IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake - PubMed (original) (raw)

. 2017 Jun 5;214(6):1607-1618.

doi: 10.1084/jem.20160770. Epub 2017 May 16.

Takashi Kanaya 1 2, Koji Hase 3 4, Sayuri Sakakibara 1, Tamotsu Kato 1, Naoko Tachibana 1, Takaharu Sasaki 1, Yusuke Hashimoto 1 2, Toshiro Sato 5, Hiroshi Watarai 6, Jun Kunisawa 7 5, Naoko Shibata 7, Ifor R Williams 8, Hiroshi Kiyono 7 9, Hiroshi Ohno 10 2

Affiliations

IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake

Toshi Jinnohara et al. J Exp Med. 2017.

Abstract

Interleukin-22 (IL-22) acts protectively and harmfully on intestinal tissue depending on the situation; therefore, IL-22 signaling needs to be tightly regulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intestinal and lymphoid tissues, although its precise distribution and roles have remained unclear. In this study, we show that IL-22BP is highly expressed by CD11b+CD8α- dendritic cells in the subepithelial dome region of Peyer's patches (PPs). We found that IL-22BP blocks IL-22 signaling in the follicle-associated epithelium (FAE) covering PPs, indicating that IL-22BP plays a role in regulating the characteristics of the FAE. As expected, FAE of IL-22BP-deficient (Il22ra2-/-) mice exhibited altered properties such as the enhanced expression of mucus and antimicrobial proteins as well as prominent fucosylation, which are normally suppressed in FAE. Additionally, Il22ra2-/- mice exhibited the decreased uptake of bacterial antigens into PPs without affecting M cell function. Our present study thus demonstrates that IL-22BP promotes bacterial uptake into PPs by influencing FAE gene expression and function.

© 2017 Jinnohara et al.

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Figures

Figure 1.

Figure 1.

IL-22BP is expressed by DCs located on SED of PPs. (A) Relative Il22ra2 mRNA expression of intestinal tissue, small intestine (SI), colon, cecum, and PPs (normalized with Actb; n = 10). Data are pooled from two independent experiments. (B) In situ hybridization analysis of PPs with a digoxigenin-labeled specific RNA probe for Il22ra2 mRNA. Nuclei were counterstained with Nuclear Fast Red. (C) Immunohistochemistry of PP tissues with anti–IL-22BP antibody. Green colors show IL-22BP–positive cells, and blue colors show nuclei. Dotted lines indicate FAE. Data are representative of three independent experiments. (D) CD11c-enriched cells were stained with antibodies to isolate DCs. CD3−B220−CD45+ PP cells were selected and analyzed by the expression of CD11c and MHCII. CD11chighMHCIIhigh cells were isolated as DCs, and CD11b+CD8α−, CD11b−CD8α+, and CD11b−CD8α− (DN) DCs were sorted from this DC population. Data are representative of four independent experiments. (E) Relative Il22ra2 mRNA expression of sorted DC and macrophage (Mac) populations from PPs (n = 5), MLNs (n = 5), spleens (SPs; n = 5), and LPs (n = 4). Data were normalized with Actb. Data are pooled from four independent experiments. Means ± SD are shown. (F) Recombinant IL-22 was intravenously injected to a WT mouse. After 15 min, PPs were collected and immunostained with anti-pSTAT3 antibody. Green colors show pSTAT3, and blue colors show nuclei. Dotted lines indicate FAE. Data are representative of at least four independent experiments. Bars: (B) 100 µm; (C and F) 50 µm.

Figure 2.

Figure 2.

IL-22BP deficiency releases IL-22 signaling on the FAE. (A) PP tissues from WT and Il22ra2−/− mice were immunostained with anti–IL-22BP antibody. Green colors show IL-22BP–positive cells, and blue show nuclei. (B) Both Il22ra2+/− and Il22ra2_−/−_ mice were intravenously administered with recombinant IL-22 protein. After 15 min, PP tissues were collected. Tissue sections prepared from these mice were immunostained with anti-pSTAT3 antibody. Green colors show pSTAT3, and blue show nuclei. Dotted lines indicate FAE. Bars, 40 µm. Data are pooled from at least three mice of each genotype. (C) Relative Il22ra1 mRNA expression of FAE and VE from both Il22ra2+/− and Il22ra2−/− mice. Circles and squares show VE and FAE, respectively. Solid symbols indicate expression profiles of Il22ra2+/− mice, and open ones indicate those of Il22ra2−/− mice. Data were normalized with Gapdh (n = 7). Data are representative of three independent experiments. (D) Relative Il22ra1 mRNA expression of intestinal organoids stimulated with RANKL and LT α1β2 for 24 h (n = 4). Data are representative of two independent experiments. Means ± SEM. are shown. One-way ANOVAs with Bonferroni’s (C) or Dunnett’s (D) post hoc test were used for statistical analyses. **, P < 0.01.

Figure 3.

Figure 3.

Excessive expression of IL-22 signaling in the FAE of Il22ra2−/− mice decreased the uptake of bacterial antigens into PPs. (A and B) Relative expressions of IL-22–responsive genes (A) and those of FAE-associated genes (B) on the FAE and VE from both Il22ra2+/− and Il22ra2−/− mice are shown. Circles and squares indicate VE and FAE, respectively. Solid symbols indicate the expression profile of Il22ra2+/− mice, and open ones indicate those of Il22ra2−/− mice. Data were normalized with Gapdh (n = 7). *, P < 0.05; ***, P < 0.001. Data are representative of three independent experiments. (C) PP tissues from both Il22ra2+/− and Il22ra2−/− mice were fixed and stained with fluorescent-labeled lectins. Red colors show UEA-I, and blue show WGA. Dotted lines indicate FAE. Data are pooled from three mice of each genotype. Fucosylated area of FAE was determined by MetaMorph software (n = 7). **, P < 0.01. (D) Both Il22ra2+/− and Il22ra2−/− mice were fed with S. Typhimurium (χ3306), and 24 h after the infection, PPs were collected. Then, PPs were homogenized and plated on Luria broth plates to calculate the number of S. Typhimurium taken up into PPs (n = 10 of each genotype). Data are representative of three independent experiments. (E) Both Il22ra2+/− and Il22ra2−/− mice were gavaged with 5 × 109 CFUs of Δ_aroA S_. Typhimurium (UF20). After 2 and 3 wk, feces were collected, and S. Typhimurium–specific IgA was analyzed by ELISA (n = 5 of each genotype). Data are representative of four independent experiments. (F) The presence of Alcaligenes spp. was visually analyzed by wholemount fluorescent in situ hybridization on the interior of PPs. Small dots indicate Alcaligenes spp. The left graph shows the dot count of Alcaligenes spp. (n = 5). Data are representative of two independent experiments. Bars: (B) 40 µm; (F) 10 µm. Means ± SD are shown. One-way ANOVA with Bonferroni’s post hoc test (A and B) and Student’s t test (C, D, and F) were used for statistical analysis.

Figure 4.

Figure 4.

IL-22BP deficiency does not affect the differentiation and function of M cells. (A) The FAE isolated from both Il22ra2+/− and Il22ra2−/− mice were immunostained with anti-GP2 (green), and samples were counterstained with fluorescent-labeled phalloidin (blue). Bar, 40 µm. Data are pooled from three mice of each genotype. (B) Relative mRNA expression of M cell–marker genes on the FAE and VE from both Il22ra2+/− and Il22ra2−/− mice are shown. Circles and squares indicate VE and FAE, respectively. Solid symbols indicate the expression profile of Il22ra2+/− mice, and open ones indicate those of Il22ra2−/− mice. Data were normalized with Gapdh (n = 7). Data are representative of three independent experiments. (C) Intestinal organoids were stimulated with GST-RANKL or GST-RANKL together with recombinant IL-22 protein for 3 d. Then, expressions of M cell marker genes were analyzed. Data were normalized with Gapdh (n = 4). Data are representative of two independent experiments. (D) Both Il22ra2+/− and Il22ra2−/− mice were orally gavaged with fluorescent-labeled nanoparticles (0.2-µm diameter). After 4 h, particles taken up into PPs were counted. Results show the number of nanoparticles in PPs per section. Ten sections from distal PPs from each three mice are shown (n = 3). Data are pooled by two independent experiments. Means ± SD are shown.

Figure 5.

Figure 5.

Bacterial colonization does not affect the expression of IL-22BP. (A) Relative Il22ra2 mRNA expression of PPs from SPF and GF C57BL/6N mice are shown (normalized with Actb). (B) Relative Il22 mRNA expression of PPs from SPF and GF C57BL/6N mice are shown (normalized with Actb). n = 4. Means ± SD are shown. (C) Microbiota compositions of Il22ra2+/− and Il22ra2−/− mice are shown. n = 5. Data are representative of two independent experiments. Student’s t test was used for statistical analysis.

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