A and 3B). Importantly, these changes were restricted to the gut compartment recapitulating the site-specific dysregulation of Treg cell frequencies in Tlr9/ mice. When irradiated WT congenic mice were reconstituted with Tlr9/ BM, the IFN-g recall response to oral infection with E. cuniculi was severely impaired and parasite load increased in the duodenum and liver (Figure 3C; Figure S5). In contrast, when TLR9 deficiency was mostly restricted to the intestinal epithelia in Tlr9/ mice reconstituted with WT BM, the IFN-g recall response improved significantly after oral infection

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 3. Expression of TLR9 by Hematopoietic Cells Is Sufficient to Negatively Modulate Levels of Treg Cells and Positively Favors Immune Responses in the GALTs Frequency of Foxp3+ Treg cells at homeostasis (A and B) and immune responses against E. cuniculi 11 days after oral infection (C) were analyzed in BM chimeric mice, in which the hematopoietic or the nonhematopoietic compartment lacks TLR9 expression. (A) shows comparative assessment of CD4+Foxp3+ Treg cells in the intestinal lamina propria (LP) of naive irradiated WT and Tlr9/ mice reconstituted with BM cells from WT or Tlr9/ congenic mice. Percentages of CD4+TCR-b+Foxp3+ were evaluated in the donor (CD45.1+) compartment. (B) shows Foxp3+ Treg cell percentages in naive irradiated WT or Tlr9/ mice reconstituted with BM cells from WT (closed circle) or Tlr9/ (open circle) congenic mice. Percentages of CD4+TCR-b+Foxp3+ were evaluated in the donor (CD45.1+) compartment. Each dot represents one mouse and each bar represents the mean of three mice analyzed. (C) shows ELISA measurement of IFN-g production by spleen and IEL compartments after in vitro restimulation with uninfected (DC) or E. cuniculi-infected (inf. DC) BMDC. Histograms represent the mean cytokine concentration from triplicate wells ± SD. Data shown are representative of two independent experiments with similar results (*p < 0.05; **p < 0.01; ***p < 0.001; ND, not detected).

than peripheral lymph nodes (less than 40% versus greater than 80% chimerism, respectively; data not shown). These findings indicate that TLR9 engagement by hematopoietic-derived cells is sufficient for modulation of Treg cell frequencies and priming the gut for immune responsiveness to oral infection with E. cuniculi.

with E. cuniculi and parasite control was enhanced (Figure 3C; Figure S5). Notably, the reciprocal chimeras (WT / Tlr9/ and Tlr9/ / WT) resulted in an intermediate phenotype, in terms of parasite burden. Although we cannot rule out a role for nonhematopoietic cells in regulating effector priming, it is important to note that chimerism was less complete in GI tissues

TLR9 Signaling Limits Treg Cell Conversion We and other groups recently demonstrated that oral exposure to antigen could induce conversion of naive CD4+Foxp3 T cells into Treg cells in the GALTs (Coombes et al., 2007; Mucida et al., 2005; Sun et al., 2007). Our findings indicated that a specialized population of DCs originating from the small intestinal LP (LpDCs) mediated this process through endogenous TGF-b and RA, in that TGF-b was critical for Treg cell differentiation and RA enhanced conversion (Benson et al., 2007; Coombes et al., 2007; Mucida et al., 2007; Sun et al., 2007). The involvement of the commensal flora in Treg cell conversion was not addressed in these studies. On the basis of the increased proportion of intestinal Treg cells in Tlr9/ mice and in the Tlr9/ hematopoietic compartment of chimeric WT Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc. 641

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 4. Engagement of TLR9 Signaling Potently Limits Treg Cell Conversion FACS-sorted naive CD4+CD25CD44loFoxp3 T cells isolated from Foxp3eGFP mice were cultured in Treg cell-polarizing conditions with WT LpDCs in the presence of the indicated TLR ligands. As shown in (A), dot plots gated on viable CD4+7-AAD T cells illustrate a4b7 versus Foxp3 expression after culture in presence or absence of CpG (10 mg/ml). As shown in (B), PGN (TLR2), LPS (TLR4), Flgn (TLR5), or CpG (TLR9) were added in the culture at starting concentrations of 2 mg/ml, 10 mg/ml, 1 mg/ml, and 10 mg/ml, respectively. Two subsequent 5-fold dilutions of ligand were tested (gray wedge). Results were normalized to conditions plated in the absence of TLR ligands with 100% equaling conversion in Treg cell-polarizing conditions. Cross bars indicate the high and lows of duplicate cultures. (C) shows CFSE-dilution analysis of naive CD4+CD25CD44lo T cells isolated from Tlr9/ mice, cultured as in (A), and analyzed for CD4 and Foxp3 expression at the indicated time points. An overlay of the histograms for the CFSE-dilution profiles of the CD4+Foxp3 T cells, indicated in the boxed in regions (solid line: control; dotted line: in presence of CpG), is shown. Data shown are representative of three independent experiments with similar results.

mice, we hypothesized that CpG-containing DNA motifs derived from the commensal flora (gfDNA) would restrict Treg cell conversion. To test this possibility, we first examined the effect of TLR9 engagement on in vitro conversion. The synthetic TLR9 agonist ODN 1826 (CpG) was added to purified LpDCs and CD4+CD25CD44loFoxp3 T cells cocultured in Treg cell-polarizing conditions, namely TGF-b and a-CD3. As shown previously, after 5 days of coculture in the absence of CpG, we observed both a high proportion of Foxp3+ cells and induction of the intestinal homing integrin heterodimer, a4b7, owing to RA-instructing signals (Figure 4A). Stimulation with CpG, however, led to a dose-dependent decrease in the frequency of Foxp3+ cells, whereas expression of a4b7 was maintained on Foxp3-negative cells (Figure 4A; Figure S6A). This observation suggested a decoupling of the tolerogenic and homing effects of RA in the presence of a strong adjuvant such as CpG (Johansson-Lindbom et al., 2005). The dramatic impact of CpG on Treg cell conversion contrasted with other bacterially derived TLR ligands, including peptidoglycan (PGN), lipolysaccharide (LPS), and flagellin (Flgn), which engage TLR2, TLR4, and TLR5, respectively. Even at high concentrations, these ligands exerted very little effect on Treg cell conversion (Figure 4B). LpDCs were not refractory to PGN, LPS, or Flgn stimulation as evidenced by cytokine production and or upregulation of CD40, CD80, and CD86 expression after overnight incubation (data not shown). To confirm that the effects observed on Treg cell conversion were the result of TLR engagement by LpDCs, we also performed this experiment with T cells that cannot respond to TLR9 (Myd88/) and found similar results (Figure S6B). For the remainder of these experiments, we used Tlr9/ or Myd88/ T cells to focus our study on the effects of TLR engagement on LpDCs. Cells failing to upregulate Foxp3 may have outgrown Treg cells in CpG-stimulated cocultures. However, CFSE-dilution analysis indicated that the Foxp3 cells proliferated equivalently in both CpG-stimulated and nonstimulated cocultures (Figure 4C). Moreover, before the cells that had upregulated Foxp3 began to divide, 642 Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc.

the frequency of Foxp3+ cells was already 50% lower in cocultures containing CpG. Together, these observations suggested that TLR9 signaling influenced Treg cell conversion at least in part through the inhibition of naive T cell differentiation into Treg cells. TLR9 Signaling Limits Treg Cell Conversion In Vitro through Promotion of Teff Cell Cytokines IL-6 and TGF-b in tandem can direct the production of IL-17secreting T cells (Th17 cells) over Treg cell conversion (Bettelli et al., 2006; Veldhoen et al., 2006). Importantly, LpDCs produce far more IL-6 than SpDCs did in response to CpG stimulation (Figure 5A). Thus, in CpG-stimulated cocultures, in lieu of Treg cells we observed T cells producing IL-17, as well as IL-4 and IFN-g (Figure 5B). On the basis of the large amount of IL-17 secreted into these cocultures (Figure 5C), we hypothesized that inhibiting IL-6 signaling may restore Treg cell conversion. However, despite substantial IL-17 abrogation, blockade of IL-6 (a-IL-6) with dual antireceptor and neutralizing Abs (Figure 5D) or the use of Il-6/ LpDCs (data not shown) failed to reverse the effect of CpG on Treg conversion. Moreover, additional measures to inhibit the IL-17 pathway, including using IL21r/ T cells (Korn et al., 2007; Nurieva et al., 2007) and adding neutralizing antibodies against IL-12-23p40 (Zhou et al., 2007) and IL-27p28 (Stumhofer et al., 2006) in addition to a-IL-6, also failed to restore Treg cell conversion (data not shown). Intriguingly, blockade of IL-6 treatment consistently enhanced IFN-g and IL-4 production (data not shown). In accord with this observation, adding neutralizing Abs against both IL-4 and IFN-g, in addition to IL-6-blocking Abs, resulted in a 70% rebound in the frequency of Treg cells (Figures 5D and 5E). The rescue afforded by IL-4 neutralization was greater than that resulting from IFN-g neutralization. In the presence of LpDCs, the addition of each cytokine alone blocked the induction of Foxp3 in vitro with IL-6 being the most potent. Maximal suppression was observed when cytokines were combined (Figure 5F). These findings complement a recent report demonstrating the antagonistic effect of Th1

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 5. Engagement of TLR9 Limits Treg Cell Conversion and Enhances Teff Cell Activity (A) A total of 5 3 104 purified LpDCs or SpDCs were stimulated for 18 hr in the presence or absence of CpG (10 mg/ml) in complete media containing 40 ng/ml of GM-CSF (CM). Recovered supernatant was assessed for IL-6 by ELISA. (B) CD4+CD25CD44loFoxp3 T cells from Tlr9/ Foxp3eGFP mice were cultured in Treg cell-polarizing conditions in the presence or absence of CpG for 6 days and then restimulated for assessment of intracellular cytokine production. (C–E) Naive CD4+CD25CD44lo T cells from Myd88/ mice were cultured in Treg cell-polarizing conditions for 6 days in the presence or absence of CpG. In wells containing CpG, antibodies to IL-6 and IL-6 receptor a (aIL-6), IL-4 (a-IL-4), and IFN-g (a-IFN-g) were added at the start of culture as indicated. (C) shows the measurement of IL-17 by ELISA for previously described conditions. (D) shows dot plots gated on viable CD4+ T cells and the percentages of Foxp3+ cells. (E) shows the summary of results of (D) normalized to baseline Treg cell conversion. Cross bars indicate the high and lows of duplicate cultures. Data shown are representative of at least three independent experiments with similar results. (F) Blockade of LpDC-induced Treg cell conversion by IL-6, IL-4, and IFN-g. Naive CD4+CD25CD44lo T cells were cocultured with LpDCs in Treg cell-polarizing conditions and with various doses of IL-6, IL-4, or IFN-g or a combination of all three cytokines. Cytokine concentration is provided on the x axis. In wells containing the combined cytokines, the same concentration of each cytokine was used. Data are representative of two independent experiments.

and Th2 cell development on Treg cell conversion (Wei et al., 2007). Thus, in addition to limiting Treg cell conversion, TLR9 signaling in the gut may promote local Teff cell activity, which in turn could enhance immune responses in the gut. Engagement of TLR9 by Commensally Derived gfDNA Inhibits Treg Cell Conversion In Vitro To test whether DNA derived from conventional gut flora (gfDNA) would have an effect on Treg cell conversion that would be

comparable to that of CpG, we extracted and purified gfDNA and added it to Treg cell-polarizing cocultures. Without a defined sequence length of gfDNA, we included a control condition that would facilitate its access into the LpDC endosomal compartment, where TLR9 stimulation occurs (Schalasta and Doppler, 1990). In this condition, gfDNA was complexed to the monocationic lipid transfection reagent, DOTAP, prior to stimulation (Bucci et al., 1992). Regardless of whether DOTAP was present, gfDNA consistently and significantly inhibited Treg cell Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc. 643

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

to sustain gut immune responses (Figure 7A). The addition of CpG alone significantly corrected this impairment, arguing that TLR9 signals serve as preconditioning stimuli for immune responses. Strikingly, gfDNA also restored the intestinal response in ATBtreated mice (Figure 7A). Impaired immune responses after ATB treatment led to increased parasite burden (Figure 7B). The increase of effector responses after CpG or gfDNA treatment correlated with restoration of parasite control (Figure 7B). In contrast, treatment of mice with LPS failed to restore immune response in the GI tract and parasite control (Figure S7C and Figure 7B). Similar data were obtained when ATB-treated mice receiving CpG or gfDNA were subsequently vaccinated (Figure S8A). To test the specificity of gfDNA for TLR9, we placed Tlr9/ mice on ATB and received gfDNA prior to infection with E. cuniculi. Antibiotic treatment also reduced immune responses to oral infection in Tlr9/ mice, but CpG or gfDNA did not rescue effector responses in these mice (Figure S8B). Thus, by means of TLR9 signaling, gfDNA serves as a natural adjuvant for priming intestinal immune responses. DISCUSSION

Figure 6. DNA Enriched from the Gut Flora Suppresses Treg Cell Conversion in a TLR9-Dependent Manner (A) CD4+CD25CD44lo T cells isolated from WT or Tlr9/ mice were cultured in Treg cell-polarizing conditions with LpDCs from WT or Tlr9/ mice, respectively. In some culture wells, 10 mg/ml of CpG, DNA enriched from murine gut flora (gfDNA), or gfDNA formulated with the cationic liposome, DOTAP, was added. CD4, Foxp3, and a4b7 expression was analyzed on day 6. (B) Summary of results from (A) normalized to baseline Treg cell conversion. Error bars represent the SD of triplicate cultures (**p < 0.01; ***p < 0.001).

conversion in a manner similar to CpG, while still inducing a4b7 (Figures 6A and 6B). Thus, LpDC can efficiently take up gfDNA. Addition of CpG or gfDNA to splenic DC also inhibited Treg cell induction in vitro although not to the same extent as LpDCs (Figure S6C). To assess whether gfDNA inhibited Treg cell generation via TLR9, we performed similar experiments using LpDCs purified from Tlr9/ mice. Under these conditions, the suppressive effect of both CpG and gfDNA was largely abolished (Figures 6A and 6B). Thus, gfDNA can inhibit Treg cell induction by LpDCs in a TLR9-dependent manner. Gut Flora DNA from Conventional Gut Flora Is a Natural Adjuvant of Intestinal Immune Responses The preceding results suggested that gfDNA have a direct adjuvant effect on the induction of intestinal immune responses through engagement of TLR9. To test this, we first minimized the impact of other gut floral signals by placing mice on a cocktail of antibiotics (ATB) (Pasare and Medzhitov, 2004) and at the same time provided CpG, gfDNA, or LPS. After 6 weeks of ATB treatment, mice were orally infected with E. cuniculi. The frequencies and absolute number of CD4+ and CD8+ T cells were comparable between the different treatment groups (Figures S7A and S7B). The reduction of the gut flora via ATB treatment was sufficient to significantly impair IFN-g and IL-17 responses in infected mice, supporting the idea that the presence of gut flora is required 644 Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc.

The tissues of the GI tract are constantly exposed to TLR ligands harbored by the commensal gut flora (Pamer, 2007). In our present study, we found that gut-floral-derived DNA primed gastrointestinal immune responses to foreign antigen through an intrinsic adjuvant activity. TLR9 signaling lowered the activation threshold in the gut through negative and positive expansion of Treg and Teff cells, respectively, and favored development of protective responses upon oral infection or during oral vaccination. Concordant with this finding, gfDNA potently limited the Treg cell-polarizing ability of LpDCs. To our knowledge, this study provides the first demonstration of a role for a gut flora component in initiating protective immune responses at sites of mucosal challenge. During conditions of floral translocation, peripheral TLR9 signaling is a crucial mediator of polymicrobial sepsis (Plitas et al., 2008). Moreover, in other conditions in which floral translocation occurs, peripheral TLR4 signals enhance the activation status of T cells (Brenchley et al., 2006; Paulos et al., 2007). However, under most circumstances, it is the tissues of the GI tract that are constantly exposed to TLR ligands harbored by the commensal gut flora (Pamer, 2007). Regulatory T cells are crucial to the integrity of the GI tract (Izcue et al., 2006). Recent evidence suggests that TLR signaling can impact Treg cell homeostasis (Sutmuller et al., 2006b). Treg cells themselves selectively express various TLRs (Sutmuller et al., 2006b). Interaction with some of these ligands can favor Treg cell expansion (Liu et al., 2006; Sutmuller et al., 2006a) Accordingly, in Tlr2/ mice Treg cell frequencies are decreased (Liu et al., 2006; Sutmuller et al., 2006a). In Tlr9/ mice, on the other hand, we observed increased Treg cell frequencies in small intestine effector tissues. Together, these findings indicate that TLR ligands can exert differential effects on Treg cell homeostasis. This could explain why Myd88/ mice or germ-free mice have unaltered Treg cell frequencies (Min et al., 2007; Rakoff-Nahoum et al., 2004; Sutmuller et al., 2006a). Our observation that Treg cells were selectively increased in gut effector sites was mirrored by a decrease in constitutive Teff cell proinflammatory secretion. Thus, gfDNA

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Figure 7. Gut Floral DNA Restores Immune Responses in AntibioticTreated Mice Orally Infected with E. cuniculi A cocktail of antibiotics (ATB) in the drinking water was given to 3-week-old mice for 6 weeks in conjunction with oral weekly treatments consisting of PBS alone or containing 100 mg of CpG, 500 mg gfDNA, or 25 mg/kg of lipopolysaccharide from Escherichia coli (LPS). Control mice received no ATB treatment. At 6 weeks posttreatment, mice were infected orally with E. cuniculi. (A) shows ELISA of IFN-g and IL-17 in supernatants of bulk leukocyte preparations from IEL and LP of 11 days postinfected WT and Tlr9/ mice restimulated with E. cuniculi-infected BMDC for 72 hr. Histograms represent the mean cytokine concentration ± SD. This experiment is representative of two independent experiments with similar results (*p < 0.05; **p < 0.01; ***p < 0.001). As shown in (B), 8-week-old mice were treated and infected as described in (A) and parasite burden was evaluated in duodenum and liver 11 days postinfection by quantitative real-time PCR. Each dot represents one mouse and each bar represents the mean of three or four mice analyzed (*p < 0.05; **p < 0.01; ***p < 0.001; n.s., nonsignificant).

interactions with TLR9 may be one of the factors that dictate the balance between Treg cells and Teff cells in the GI tract at steady-state conditions. Our finding that Tlr9/ mice mounted an impaired response to E. cuniculi only during the oral route of infection suggested that the enhancement of intestinal Treg cell and/or impaired Teff cell priming in these animals may predispose them to less efficient mucosal responses. A role for TLR9 has been previously reported against various infections that occur at mucosal sites such as Toxoplasma gondii or Herpes simplex virus (Lund et al., 2003; Minns et al., 2006). Thus, although TLR9 may be critical for recognition of these microbes, we further propose that the dysregulated Treg/Teff cell ratio within the gut tissues would have additional bearing on the phenotype observed. It is still unclear by what means gfDNA becomes accessible for interaction with TLR9. Epithelia in the GI tract basally express TLR9 on their surface (Ewaschuk et al., 2007; Lee et al., 2006). However, TLR9 signaling on the apical side can mediate anti-inflammatory effects, thereby linking lumenal signals from gfDNA to intestinal homeostasis (Lee et al., 2006). Our observation that Treg cell frequencies were unaffected in the large intestine suggests a compartmentalization of the effect of TLR9 signaling on the control of gut homeostasis. In this site, the adjuvant effect of bacteria DNA may be lost for the prevention of detrimental inflammation. In support of this, oral administration of CpG was shown to ameliorate the severity of dextran sodium sulfate (DSS)-induced colitis via TLR9 (Katakura et al., 2005). Along these lines, the absence of TLR9 exacerbated the severity of DSS-induced colitis (Lee et al., 2006). Thus, in a context of a highly inflammatory setting, TLR9 signaling on epithelial cells can mediate a protective function. In contrast, at steady-state conditions or during oral exposure to pathogens, our results demonstrate that luminal gfDNA may also have potent immunogenic potential. Although gfDNA similarly restored protective immune responses to infection with E. cuniculi in ATB-treated mice, the impact of LPS conditioning was minor. The inability of LPS to efficiently adjuvant intestinal immune responsiveness is reminiscent of a recent report, which found that LPS tolerance in the intestine is established shortly after birth (Lotz et al., 2006). Our results obtained with chimera mice suggested that TLR9 expression by the hematopoietic compartment was sufficient to constrain intestinal Treg cell frequencies and enhance protective immune response. Thus, gfDNA makes essential contributions to intestinal homeostasis through two seemingly disparate pathways with the establishment of this dichotomy potentially resting on the cellular target, the intensity of the tissue disruption, or even the gut compartment involved. We have previously shown that peripheral conversion of CD4+ T cells to Treg cells occurs primarily in the GALTs. DCs purified from these compartments were found to promote a high level of Treg cell conversion. Enhanced conversion of Treg cells by LpDCs was dependent on TGF-b and RA, a vitamin A metabolite. One possible explanation for the phenotype observed in Tlr9/ mice could be associated with a dysregulation of this pathway. Indeed, we found that TLR9 signaling by gfDNA potently limited Treg cell induction by LpDCs. Notably, other representative bacterial TLR ligands did not markedly affect LpDC-induced Treg cell conversion in vitro. When added to Treg cell-polarizing cocultures, TLR5, TLR4, or TLR2 ligation had little effect on Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc. 645

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

conversion, supporting previous reports suggesting regulatory properties for TLR4 and TLR5 in this environment (Bashir et al., 2004; Rakoff-Nahoum et al., 2004; Vijay-Kumar et al., 2007). Several proinflammatory signals have been shown to limit Foxp3 induction (Bettelli et al., 2006; Korn et al., 2007; Nurieva et al., 2007; Stumhofer et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007). For instance IL-6, IL-4, and IFN-g can limit Treg cell conversion (Bettelli et al., 2006; Veldhoen et al., 2006; Wei et al., 2007). We found that LpDCs responded robustly to TLR9 stimulation and secreted several proinflammatory cytokines, including large amounts of IL-6. Accordingly, in addition to restricting Treg cell conversion, TLR9 activation induced Teff cell cytokines including IL-17, IFN-g, and IL-4. The difficulty in reversing this effect underscored TLR9’s potent antagonizing influence on Treg cell conversion. Effective rescue required inhibition of IL-6 in conjunction with neutralization of IL-4 and IFN-g. These data are in line with the augmented Treg/Teff cell ratio that we observed in the intestinal tissues of Tlr9/ mice. In addition to limiting Treg cell conversion, TLR9 signaling may be required for sustaining effector activity and/or priming in the GI tract. Interestingly, temporary ablation of LpDCs was recently shown to reduce constitutive Th17 cell production in the lamina propria (Denning et al., 2007). The reduction of both IFN-g and IL-17 in TLlr9/ mice suggested that gfDNA signaling on LpDCs was involved in this basal cytokine production. In addition to cytokine induction, we propose that TLR9 signals may also promote Teff cell migration into the gut via RA release from DCs. Although our data suggest that RA contributes to tolerance mechanisms, RA is also required for migration of lymphocytes into intestinal effector sites via induction of a4b7 and CCR9 (Johansson-Lindbom et al., 2005; Johansson-Lindbom et al., 2003; Mora et al., 2006; Siewert et al., 2007). Additionally, a recent report demonstrated that low doses of RA have a proinflammatory effect on T cell polarization when coupled with microbial stimulus (Uematsu et al., 2008). We observed strong a4b7 induction on Teff cells in cocultures exposed to CpG and gfDNA. Synthesizing these findings, we found that constitutive TLR9 signaling in DCs derived from the lamina propria may therefore create a negativeregulatory circuit that mediates sensitivity to intestinal pathogens through modulation of Treg/Teff cell proportions in intestinal effector sites and intestinal homing. Previously, Pasare et al. demonstrated that CpG could render Teff cells resistant to Treg cell regulation after DC activation (Pasare and Medzhitov, 2003). In this regard, the absence of TLR9 may subject Teff cells to more regulation by Treg cells in the GI tract. The adult human intestine harbors up to 100 trillion microorganisms (Backhed et al., 2005). These microflora via their interaction with specific TLRs can mediate gut homeostasis (Bashir et al., 2004; Fukata et al., 2005; Katakura et al., 2005; Rachmilewitz et al., 2002; Rachmilewitz et al., 2004; Rakoff-Nahoum et al., 2004). Modification of gut flora was shown in areas of inflamed gut in IBD patients (Barnich et al., 2003; Darfeuille-Michaud et al., 2004; Martin et al., 2004; Masseret et al., 2001; Ott et al., 2004; Seksik et al., 2003; Swidsinski et al., 2005). Furthermore, the presence of certain bacteria can aggravate small intestinal immunopathology after oral infection with mice (Heimesaat et al., 2006). Importantly, gut flora bacteria are not equal in their capacity to stimulate TLR9 (Dalpke et al., 2006). It is tempting to speculate that alteration of Treg cell homeostasis mediated by 646 Immunity 29, 637–649, October 17, 2008 ª2008 Elsevier Inc.

TLR9 signaling, either because of genetic polymorphism or changes in gut flora composition, could also have consequences on development of gut inflammatory disorders. Manipulation of Treg cell numbers or functions offers promising therapeutic avenues. Our present data offer the possibility to target the ratio between Treg and Teff cells in a site-specific manner. We propose to exploit our current observations to test how manipulation of gut flora or gut flora signaling could become part of a rational therapeutic strategy against oral infections or to favor oral vaccination. EXPERIMENTAL PROCEDURES Mice C57BL/6 (WT) and B6.SJL mice were purchased from Taconic Farms or bred in house. B6.129P2-Tlr9tmAki (Tlr9/) (Hemmi et al., 2000) mice were obtained from S. Akira (Osaka University) via R. Seder (Vaccine Research Center, NIH) and backcrossed 11 generations onto the C57BL/6 background (Taconic). B6.129S6-Il6tm1Kopf (Il-6/) were purchased from the Jackson Laboratory, and B6.129P2-Myd88tmAki (Myd88/) were kindly provided by Dr A. Sher. Foxp3 eGFP reporter mice (Foxp3eGFP) were obtained from M. Oukka (Bettelli et al., 2006). We generated Tlr9/Foxp3eGFP mice by crossing the F1 progeny of Tlr9/ 3 Foxp3eGFP breeders. We generated Tlr9/B6.SJL mice by crossing the F1 progeny of Tlr9/ 3 B6.SJL breeders. All mice were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by the NIAID Animal Care and Use Committee. In some experiments mice were cohoused. Mice between 8 and 12 weeks of age were used. Mice were gender and age-matched for each experiment. Phenotypic Analysis Cells from spleen, MLN, IEL, LP, and PP were prepared as previously described (Sun et al., 2007). Single-cell suspensions were incubated with an anti-Fc3III/II receptor. Cells were stained with fluorochrome-conjugated antibodies against surface markers CD4 (RM4-5), CD8a (53-6.7), CD25 (PC61.5), TCR-b chain (H57-597), CD45.1 (A20), CD45.2 (104), and/or a4b7 (DATK32) in PBS containing 1% FBS for 20 min on ice and then washed. In some experiments, we used 7-amino-actinomycin D (7-AAD, eBioscience) to exclude dead cells. For Foxp3 staining, cells were subsequently stained with the Foxp3 staining set (eBioscience) according to the manufacturer’s protocol. Nuclear Ki-67 staining was performed with an antibody against human Ki-67 (clone B56) from BD PharMingen. Cell acquisition was performed on an LSRII machine with FACSDiVa software (BD Biosciences). For each sample, at least 300,000 events were collected. Data were analyzed with FlowJo software (TreeStar). Intracellular Cytokine Detection For basal cytokine detection, IEL, LP, and PP single-cell suspensions were stained with the CD90-positive selection kit and enriched for T cells with an autoMACs (Miltenyi Biotec). Cells were then cultured in triplicate at 1 3 106 cells/ml in a 96-well U-bottom plate and stimulated with 50 ng/ml PMA (Sigma) and 5 mg/ml ionomycin (Sigma), in the presence of brefeldin A (GolgiPlug, BD Biosciences) at 37 C in 5% CO2. After 5 hr, cells were fixed in 4% paraformaldehyde for 10 min at room temperature or with the BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Cells were then stained with fluorochrome-conjugated antibodies against CD4 (RM4-5), CD8a (53-6.7), IFN-g (XMG1.2), IL-10 (JES5-16E3), IL-4 (11B11), and IL-17 (clone TC11-18H10) or with isotype controls rat IgG1 (clone R3-34), rat IgG2a (clone eBR2a), rat IgG2b (A95-1), mouse IgG2a (eBM2a), and hamster IgG2 (Ha4/8) for 30 min in FACS buffer containing 0.1% saponin or permabilization buffer supplied with the BD Cytofix/Cytoperm kit. All antibodies were purchased from eBioscience or BD Biosciences.

Immunity Role of Flora DNA in Regulation of Gut Treg Cells

Vaccination and Infection Protocol C57BL/6 and Tlr9/mice were immunized orally with OVA protein (grade V; Sigma-Aldrich; 1 mg/mouse for each immunization) on day 0 and 7 in combination with 20 mg/mouse of the mutant form of E. coli LT(R129G) (Chong et al., 1998). Both OVA and LT(R129G) were prepared in an isotonic bicarbonate buffer. Immune responses were analyzed 2 weeks after the first immunization. A rabbit isolate of E. cuniculi obtained from Waterborne was used throughout the study. The parasites were maintained by continuous passage in rabbit kidney (RK13) cells obtained from the American Type Culture Collection (ATCC #CCL37) and maintained as previously described (Bouladoux et al., 2003). Spores were resuspended in sterile PBS and immediately used for inoculation of mice or cell cultures. Mice were infected by intragastric gavage with 5 3 106 fresh spores in a volume of 200 ml. In some experiments, mice were infected i.p. with the same number of spores of E. cuniculi. For CD25+ cell depletion, C57BL/6 and Tlr9/ mice were injected i.p. with 0.5 mg of anti-CD25 (clone PC61.5) or the corresponding isotype control (clone GL113) 3 days before the first oral immunization with OVA and then on the day of each immunization. In Vitro Restimulation Single-cell suspensions of spleen, MLN, IEL, LP, and PP from vaccinated or E. cuniculi-infected mice were prepared as described above. BMDC were generated as previously described (Lutz et al., 1999). Leukocytes (5 3 105) were incubated with 1 3 105 BMDC in 250 ml RPMI, 55 mM 2-b mercaptoethanol, and 10% FBS per well of a 96-well U-bottom plate. BMDC were previously incubated overnight with or without recombinant vaccinia virus expressing OVA protein (10 pfu / BMDC), E. cuniculi spores (parasite:BMDC ratio, 10:1), or OVA peptide (1 mg/ml) in the presence of 20 ng/ml GM-CSF (Peprotech) and washed before culture with leukocytes. After 3 days at 37 C in 5% CO2, culture supernatants were collected for cytokine assays. IFN-g, IL-4, IL-6, IL-10, and IL-17 were quantitated in culture supernatants of restimulated leukocytes with the DuoSet ELISA system (R&D Systems) according to the manufacturer’s protocol. Quantitation of Parasite Tissue Loads Duodenum and liver were removed from infected mice and digested with proteinase K (Invitrogen or QIAGEN) after homogenization. DNA was subsequently extracted either with phenol-chloroform-isoamyl alcohol and then underwent ethanol precipitation or with the DNeasy Tissue kit from QIAGEN. Quantitative real-time PCR was performed in triplicates with 50 ng of total tissue DNA, the iQ SYBR Green Supermix (BioRad), and the following primers specific for a 268-bp DNA sequence of the SSU rRNA gene from E. cuniculi (Weiss and Vossbrinck, 1998): forward 50 -GTGAGACCCTTTGACGGTGT-30 and reverse 50 -CTCAGACCTTCCGATCTTCG-30 . Real-time PCR was conducted on a Bio-Rad iCycler under the following conditions: 3 min at 95 C, 40 cycles of 45 s at 95 C, 60 s at 60 C, and 45 s at 72 C. Genomic DNA were extracted from known amounts of E. cuniculi with the QIAamp DNA stool mini-kit (QIAGEN) and used as PCR standards. A standard curve was generated by linear regression on plotted cycle threshold (CT) values of the standards against the logarithms of parasite numbers with iCycler iQ Optical System software (version 3.1; Bio-Rad). Lamina Propria DC and T Cell Purification After LP digests were passed through 70- and 40-mm cell strainers, cells were resuspended in 1.077 g/cm3 iso-osmotic NycoPrep medium (Accurate Chemical & Scientific Corp.), and overlaid with RPMI 1640. The low-density fraction was collected after centrifugation at 1650 g for 15 min. Cells were washed and incubated with a mixture of mAb containing a-CD16/32 (2.4G2), 7-AAD viability staining solution, a-CD11c (HL-3), and a-MHCII (AF6-120.1), as well as the non-DC components a-DX5 (DX5), a-NK1.1 (PK136), and a-B220 (RA3-6B2) (all from eBioscience). DCs were defined as CD11chiMHCII+ cells and nonDCs were excluded when sorted by flow cytometry on a FACSAria. CD11c+MHCII+ cells were >90% and used for in vitro conversion assays. For T cell purification, single-cell suspensions of peripheral LNs extracted from Foxp3eGFP, Tlr9/Foxp3eGFP, WT, Tlr9/, Myd88/, or IL21r/ mice were enriched for CD4+ T cells by a negative selection kit with an autoMACs (Miltenyi Biotec). The enriched fraction was further labeled with fluorescent dye-conjugated mAbs, including CD4 (RM4-5), CD25 (7D4), and CD44 (IM7)

(all from eBioscience), and sorted by flow cytometry on FACSVantage or FACSAria (BD Biosciences). Purified CD4+CD25CD44loFoxp3 T cells or CD4+CD25CD44lo (<1% Foxp3+) were used for in vitro conversion assays. In proliferation assays, purified CD4+ T cells were labeled with 1.25 mM CFSE (Invitrogen) in HBSS (Mediatech) for 5 min at room temperature. Cells were washed twice in media containing 3% FBS. In Vitro Conversion Assay Our conversion protocol was performed as previously described (Bettelli et al., 2006) with T cells and LpDCs obtained as described above. In brief, FACS-purified LpDCs and CD4+ T cells were cocultured at a 1:10 ratio (1 3 105 CD4+ T cells) in complete medium (RPMI-1640 containing 10% FBS, 20 mM of 2b mercaptoethanol, and antibiotics) and under conditions favoring Treg cell generation, soluble a-CD3 (1 mg/ml) (BD Bioscience), and human rTGF-b (0.6 ng/ml) (Cell Science). We supplemented 5 ng/ml of IL-2 in cocultures every 2 days. In experiments detailed in Figure 4, the following were added to co-coculture conditions: peptidoglycan (PGN), ultra-pure lipopolysaccharide (LPS), flagellin (Flgn), CpG ODN 1826 (CpG) (all from Invivogen) at the indicated concentrations. In experiments detailed in Figure 5, various combinations of mAbs including a-IL-6 (MP5-20F3), a-IL-6R (D7715A7), a-IL12/ 23p40 (C17.8), a-IL27p28 (RaD), and/or a-IFN-g (XMG1.2 or 11B11) or isotype controls a-IgG1k (R3-34) and/or a-IgG2a (R35-95) were added at the start of cocultures in Treg cell-polarizing conditions containing CpG (10 mg/ml). All antibodies were purchased from BD Biosciences, excluding a-IL27p28, which was purchased from R&D Systems. On day 5, cells were surface stained with antibodies to CD4 (RM4-5), a4b7 (DATK32), CD25 (PC61.5), and the viability marker 7-AAD (all from eBioscience). Foxp3+ cells were detected on an LSRII by eGFP expression and/or a-Foxp3 (FJK-16) after fixation and permeabilization with the kit provided by eBioscience in accordance with the manufacturer’s protocol. For intracellular-cytokine staining, supernatants were removed and replaced with complete media containing PMA, ionomycin, and brefeldin A, as described above. Gut Flora DNA Extraction Gut contents from the caecum and the colon of naive C57BL/6 mice were collected and washed in cold PBS. The pellet was resuspended in lysis buffer (10 mM Tris/HCl and 50 mM EDTA [pH 8.0]) containing lysozyme (0.5 mg/ml; Sigma-Aldrich). After incubation at 37 C for 2 hr, 2 mg/ml of proteinase K and 1% SDS were added and the sample was incubated at 60 C for 3 hr. DNA was then purified by a series of seven consecutive phenol-chloroformisoamyl alcohol affinity extractions. Antibiotic Treatment Male 3- or 8-week-old C57BL/6 mice were provided ampicillin (1 g/l), vancomycin (500 mg/l), neomycin trisulfate (1 g/l), and metronidazole (1 g/l) in drinking water as previously described (Rakoff-Nahoum et al., 2004). All antibiotics were purchased from Sigma-Aldrich. Mice also received orally 100 mg CpG ODN 1826 in sterile PBS (Coley Pharmaceutical) or 500 mg of DNA from the gut content in sterile water or 25 mg/kg of LPS from Escherichia coli (serotype 026:B6 from Sigma-Aldrich) once weekly from the start of the antibiotic course. Statistical Analysis Groups were compared with Prism software (GraphPad) with the unpaired or paired Student’s t test. SUPPLEMENTAL DATA Supplemental Data include eight figures and can be found with this article online at http://www.immunity.com/cgi/content/full/29/4/637/DC1/. ACKNOWLEDGMENTS This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank the NIAID sorting facility and K. Beacht for technical assistance. We would like to thank A. Sher and D. Chou for critical reading of the manuscript.

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