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Absence of MHC class II on cDCs results in microbial-dependent intestinal inflammation

Nussenzweig et al. use a novel mutant mouse lacking MHC class II expression on conventional dendritic cells (cDCs) to demonstrate the importance of cDCs in the maintenance of intestinal homeostasis.

Conventional DCs (cDCs) are specialized immune cells that link the innate and adaptive immune system. Innate features of cDCs allow them to recognize and respond to pathogens by producing essential cytokines such as IL-6, IL-12, IL-23, and TNF. These cytokines contribute to the activation of other immune cells, including T and B cells and cells of the innate immune system. For example, in the intestine, cDCs sense bacteria and produce IL-23, which induces type III innate lymphoid cells (ILC3s) to produce IL-22, which in turn stimulates production of antimicrobial peptides (AMPs; Sonnenberg et al., 2011;Kinnebrew et al., 2012;Satpathy et al., 2013;Bernink et al., 2015). In addition to their innate functions, cDCs initiate adaptive immune responses by ingesting, processing, and presenting antigens to T cells (Nussenzweig et al., 1980;Steinman et al., 2003). In the intestine, cDCs are responsible for transport of antigen to the draining mesenteric LNs (mLNs). Under physiological conditions, the capacity of cDCs to migrate from tissue to draining LNs distinguishes them from more sessile macrophages (Schreiber et al., 2013). The importance of cDCs in adaptive immune function is exemplified by the fact that cDC depletion during viral and bacterial infection results in impaired T cell immunity and increased susceptibility to infection (Jung et al., 2002;Kas-sim et al., 2006;Hildner et al., 2008;Satpathy et al., 2013;Schreiber et al., 2013).
In mice, expression of Itgax (CD11c) is a hallmark of the DC lineage, and its expression has been used to label (CD11c YFP ), deplete (CD11c DTR ), and conditionally target (CD11c Cre ) cDCs (Jung et al., 2002;Lindquist et al., 2004;Caton et al., 2007;Stranges et al., 2007). However, CD11c is also expressed by plasmacytoid DCs (pDCs), activated monocytes, macrophages, and some NK cells, and therefore CD11c-based labeling and targeting strategies are not entirely cDC specific (Serbina et al., 2003;Hohl et al., 2009;Meredith et al., 2012;Schreiber et al., 2013). Higher levels of specificity can be achieved by deletion of genes that regulate the development of specific subsets of cDCs, such as Batf3, Irf4, and Notch2; however, these methods have yet to be applied to dissect the relative contributions of the innate and adaptive functions of cDCs to immune homeostasis (Caton et al., 2007;Hildner et al., 2008;Persson et al., 2013;Schlitzer et al., 2013).
To investigate the adaptive functions of cDCs independent of innate activities, we produced a cDC-restricted Cre mouse wherein Cre expression is directed by the Zbtb46 gene (zDC Cre ) and used it to delete MHC II in cDCs in vivo. These mice exhibited profound intestinal inflammation that was directly related to the presence of intestinal bacteria, as antibiotictreated or germ-free mice lacking MHC II on cDCs showed no signs of intestinal inflammation. Colonization of germ-free Conventional dendritic cells (cDCs) play an essential role in host immunity by initiating adaptive T cell responses and by serving as innate immune sensors. Although both innate and adaptive functions of cDCs are well documented, their relative importance in maintaining immune homeostasis is poorly understood. To examine the significance of cDC-initiated adaptive immunity in maintaining homeostasis, independent of their innate activities, we generated a cDC-specific Cre mouse and crossed it to a floxed MHC class II (MHC II) mouse. Absence of MHC II on cDCs resulted in chronic intestinal inflammation that was alleviated by antibiotic treatment and entirely averted under germ-free conditions. Uncoupling innate and adaptive functions of cDCs revealed that innate immune functions of cDCs are insufficient to maintain homeostasis and antigen presentation by cDCs is essential for a mutualistic relationship between the host and intestinal bacteria. , pDCs (CD11c low BST2 + ), RPMs (Lin − CD11b − F4/80 + ), and monocytes (Lin − CD11b + Ly6C + CD115 + ). Histograms show percentage of cells expressing YFP in zDC Cre Rosa lSlYFP mice (open histograms) or Rosa lSlYFP control mice (filled gray histograms). (D) Graph depicts percentage of various immune cell populations in the spleen expressing YFP in zDC Cre Rosa lSlYFP mice (blue symbols) and CD11c Cre Rosa lSlYFP mice (black symbols). Each data point represents one individual mouse (n = 4-13). (E) Splenocytes were isolated 16 h after i.p. administration of 10 µg pIC. Graph depicts percentage of various immune cell populations expressing YFP in zDC Cre Rosa lSlYFP mice (black symbols) and zDC Cre Rosa lSlYFP mice that received pIC (red symbols). Each data point represents one individual mouse (n = 3-6). (F) Flow cytometric analysis of small intestine lamina propria mononuclear phagocytes (Lin − CD11c + MHC II + ). Histogram shows YFP expression of DC subsets mice allowed us to monitor adaptive immune responses against intestinal bacteria and revealed that mice lacking MHC II on cDCs have a defect in inducing proper adaptive immune responses against commensals. Collectively, our studies reveal the importance of the adaptive function of cDCs in maintaining intestinal homeostasis.

RES ULTS Generation of zDC Cre mouse
Zbtb46 (zDC) is expressed in pre-DCs and their progeny, but not in monocytes, macrophages, and other myeloid cells (Fig. 1 A; Meredith et al., 2012;Satpathy et al., 2012). To express Cre recombinase in cells that actively transcribe zDC, we inserted Cre into the 3′ UTR of zDC ( Fig. 1 B ;Meredith et al., 2012). Cell type-specific expression of Cre was confirmed by crossing zDC Cre to Rosa26 lSlYFP mice (zDC Cre Rosa lSlYFP ), wherein Cre-mediated excision of a transcriptional Stop element results in YFP expression. The majority of cDCs (Lin − CD11c high MHC II + ) in the spleen of zDC Cre Rosa lSlYFP mice were YFP + . In contrast, B and T lymphocytes, pDCs (CD11c low BST2 + ), red pulp macrophages (RPMs; Lin − CD11b low F4/80 + ), and monocytes (Lin − CD11b + Ly6C + CD115 + ) expressed little or no YFP (Fig. 1,C and D). In contrast, in CD11c Cre Rosa lSlYFP mice, cDCs, as well as the majority of macrophages, and pDCs were labeled in the spleen (Fig. 1 D). Similar results were also obtained after administration of polyinosinic :polycytidylic acid (pIC; Fig. 1 E) and with zDC Cre+ IRF4 wt/fl mice that express GFP upon Cre-mediated IRF4 excision (Klein et al., 2006;Fig. S1).
In the intestine, the phenotype of monocytes and cDCs is altered (Caballero and Pamer, 2015). For example, small intestine lamina propria CD11c + MHC II + cells represent a mixture of cDCs and monocytes that can be further subdivided based on expression of CD103, CD11b, and CD64 (Fig. 1 F;Bogunovic et al., 2009;Tamoutounour et al., 2012;Schreiber et al., 2013). Whereas all of these cell types are labeled in CD11c Cre Rosa lSlYFP mice, only pre-DC-derived CD64 − cells were YFP + in zDC Cre Rosa lSlYFP indicator mice (Fig. 1, G and H). We did not observe a difference in YFP expression in CD103 + /CD11b − and CD103 + /CD11b + CD64 − cells in zDC Cre Rosa lSlYFP mice ( Fig. 1 F). We conclude that zDC Cre mice are suitable for cDC-specific conditional gene deletion. zDCΔMHC II mice exhibit preserved innate functions but impaired adaptive functions In addition to antigen processing and presentation, cDCs are essential innate sensors of pathogens and other stimuli. To distinguish between innate and adaptive functions of cDCs, we produced a cDC-specific MHC II knockout by crossing zDC Cre with MHC II fl/fl mice (zDCΔMHC II; Hashimoto et al., 2002). As expected, zDCΔMHC II mice expressed MHC II on the surface of pDCs, but not on cDCs (Fig. 2 A). Absence of MHC II expression by cDCs did not change the frequency of cDCs among splenocytes (Fig. 2 B), nor did it alter the ratio of CD8α + and CD11b + cDCs (Fig. 2 C). The same was true for the mLNs (Fig. 2, D and E). Moreover, the levels of circulating Flt3L in the serum, which are increased upon cDC depletion (Birnberg et al., 2008;Meredith et al., 2012), were normal in zDCΔMHC II mice (Fig. 2 F).
To determine whether adaptive responses are altered by cDC-specific MHC II deletion, we transferred OVA-specific CD4 + T cells (OT-II cells) to control and zDCΔMHC II mice and compared their responses to immunization with OVA. OT-II cells responded by proliferating and up-regulating CD44 expression in control but not in zDCΔMHC II mice (Fig. 2,G and H). In contrast, proliferation and activation of CD8 + OT-I T cells in response to immunization with OVA was not altered in zDCΔMHC II mice (Fig. 2, G and H). Thus, deletion of MHC II on the surface of cDCs is sufficient to impair priming of naive CD4 + T cells.
To confirm that the innate responses of cDCs are intact in zDCΔMHC II mice, we purified cDCs from the spleen, stimulated them with CpG 1668, LPS, or pIC, and measured expression of co-stimulatory molecules (CD80 and CD86) and production of cytokines (IL-6 and TNF). We found no differences between control and zDCΔMHC II cDCs, indicating that their responses to innate stimuli are intact ( Fig. 2 I-M). Thus, zDCΔMHC II cDCs show intact responses to innate stimuli, but are unable to initiate MHC IIrestricted adaptive immune responses.

Intestinal inflammation develops in mice lacking MHC II on cDCs
Although zDCΔMHC II mice were born at the expected Mendelian ratio, they showed significantly reduced weight gain ( Fig. 3 A) and developed rectal prolapse starting at 6 wk of age ( Fig. 3 B). In addition, the mutant mice concomitantly developed splenomegaly and showed increased circulating levels of the acute phase-reactant Csf3 and neutrophilia, which are indicative of a systemic process (Fig. 3, C-E). The levels of Csf1 and Csf2 remained within normal limits (unpublished data).
To quantitate intestinal inflammation, we measured Lipocalin-2, a sensitive biomarker for intestinal inflammation, in the feces of control and zDCΔMHC II mice (Chassaing et al., 2012). zDCΔMHC II mice showed a 100-fold increase in Lipocalin-2 compared with controls ( Fig. 3 F). Consistent with this observation, histopathological analysis revealed inflammation that was primarily restricted to the large intestine, with inflammatory infiltrates, crypt hyperplasia, and villus destruction (Fig. 3 G). Neutrophil infiltration in the large intestine was also detected by flow cytometry (Fig. 3 H). In addition, we found elevated levels of the inflammatory cytokine IL-23 in the large intestine of zDCΔMHC II, but not in control mice (Fig. 3 I). To confirm that intestinal innate immune responses were intact, we measured the expression of AMPs in the intestine of zDCΔMHC II and control mice. Consistent with the inflammation found in the intestines of these mice, RegIIIγ and S100A9 were found at higher levels in zDCΔMHC II mice compared with controls ( Fig. 3 J). ILC3s, whose IL-22 production is crucial for AMP expression (Sonnenberg et al., 2011;Kinnebrew et al., 2012;Satpathy et al., 2013;Bernink et al., 2015), were not affected and actually occurred at higher frequencies in zDCΔMHC II mice compared with controls ( Fig. 3 K). We therefore conclude that intestinal inflammation in zDCΔMHC II mice occurs even in the presence of active cDC-dependent innate immune responses.
Normally, intestinal bacteria are separated from epithelial cells by a mucous layer. Innate immune responses and AMPs are essential for this spatial segregation (Vaishnava et al., 2011;Gallo and Hooper, 2012). To visualize bacteria in the large intestine (colon and cecum) of zDCΔMHC II mice and determine their location and proximity to the epithelial barrier, we performed 16S ribosomal RNA (rRNA) fluorescence in situ hybridization (16S FISH). We found that, in zDCΔMHC II mice, intestinal microbes and gut epithelial cells were in direct contact (Fig. 3 L). However, Muc2 expression in the large intestine was unaltered, and therefore zDCΔMHC II mice do not have a defect in mucus production ( Fig. 3 M). Finally, neither orally administrated FITC-Dextran nor LPS were detectable at higher levels in the serum of zDCΔMHC II compared with control mice, indicating no gross defect in the epithelial barrier (unpublished data).
A similar phenotype was also observed in CD11cΔMHC II mice ( Fig. 4 A) and in zDCΔMHC II, but not control BM chimeras (Fig. 4, B and C). Finally, BM chimeras reconstituted with a 1:1 mixture of control and zDCΔMHC II BM did not develop disease (Fig. 4 C). Therefore, intestinal inflammation in zDCΔMHC II mice is BM cell-dependent and can be rescued by WT BM cells.
Despite the changes in the anatomical distribution of the microbiome in the intestine, we did not observe dramatic differences in the composition of the microbiome (determined by 16S sequencing of fecal bacteria) between control and zDCΔMHC II mice that were cohoused. UniFrac analysis showed that the microbiome in individual mice clustered by the cage in which they were housed and not by genotype, suggesting that the composition of the microbiome is not dramatically altered in zDCΔMHC II mice compared with controls ( Fig. 4 D). These data indicate that in addition to AMPs, MHC II expression by cDCs is required to maintain the spatial segregation between microbes and gut epithelial cells.

CD4 + T cell activation is impaired in zDCΔMHC II mice
DCs and thymic epithelial cells are both involved in the selection of mature T cells in the thymus; therefore, we examined thymic CD4 + T cells in zDCΔMHC II mice. Although there was a small increase in mature single-positive CD4 + thymocytes in zDCΔMHC II mice compared with control mice, their Vβ chain usage was comparable (Fig. 5, A and B).
To check for the presence of autoreactive CD4 + T cells in the periphery, we analyzed T cells in the spleen, mLN, and lamina propria of the large intestine for the presence of activated and cytokine-producing CD4 + T cells. zDCΔMHC II mice showed no evidence of increased levels of such cells (Fig. 6). To the contrary, we found reduced frequencies of total CD4 + T cells, as well as increased frequencies of naive CD4 + T cells and reduced frequencies of activated CD4 + T cells, in zDCΔMHC II mice when compared with controls ( Fig. 6). In addition, zDCΔMHC II mice showed lower frequencies of peripherally induced regulatory T cells (iT reg cells; defined as Nrp1 − ) in the mLN and lamina propria of the large intestine compared with controls. This is in accordance with a previously described cDC-mediated feedback mechanism controlling T reg cell numbers (Darrasse-Jèze et al., 2009). Despite reduced iT reg cells, the relative frequency of CD4 + T cells producing IL-2, IL-17, or IFN-γ was comparable or reduced in zDCΔMHC II mice compared with controls ( Fig. 6, D-F). This was also confirmed by quantitative PCR (qPCR) analysis of the large intestine, where we found similar expression levels of IL-17 and IFN-γ ( Fig. 3 I). Thus, in the absence of MHC II expression by cDCs, the defect in peripheral T reg cell induction did not result in aberrant levels of CD4 + T cell activation.
In contrast to CD4 + T cells, we observed a higher frequency of activated CD8 + T cells (CD3 + CD8 + CD62L − CD44 + ) in the spleen and mLN of zDCΔMHC II mice (Fig. 6,D and E). Furthermore, zDCΔMHC II mice showed unaltered or slightly reduced frequencies of γδ T cells in spleen, mLN, and large intestine compared with control mice, ruling out the involvement of pathogenic γδ T cells (Park et al., 2010;not depicted). To investigate whether the phenotype in zDCΔMHC II mice might be a result of insufficient CD4 + T cell activation, we depleted CD4 + T cells in control (MHC II sufficient) mice. Within 4 wk of depletion, mice developed neutrophilia, and elevated levels of Lipocalin-2 were detectable in their feces, although mice did not develop rectal prolapse within the 4 wk of depletion (Fig. 7 A; and not depicted). To test whether the observed increase in activated CD8 + T cells contributed to the phenotype in zDCΔMHC II mice, we depleted CD8 + T cells for 4 wk. However, CD8 + T cell-depleted zDCΔMHC II mice did not differ from nondepleted zDCΔMHC II mice (Fig. 7 B). Collectively, these data suggest that the phenotype in zDCΔMHC II mice is associated with insufficient adaptive CD4 + T cell responses but not pathogenic γδ or CD8 + T cells.

Microbiota is required for intestinal inflammation
To test the possibility that zDCΔMHC II mice develop intestinal inflammation as a result of inefficient effector CD4 + T cell responses to intestinal bacteria, we treated the mice with broad-spectrum antibiotics. Lipocalin-2 in the feces, Csf3 in serum, and neutrophil levels in the spleen of zDCΔMHC II mice dropped to baseline levels after 4 wk of antibiotic treatment (Fig. 8, A-C). Consistent with these findings, antibiotic-treated zDCΔMHC II mice were free of intestinal inflammation and inflammatory infiltrate as determined by histology (Fig. 8 E). Moreover, maintaining zDCΔMHC II mice in a germ-free environment completely prevented the development of all signs of disease, including intestinal inflammation, high levels of Lipocalin-2, elevated Csf3 levels, neutrophilia, and increased numbers of activated CD8 + T cells (Fig. 8, A-E). These results indicate that the intestinal inflammation in zDCΔMHC II mice is dependent on the microbiota. They also suggest that rather than causing the disease, the activated CD8 + T cells in specific pathogen-free (SPF) zDCΔMHC II mice develop in response to microbiota-mediated inflammation.

zDCΔMHC II mice exhibit impaired antimicrobial adaptive immune responses
IgA, the most abundant antibody isotype at mucosal surfaces, is secreted into the intestinal lumen, where it coats bacteria that enter the host and elicit adaptive immune responses (Palm et al., 2014). mLNs, Peyer's patches (PPs), and isolated lymphoid follicles are the primary sites of adaptive immune responses against intestinal antigens. zDCΔMHC II mice showed normal levels of B cells in the mLNs, PPs, and lamina propria of the large intestine. Whereas GC B cells were reduced in zDCΔMHC II mice, the relative frequency of IgA + B cells was not; the only significant defect in this compartment was in the lamina propria, suggesting that zDCΔMHC II mice do not have an overall defect in class switching. Consistent with the relative paucity of GC B cells, zDCΔMHC II mice showed significantly reduced frequencies of follicular T helper (TFH) cells in both mLNs and PPs (Fig. 9, A-D). These results support the idea that zDCΔMHC II mice have aberrant antimicrobial adaptive immune responses due to insufficient CD4 + T cell help (Grajales-Reyes et al., 2015).
To directly measure adaptive immune responses against intestinal bacteria, we colonized germ-free control and zDCΔMHC II mice with a mixture of six different commensal intestinal bacteria (Escherichia coli, Enterococcus faecalis, Bifidobacterium longum, Lactobacillus plantarum, Bacteroides vulgatus, and Ruminococcus gnavus). Colonization was similar for control and zDCΔMHC II mice as determined by PCR 8 wk after colonization (Fig. S2). Although germ-free mice have no detectable GC B or TFH cells in gut-associated lymphoid tissues, newly colonized control mice showed robust GC B and TFH cell responses in mLNs and PPs. In contrast, these responses were barely detectable in colonized zDCΔMHC II mice, even 8 wk after colonization (Fig. 10, A and B).
Consistent with the near absence of GC responses, IgA levels in the feces and serum of colonized zDCΔMHC II mice were significantly lower compared with colonized control mice. Fecal IgA increased progressively in control mice starting after day 7, reaching a mean of 200-400 ng/mg feces. In contrast, fecal IgA reached a plateau of around 10-20 ng/mg feces by day 12 and remained relatively constant thereafter in zDCΔMHC II mice (Fig. 10, C and D). Consistent with the overall lower levels of serum and fecal IgA, bacteria isolated from feces of zDCΔMHC II mice were coated with far lower levels of IgA than bacteria from feces of control mice (Fig. 10 E).
Collectively, these data reveal that zDCΔMHC II mice have defective adaptive immune responses against intestinal commensals, and this defect is associated with a loss of bacterial segregation from the epithelium and severe colitis.

DIS CUS SION
cDCs link innate and adaptive immunity by sensing pathogens and initiating adaptive immune responses. Although the two physiological functions of cDCs are likely to play distinct roles in immune homeostasis, they have not previously been evaluated independently. In the gut, cDCs sense and capture gut microbes in part by extending their processes into the gut lumen (Macpherson and Uhr, 2004;Niess et al., 2005;Chieppa et al., 2006;Vallon-Eberhard et al., 2006). Microbial sensing induces cDCs to produce cytokines such as IL-23, which are required to activate innate lymphoid cells (Kinnebrew et al., 2012;Satpathy et al., 2013). In addition, the ingested microbes are carried to local lymphoid organs, such as the mLNs, processed, and presented to T cells to initiate adaptive immune responses (Macpherson and Uhr, 2004;Niess et al., 2005).
Ablation experiments using CD11c DTR mice, conditional deletion of genes (e.g., Irf4, Irf8, or Notch2) with CD11c Cre mice, and Batf3-deficient mice result in loss of cDCs and cDC subsets, and therefore concomitant loss of both innate and adaptive immune functions (Hildner et al., 2008;Lewis et al., 2011;Persson et al., 2013;Schlitzer et al., 2013). For example, CD11c Cre -mediated DC deletion has been described to activate a feedback loop that produces high levels of circulating Flt3L, resulting in a myeloproliferative syndrome characterized by extramedullary hemacells that proliferated in response to OVA + pIC. Each data point represents one individual mouse (n = 5-6). (I-M) cDCs were isolated from spleens of control and zDCΔMHC II mice. 250,000 cells were incubated with 1 µM CpG 1668, 1 µg/ml LPS, and 1 µg/ml pIC. 16 h later, expression of CD80 and CD86 was determined by flow cytometry. Cytokines produced by cDCs after stimulation were measured in the supernatant. *, P < 0.05. (I) Histograms show expression of CD80 and CD86 on cDCs. Filled histograms show expression of CD80 and CD86 on unstimulated cells. Open histograms show levels of CD80 and CD86 after stimulation. (J and K) Graphs depict levels of expression (MFI) of CD80 and CD86 on cDCs. (L and M) Graphs depict production of IL-6 and TNF by cDCs. n = 3. (G) Histopathological analysis of the large intestine of control (left) and zDCΔMHC II mice (right) that were maintained under SPF conditions. The large inset represents enlarged view of small inset and shows cellular infiltrates. (H) Graph depicts frequency of neutrophils among living cells in the lamina propria of the large intestine. Each data point represents one individual mouse (n = 5-7). (I) Expression of cytokines in the large intestine (whole tissue) was determined by qPCR. Graph depicts expression as relative units. Each data point represents one individual mouse (n = 5). (J) Expression of RegIIIγ, S100A8, and S100A9 in epithelial cells isolated from the small intestine was determined by qPCR. Graphs depict expression as relative units. Each data point represents one individual mouse (n = 15-17). (K) Graph depicts frequency of ILC3s (CD45 + /CD19 − /B220 − /NK1.1 − /Gr1 − /CD3 − /Thy1.2 + /RORγt + ) among living cells in the topoiesis, splenomegaly, and neutrophilia (Birnberg et al., 2008). Depletion of cDCs in this model likely has additional consequences beyond elevated Flt3L levels. Our data suggests that the phenotype may have been mediated in part by intestinal microbes. In a different study, however, CD11c Cremediated DC depletion was associated with colitis and increased numbers of activated CD4 + T cells (Birnberg et al., 2008;Ohnmacht et al., 2009). Although cDCs play an important role for T cell selection in the thymus (Perry et al., 2014), in our model we found no evidence of activated or autoreactive CD4 + T cells. In addition to being unable to distinguish between the innate and adaptive functions of cDCs, interpretation of these earlier studies was confounded in part by deletion of CD11c expressing monocytes, macrophages, pDCs, and NK cells. In contrast, deletion of MHC II in cDCs using zDC Cre selectively ablates the ability of this cell type to initiate adaptive immunity while leaving its innate activity intact. The described phenotype was specific for cDCs because zDC expression is restricted to cDCs and their precursors. zDC is first expressed in committed cDC progenitors (pre-DCs) in the BM and is maintained in all subsequent stages of cDC development, including all major subsets of cDCs (Meredith et al., 2012;Satpathy et al., 2012).
Our experiments show that cDC-induced adaptive immune responses are required to maintain homeostasis between intestinal flora and the host. Other MHC IIexpressing antigen-presenting cells, such as activated monocytes, macrophages, or monocyte-derived DCs, are unable to compensate for this requirement, because they are not specialized for CD4 + T cell priming. Absence of MHC II on cDCs in zDCΔMHC II mice is associated with intestinal inflammation and elevated serum levels of Csf3, an acute phase reactant. This phenotype is dependent on bacteria, as Csf3 levels dropped to normal levels when the mice were treated with antibiotics or housed under germ-free conditions. These results highlight a previously underappreciated role for cDC-mediated adaptive immune responses to commensal bacteria. Mice lacking MHC II on cDCs may therefore be considered immunocompromised, as they are unable to efficiently prime CD4 + T cells. Consistent with our observations, mice lacking CD4 + T cells and HIV-infected humans that suffer CD4 + T cell loss show increased microbial translocation and systemic immune activation (Brenchley et al., 2006;Sandler and Douek, 2012).
cDC-initiated CD4 + T cell responses are likely to help keep the gut microbiome in check by at least several distinct mechanisms. They are required to activate and induce differentiation of T cells, such as protective TH17/TH22 cells, which prevent invasion of enterobacteria (Mangan et al., 2006;Curtis and Way, 2009;Ivanov et al., 2009;Basu et al., 2012). Consistent with this idea, T cell-deficient mice and humans show increased risk for intestinal inflammation (Tamura et al., 2005). In addition, cDCs are also essential in initiating TFH responses that are required to produce germinal centers and high-affinity IgA (Crotty, 2011;Maruya et al., 2013;Kato et al., 2014;Kubinak et al., 2015). Finally, MHC II expression by cDC is required to maintain the physiological number of induced T reg cells (Darrasse-Jèze et al., 2009). In zDCΔMHC II mice, reduced numbers of T reg cells did not result in increased numbers of activated CD4 + T cells because their activation requires MHC II expression on cDCs. Nevertheless, decreased numbers of induced T reg cells could contribute to the severity of the phenotype by altering innate immune responses.
Colonization of germ-free mice allowed us to follow the immune response against a limited number of commensal bacteria. This controlled experiment showed that zDCΔMHC II mice have diminished TFH, GC, and IgA responses to intestinal bacteria. Mice that are unable to secrete polymeric antibodies into the intestinal lumen show elevated serum IgG levels specific for commensal bacteria, suggesting more translocation of intestinal bacteria in the absence of luminal IgA (Johansen et al., 1999). In addition, translocation of bacteria was observed in germ-free B celldeficient mice that were colonized with SPF microbiota (Macpherson and Uhr, 2004), emphasizing the importance of the adaptive response in containing the microbiota. However, the phenotype observed in zDCΔMHC II mice cannot be mediated solely by a defective IgA response against intestinal bacteria. AID −/− mice, which are unable to switch to IgA, do not develop the phenotype in our SPF facility, possibly because they have unimpaired TFH and germinal center responses and IgM can compensate in part for IgA. In zDC Cre+ MHC II fl/fl mice, however, CD4 + TFH cell priming is defective, resulting in diminished GCs. This idea is supported by the observation that CD4 + T cell depletion partially phenocopies zDC Cre+ MHC II fl/fl mice. cDCs in zDCΔMHC II mice show intact innate microbial sensing and cytokine production. These features are essential for activation and normal function of innate lymphoid cells (Bernink et al., 2015). The observation that zDCΔMHC II mice develop intestinal inflammation indicates that intact innate mechanisms are insufficient to maintain the normal balance between host and intestinal commensals. Transferable risk for colitis has been described for mice lacking components of the inflammasome, which has been attributed to alterations in intestinal microbiota (Elinav et al., 2011;Palm et al., 2014). In our experiments, control mice did not develop any signs of inflammation, although they were cohoused with zDCΔMHC II mice. The fact that cohoused control mice did not get sick, along with the finding that they share a similar microbiome, indicates that commensal bacteria that can be controlled and are usually harmless in mice with an unimpaired immune system may be responsible for disease in zDCΔMHC II mice that have defective adaptive immune responses. Collectively, the data indi-cate that in the steady state, cDC-mediated CD4 + T cell priming and subsequent adaptive immune responses are essential to maintain homeostasis and prevent intestinal commensals from producing pathology.

MAT ERI ALS AND MET HODS
Mice. zDC Cre knock-in mice were generated by homologous recombination in C57BL/6 albino embryonic stem cells at The Rockefeller University Gene Targeting Resource Cen- (A) Graphs depict serum levels of Csf3, frequency of neutrophils among living cells in the spleen, and concentration of Lipocalin-2 in the feces of control and CD11cΔMHC II mice. Each data point represents one individual mouse (n = 6). Mice were analyzed at the age of 6-8 wk. (B and C) WT B6 mice were irradiated and reconstituted with BM from control or zDCΔMHC II mice. A third group received a 1:1 mix of BM from control and zDCΔMHC II mice. (B) Graph depicts percentage of cDCs expressing MHC II. (C) Graphs depict serum levels of Csf3, frequency of neutrophils among living cells in the spleen and concentration of Lipocalin-2 in the feces of mice reconstituted with BM from control, zDCΔMHC II mice, or a 1:1 mix of BM from control and zDCΔMHC II mice, respectively. Each data point represents one individual mouse (n = 10). Mice were analyzed 10 wk after irradiation. *, P < 0.05. (D) Feces was collected from control and zDCΔMHC II mice (10-12 wk old) and microbial composition was analyzed by 16S rRNA sequencing. UniFrac analysis is shown. Cage numbers indicate which mice were cohoused in the same cage.
ter (New York, NY). The targeting strategy was the same as for the previously described zDC DTR mice (Meredith et al., 2012). zDC Cre mice were maintained on a C57BL/6 background. Itgax Cre (CD11c Cre ; stock number 008068), Rosa26 lSlYFP (Rosa26 lSlYFP ; stock number 006148), iab neo (MHC II fl/fl ; stock number 013181), and Irf4 fl/fl (IRF4 fl/fl ; stock number 009380) mice were purchased from The Jackson Laboratory. Mice were housed in The Rockefeller University Comparative Bioscience Center (New York, NY) under SPF and germ-free conditions, respectively. All experiments were performed in accordance with the National Institutes of Health guidelines and approved by The Rockefeller University Animal Care and Use Committee. Germ-free mice were obtained by transfer of newborn pups (Cesarean section) into germ-free isolators. Germ-free status was confirmed by plating feces on a broad range of nutrient agars, as well as by qPCR analysis (16S rRNA). zDCΔMHC II mice were bred with MHC II fl/fl mice. This way we could compare Cre + (zDCΔMHC II) and Cre − (control) littermates. It is also important to note that zDCΔMHC II males were bred with MHC II fl/fl females. This way, the fostering female is healthy and does not develop the described phenotype, and development of pups is not affected by the genotype of the fostering female. Littermates were cohoused during all experiments. Mice were generally analyzed at 8-12 wk of age. BM chimeras. BM chimera recipients were irradiated with two doses of 525 rad 3 h apart and injected with 5-10 × 10 6 BM cells. BM chimeras were analyzed 10 wk after irradiation. . PI (Sigma-Aldrich), DAPI (Invitrogen), or Aqua live/ dead (Invitrogen) was added to exclude dead cells from analysis. For intracellular cytokine staining, cells were stimulated for 4 h using Leukocyte Activation Cocktail with GolgiPlug (BD). Surface stained cells were fixed with Cytofix/Cytoperm (BD) and washed with Perm/Wash buffer (BD). Cells were analyzed on a LSR Fortessa flow cytometer (BD). Analysis was performed using FlowJo software (Tree Star).
Spleen, LN, thymus, and PP cell isolation. For isolation of mononuclear phagocytes, spleen and LNs were mechanically disrupted and incubated with HBSS (Gibco) + 5% FBS + 0.5 mg/ml Collagenase D (Roche) at 37°C for 30 min. Cells were filtered through 100 µM cell strainers and washed. Erythrocytes were lysed with ACK Lysing buffer (Gibco). For isolation of T and B cells, spleen, LNs, thymi, and PP were mechanically disrupted, filtered through 100-µM cell strainers, and washed. Erythrocytes were lysed with ACK Lysing buffer (Gibco).
In vitro stimulation of DCs. 250,000 cDCs (CD11c MACS enrichment) were stimulated with 1 µM CpG 1668, 1 µg/ml LPS, and 1 µg/ml pIC, respectively. Expression of CD80 and CD86 were determined by flow cytometry 16 h later. Cytokines were measured using the Cytometric Bead Array kit (BD).  Intestinal cell isolation. Small and large intestine were excised and freed of mesentery and fat. Feces were removed and PPs were excised from the small intestine. Both small and large intestine were opened longitudinally and washed in HBSS, followed by HBSS + 1 mM DTT (Sigma-Aldrich). Tissue was cut into 1-cm pieces and incubated with 25 ml of HBSS + 5% FBS + 5 mM EDTA for 15 min at 37°C at 230 rpm. Supernatant containing intraepithelial lymphocytes was decanted and spun down (for RNA isolation from epithelial cells, see next section). Tissue was washed with 10 ml of HBSS + 5% FBS for 15 min at 37°C at 230 rpm. The gut tissues were then finely chopped and digested in HBSS + 5% FBS + 1X Sodium Pyruvate + 25 mM Hepes + 50 µg/ml DNaseI + 0.05 mg/ml Collagenase VIII for 1 h at 37°C at 100 rpm. Cells were separated by discontinuous Percoll gradient (70%/35%) centrifugation. Cells were isolated from the interphase.
Histology. Tissue was fixed in 10% Formalin. Tissue was embedded, cut, and stained by the Tri-Institutional Laboratory of Comparative Pathology (New York, NY). A pathologist evaluated the tissue sections in a blinded fashion and took pictures representing the phenotype of the respective group.

Figure 7. CD4 + T cell depletion induces neutrophilia and intestinal inflammation.
CD8 + T cell depletion does not affect disease. (A) Cre − control mice were injected with PBS or 100 µg anti-CD4 antibody (clone GK1.5) every 2-3 d for 4 wk. Graphs depict frequency of neutrophils among living cells in the spleen and concentration of Lipocalin-2 in the feces. Each data point represents one individual mouse (n = 9-13). (B) Cre − control and zDCΔMHC II mice were injected with PBS or 100 µg anti-CD8 antibody (clone 53-6.72) every 2-3 d for 4 wk. Graphs depict concentration of Lipocalin-2 in the feces, serum levels of Csf3, and frequency of neutrophils among living cells in the spleen. Each data point represents one individual mouse (n = 5). *, P < 0.05. markers was analyzed by flow cytometry (pooled mesenteric and inguinal LN).
Time course/aging experiments. Body weight was measured weekly, as well as occurrence of rectal prolapse. Feces and serum samples were collected and stored at -20°C until further use.
Bacteria isolation and IgA staining. Bacteria were isolated from feces as described previously (Palm et al., 2014). Coating of bacteria with endogenous IgA was determined by staining bacteria with anti-mouse-IgA-PE (clone mA-6E1). Where indicated, MFIs were normalized to the sample with the lowest MFI to be able to compare different time points.
DNA extraction, V4-V5 16S rRNA gene amplification, multiparallel sequencing, and sequence analysis. Fecal samples and intestinal contents were frozen immediately after collection in a dry ice/ethanol slurry and stored at −80°C. DNA was extracted using the phenol-chloroform and bead beating method described previously (Ubeda et al., 2010). The V4-V5 region of the 16S rRNA gene Figure 8. Treatment of zDCΔMHC II mice with antibiotics reduces intestinal inflammation. (A) Graph depicts concentration of Lipocalin-2 in the feces of control and zDCΔMHC II mice that were maintained under SPF conditions, zDCΔMHC II mice that received antibiotics with the drinking water for 4 wk, and zDCΔMHC II mice that were maintained under germ-free conditions. Each data point represents one individual mouse (n = 4-12). (B) Graph depicts serum levels of Csf3. Each data point represents one individual mouse (n = 4-12). (C) Graph depicts frequency of neutrophils among living cells in the spleen. Each data point represents one individual mouse (n = 4-12). (D) Graph depicts frequency of CD62L − CD44 + cells among CD8 + T cells in the spleen. Each data point represents one individual mouse (n = 4-8). (E) Histopathological analysis of the large intestine of control and zDCΔMHC II mice that were maintained under SPF conditions, zDCΔMHC II mice that received antibiotics in the drinking water for 4 wk, and zDCΔMHC II mice that were maintained under germ-free conditions. Large inset represents enlarged view of small inset and shows cellular infiltrates. *, P < 0.05.
was amplified and sequenced with the Illumina Miseq platform, as described previously (Buffie et al., 2015). Sequences were analyzed using version 1.33.3 of the MOT HUR pipeline (Schloss et al., 2009) as described in Buffie et al. (2015). Sequences with distance-based similarity of at least 97% were assigned the same OTU (operational taxonomic unit) and representative OTUs were classified using a modified Greengenes reference database (DeSantis et al., 2006).
Colonization of germ-free mice. Germ-free control and zDCΔMHC II mice were colonized with a mix of six in-  Germ-free control and zDCΔMHC II mice were colonized with a mixture of six different intestinal bacteria. Flow cytometric analysis of TFH cells (CD3 + CD4 + CD62L − CD44 + PD-1 + CXCR5 + ) and GC B cells (CD19 + B220 + CD38 − CD95 + ) isolated from mLNs (A) and PPs (B) of control and zDCΔMHC II mice 61 d after colonization. Graphs depict percentage of TFH cells among CD4 + T cells and percentage of GC B cells among all B cells in mLNs (A) and PPs (B). Each data point represents one individual mouse (n = 3-5). (C and D) Graphs depict amount of total IgA in feces (C) and serum (D) 61 d after colonization. Each data point represents one individual mouse (n = 3-5). (E) Bacteria were isolated from the feces of control and zDCΔMHC II mice and stained with antimouse-IgA. Graph depicts normalized MFI of IgA coated bacteria. Each data point represents one individual mouse (n = 3-5). *, P < 0.05.

Statistical analysis.
Results are shown as means + SEM. Comparisons between groups was done using Student's two-tailed t test analysis. Statistical significance was determined as p-values <0.05.