Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.


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We did not performed sample-size calculation. The sample size was empirically started at 3 or 5, and was increased at the next experiment when there was no significant difference. Ultimately at least two independent experiments were conducted to draw conclusions.
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October 2018

Validation
The specificity of anti-Tnfaip2 antibody was confirmed by Western blotting and immunohistochemistry of tissues from Tnfaip2 knockout mice. Likewise, The specificity of anti-OPG antibody was confirmed by immunohistochemistry of tissues from OPG knockout mice. All other antibodies used in this study were validated by manufactures.

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Wild animals
This study did not involve wild animals.

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Protocols approved by Hokkaido University, IMSUT, and Keio University were used for all animal experiments.
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Flow Cytometry
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Methodology Sample preparation
Single cell suspensions of gut tissues were prepared as described previously40. Cell population abundance In this study, cell sorting was not performed.

Gating strategy
For detection of lymphoid cell populations, Lymphocyte, monocyte and neutrophil populations were gated on a forward scatter (FSC)/side scatter (SSC) plot. Dead cells were excluded as SYTOX-blue positive cells in Fig. 4, Fig. 5F or Live Dead Aqua-V500 negative cells in Fig. 5. Detailed gating strategies were shown as below. Positive and negative were discriminated for each cell population by counter plot (contour line plot). In the case where no independent cell population was formed, the wide interval between contour lines was defined as boundaries between positive and negative. For Bug-Ig flow cytometry, bactreria were gated on DAPI-positive/SSC-A plot. For determining boundaries between "positive" and "negative", unstained bacteria were used as negative controls. IgG-Alexa488-positive B220-APCCy7-positive d: IgA-PECy7-positive B220-APCCy7-negative IgG-Alexa488-positive B220-APCCy7-negative e: Live Dead Aqua-V500-negative B220-V421-negative → CD3-Alexa700-negative CD4-APCH7-positive Live Dead Aqua-V500-negative B220-V421-negative → CD3-Alexa700-negative CD8a-Alexa488-positive