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Identification of subepithelial mesenchymal cells that induce IgA and diversify gut microbiota

Nature Immunology volume 18, pages 675–682 (2017)Cite this article

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Abstract

Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell–dependent antigen sampling and B cell–dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.

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Figure 1: RANKL regulates epithelial CCL20 expression in the FAE of GALTs.
Figure 2: Membrane-bound RANKL induces M cell differentiation and CCL20 expression in the FAE of GALTs.
Figure 3: RANKL+ mesenchymal cells in contact with the FAE show distinct gene expression profiles.
Figure 4: Subepithelial mesenchymal cells have a role as MCi cells.
Figure 5: RANKL produced by mesenchymal cells is essential for B cell–DC interaction and antigen sampling in the SED.
Figure 6: Mesenchymal RANKL regulates germinal center formation in PPs.
Figure 7: Mesenchymal RANKL induces bacteria-specific IgA responses in the gut.
Figure 8: RANKL supplied by mesenchymal cells facilitates diversification of the gut microbiota.

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Acknowledgements

We are grateful to T. Yamada (Tokyo Institute of Technology), H. Mori (Tokyo Institute of Technology), K. Hase (Keio University), Y. Obata (Keio University), H. Kiyono (the University of Tokyo), Y. Morishita and H. Tanaka (Okayama University) for insightful discussion and valuable technical assistance. We thank all our laboratory members for discussion. We thank S. Nitta, Y. Nakayama, K. Kaneki, Y. Ogiwara, K. Kusubata, R. Yanobu-Takanashi and K. Nakano for technical assistance. D. Kioussis (UK National Institute for Medical Research) provided Vav-iCre mice. Supported in part by a Grant-in-Aid for Specially Promoted Research from the Japan Society for Promotion of Science (JSPS) (15H05703); grants for Exploratory Research for Advanced Technology (ERATO) Program and PRESTO from JST; Yakult Bio-Science Foundation; and JSPS KAKENHI (25111503 and 16H05202). K.N. was supported by Japanese Society for Immunology (JSI) Kibou Scholarship for Doctoral Students in Immunology.

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Authors and Affiliations

  1. Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan

    Kazuki Nagashima, Shinichiro Sawa, Takeshi Nitta, Masanori Tsutsumi & Hiroshi Takayanagi

  2. Department of Molecular Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

    Shinichiro Sawa

  3. Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan

    Tadashi Okamura

  4. Department of Infectious Diseases, Section of Animal Models, Research Institute National Center for Global Health and Medicine, Tokyo, Japan

    Tadashi Okamura

  5. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria

    Josef M Penninger

  6. Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

    Tomoki Nakashima

  7. Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Tokyo, Japan

    Tomoki Nakashima

  8. Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (AMED-CREST), Tokyo, Japan

    Tomoki Nakashima

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Contributions

K.N. performed most of the experiments, interpreted the results and prepared the manuscript. S.S. provided advice on project planning and data interpretation and contributed to the manuscript preparation. T. Nitta, T.O., M.T., J.M.P. and T. Nakashima provided genetically modified mice and contributed to data interpretation and discussion. H.T. directed the project and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Generation of Tnfsf11ΔS/ΔS mice by CRISPR–Cas9-mediated gene editing.

(a) Scheme of the Tnfsf11ΔS allele. The Tnfsf11 cleavage sites were genetically trimmed by CRISPR/Cas9-mediated homology-directed repair. (b) The target sequences of sgRNA1 and sgRNA2, and the sequences of single-stranded oligodeoxynucleotides (ssODNs). (c) Flow cytometry analysis of RANKL expression in lymphocytes in PPs. Histograms show RANKL expression patterns in the ILC3s or T cells of the PPs from Tnfsf11ΔS/ΔS and Tnfsf11+/+ mice after four hours of stimulation with PMA/ionomycin (n=8,5 and 3 mice respectively). (d) Representative histological sections of RANKL+ mesenchymal cell in the SED of PPs from Tnfsf11+/+ and Tnfsf11ΔS/ΔS mice. Sections were stained for podoplanin, RANKL and DAPI. Scale bars represent 10 μm. (e) Q-PCR analysis of the Tnfsf11 transcripts in MAdCAM-1−podoplanin+CD21−CD31−CD45− mesenchymal cells in PPs (n=5 and 3 mice respectively). Statistical analyses were carried out using Student’s t test (e) or analysis of variance (ANOVA) with Turkey’s multiple-comparison test (c). Error bars denote the mean ± SEM. NS, not significant (p>0.05); *P<0.05 and **P<0.01.

Supplementary Figure 2 Gating strategy for mesenchymal stromal cells and lymphocytes in PPs.

(a) Flow cytometry analysis of CD45−EpCAM−Ter119− stromal subsets in PPs. The CD45−CD21/35− fraction was enriched with a MACS Column before staining in order to eliminate CD45+ hematopoietic cells and CD21/35+ follicular dendritic cells. LECs, lymphatic endothelial cells; BECs, blood endothelial cells; DN, double negative cells. (b) Flow cytometry analysis of RANKL expression in CD45+ lymphocytes in PPs after four hours of stimulation with or without PMA/ionomycin. The numbers indicate the mean ± SEM. The data are from three independent experiments (n=3 mice).

Supplementary Figure 3 Histological analysis of mesenchymal cell populations in PPs.

(a) MAdCAM-1 expressing mesenchymal stromal cells in PPs. Blood endothelial cells (BECs), follicular dendritic cells (FDCs) and marginal reticular cells (MRCs) expressed MAdCAM-1. Scale bars, 100 μm (left and middle), 25 μm (right). (b) Analysis of T cell-containing interfollicular zones in the PPs from Tnfsf11fl/Δ, Tnfsf11fl/Δ Twist2-Cre, Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice. Scale bars, 200 μm. (c) Identification of RANKL expressing mesenchymal cells in the SED of PPs. Scale bars, 25 μm. (d) HE staining of the small or large intestine of Tnfsf11fl/Δ, Tnfsf11fl/Δ Twist2-Cre, Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice. Scale bars, 50 μm.

Supplementary Figure 4 Histological identification of RANKL+ mesenchymal cells in the large intestine.

(a) Representative images of colonic patches. RANKL+podoplanin+ mesenchymal cells were found to be in contact with the FAE (upper panels). MAdCAM-1 was expressed by podoplanin− blood endothelial cells (BECs), but MAdCAM-1+podoplanin+ MRCs were not observed (lower panels). Scale bars, 50, 20, 20 or 20 μm (left to right). (b,c) Representative sections of colonic ILFs (b) and cecal patches (c). RANKL+podoplanin+ mesenchymal cells interacting with the FAE were observed in colonic ILFs and cecal patches, while MAdCAM-1+podoplanin+ MRCs were identified only in cecal patches. The dashed lines indicate the FAE basal membrane. Scale bars, 50 μm (b, left), 100 μm (c, left), 15 μm (middle and right). (d) Representative histological images of the FAE of colonic patches, colonic ILFs and cecal patches of Tnfsf11+/+, Tnfsf11ΔS/ΔS and Tnfsf11fl/Δ Twist2-Cre mice. Scale bars, 10 μm. (e) Representative sections of the colonic patches, colonic ILFs and cecal patches from Tnfsf11fl/Δ and Tnfsf11fl/Δ Twist2-Cre mice. Scale bars, 100 μm (upper and lower panels), 50 μm (middle panels). (f) Representative images of the large intestinal lamina propria (LILP) obtained from Tnfsf11fl/Δ, Tnfsf11fl/Δ Twist2-Cre, Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice. Scale bars, 25 μm.

Supplementary Figure 5 Mesenchymal cell–specific deletion of RANKL in Tnfsf11fl/Δ; Twist2-Cre mice.

(a ,b,c) Histological sections of the SED of the PPs from Twist2-Cre CAG-tdTomato mice. The dashed lines indicate the FAE basal membrane. Scale bars, 5 μm (a), 25 μm (b,c). (d) Q-PCR analysis of the Pigr transcripts in the FAE of PPs from Tnfsf11fl/Δ and Tnfsf11fl/Δ Twist2-Cre mice (n=8 PPs). (e) Q-PCR analysis of the Tnfsf11 transcripts in MAdCAM-1−podoplanin+CD21−CD31−CD45− mesenchymal cells (n=5 and 4 mice, respectively). (f) Flow cytometry analysis of RANKL expression in ILC3s in PPs from Tnfsf11fl/Δ and Tnfsf11fl/Δ Twist2-Cre mice after four hours of stimulation with PMA/ionomycin. (g) Mean fluorescence intensity (MFI) of RANKL expression in ILC3s and T cells (n=3 mice). (h) Q-PCR analysis of the Tnfsf11 transcripts in the MAdCAM-1−podoplanin+CD21−CD31−CD45− mesenchymal cells of Tnfsf11fl/Δ and Tnfsf11fl/Δ Vav-iCre mice (n=4 mice). (i,j) Flow cytometry analysis of RANKL expression in ILC3s (i) and T cells (j) in PPs from Tnfsf11+/+, Tnfsf11fl/Δ and Tnfsf11fl/Δ Vav-iCre mice after four hours of stimulation with PMA/ionomycin (n=23,9,7 and 4 mice respectively). Statistical analyses were carried out using Student’s t test (d,g,h) or analysis of variance (ANOVA) with Dunnett's multiple-comparison test (i,j) Error bars denote the mean ± SEM. NS, not significant (p>0.05); *P<0.01 and **P<0.001.

Supplementary Figure 6 Epithelial RANK is crucial for IgA induction in the gut.

(a) Assessment of antigen sampling in PPs. Ligated loop assay was performed in the Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice (n=12 slides). (b) The number of IgD+ pre-GC B cells interacting with CD11c+ DCs in the SED quantified by histological sections (n=8 and 7 slides respectively). (c) Q-PCR analysis of the Aicda transcripts in the B220+ B cells sorted from PPs (n=7 mice). (d) B cell-containing ILFs in the terminal ileum quantified by whole-mount immunostaining (n=12 and 13 mice respectively). (e) The size of PPs measured by stereomicroscopy (n=33 and 31 PPs respectively). (f) Histological analysis of GL7+ germinal center formation in PPs. Scale bars, 200 μm. (g,h,i) Flow cytometry analysis of GL7+ GC B cells in B220+ cells (g), IgA+ B cells in B220+ cells (h) and PD-1+CXCR5+ Tfh cells in CD3+CD4+ cells (i) in the PPs (n=8 and 9 mice). (j) IgM+ and IgA+ cells in CD45+CD3− cells in the SILP (n=9 mice). (k) The fecal IgA and serum IgA, IgG and IgM levels measured by ELISA (n=7 mice). (l) Cholera toxin-specific IgA production determined by ELISA (n=6 mice). (m) The percentages of IgA-coated bacteria in the total bacteria determined by flow cytometry (n=7 mice). The data are from 3 (m), 4 (e), 6(i), 7(g,h,j) and 10 (d) independent experiments. Statistical analyses were carried out using Student’s t test, and the data are shown as the mean ± SEM. NS, not significant (p>0.05); *P<0.05, **P<0.01 and ***P<0.001. 4-6 week old mice were used along with littermate controls. A similar reduction in IgA production was observed in 8-10 week old Tnfrsf11afl/Δ Vil1-Cre mice.

Supplementary Figure 7 Analysis of B cell homeostasis in M cell–deficient mice.

(a,b) Flow cytometry analysis of B cells in the bone marrow (BM), superficial lymph nodes (sLNs), peritoneal cavity (PC), peripheral blood (PB) or spleen obtained from Tnfsf11fl/Δ, Tnfsf11fl/Δ Twist2-Cre (a), Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice (b) (n=4 mice). (c,d) Flow cytometry analysis of CD11c+1-A/1-E+ dendritic cells in Peyer’s patches (PPs) (n=3 mice) (c) (n=7 mice) (d). Statistical analyses were carried out using Student’s t test, and the data are shown as the mean ± SEM. NS, not significant (p>0.05).

Supplementary Figure 8 Epithelial RANK is required for the diversification of the gut microbiota.

(a) Relative abundance (major order; family) in the fecal pellets from Tnfrsf11afl/Δ and Tnfrsf11afl/Δ Vil1-Cre mice (n=12 fecal pellets from 6 mice). (b) Relative abundance of the 16S rRNA gene of SFB among the total bacteria (n=17 and 24 mice respectively). (c) Differences between bacterial communities. PCoA and PERMANOVA comparisons of Bray-Curtis distances are shown (n=12 fecal pellets from 6 mice). (d) Diversity of bacterial species shown by a chao1 refraction measure based on 1,000-10,000 sequences (n=12 fecal pellets from 6 mice). Statistical analyses were carried out using Student’s t test (b,d). Error bars denote the mean ± SEM. *P<0.05 and **P<0.01. 4-7 week old mice were separately caged after weaning and used along with littermate controls. (e) Schematic model of the IgA production against the gut microbiota organized by MCi cells in GALTs. Membrane-bound RANKL provided by MCi cells induces M cell differentiation and CCL20 production by enterocytes in the FAE. CCL20 and the microbial antigens sampled by M cells cooperatively stimulate the B cell-DC interaction required for the initiation of IgA class switching. IgA is secreted into the lumen and shapes the gut microbiota. MRCs, another mesenchymal stromal subset in GALTs, express high levels of CXCL13 and may guide B cells from the SED to follicles so as to promote germinal center formation.

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Nagashima, K., Sawa, S., Nitta, T. et al. Identification of subepithelial mesenchymal cells that induce IgA and diversify gut microbiota. Nat Immunol 18, 675–682 (2017). https://doi.org/10.1038/ni.3732

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