Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid - PubMed (original) (raw)

doi: 10.1038/mi.2013.28. Epub 2013 May 8.

G Bériou 2, M Heslan 2, C Chauvin 2, L Utriainen 3, A Aumeunier 3, C L Scott 3, A Mowat 3, V Cerovic 3, S A Houston 3, M Leboeuf 4, F X Hubert 5, C Hémont 1, M Merad 4, S Milling 3, R Josien 1

Affiliations

Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid

J C J Martin et al. Mucosal Immunol. 2014 Jan.

Abstract

Interleukin-22 (IL-22) is mainly produced at barrier surfaces by T cells and innate lymphoid cells and is crucial to maintain epithelial integrity. However, dysregulated IL-22 action leads to deleterious inflammation and is involved in diseases such as psoriasis, intestinal inflammation, and cancer. IL-22 binding protein (IL-22BP) is a soluble inhibitory IL-22 receptor and may represent a crucial regulator of IL-22. We show both in rats and mice that, in the steady state, the main source of IL-22BP is constituted by a subset of conventional dendritic cells (DCs) in lymphoid and non-lymphoid tissues. In mouse intestine, IL-22BP was specifically expressed in lamina propria CD103(+)CD11b(+) DC. In humans, IL-22BP was expressed in immature monocyte-derived DC and strongly induced by retinoic acid but dramatically reduced upon maturation. Our data suggest that a subset of immature DCs may actively participate in the regulation of IL-22 activity in the gut by producing high levels of IL-22BP.

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Figures

Figure 1

Figure 1. Tissue expression pattern of IL-22BP in the rat

(A) IL-22BP gene expression was analyzed by RT-qPCR. Bars represent the mean ± SEM ratio of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt method of relative quantification from 2 experiments. (B) IL-22BP gene expression was analyzed by RT-PCR using primers allowing amplification of whole il22ra2 mRNA. Values indicate expected molecular mass of PCR products. Data are representative of two independent experiments.

Figure 2

Figure 2. IL-22BP is highly expressed in the spleen by a subset of resident cDCs

(A) The different populations of splenic hematopoietic cells were isolated by cell sorting. IL-22BP gene expression was analyzed by RT-qPCR and normalized relative to HPRT expression (n=4 for DC; n=5 for B cells; n=3 for T cells, NK cells and monocytes). (B) IL-22BP gene expression was compared by RT-qPCR for DCs (n=3) in spleen, MLN and thymus (n=2). In A and B bars represent mean ± SEM of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt method of relative quantification. (C) Hematopoietic cells were isolated by cell sorting. IL-22R1 gene expression was analyzed by RT-qPCR with several tissues being used as positive controls (n=3 for cell samples; n=2 for tissue samples). Bars represent mean ± SEM ratio of IL-22R1 gene to HPRT expression as determined by the 2−ΔΔCt method of relative quantification. * p<0.05.

Figure 3

Figure 3. IL-22BP staining on sorted subsets of rat splenic conventional DC

The two subsets of rat conventional spleen DCs were isolated by cell sorting and let to adhere on poly-L-Lysine pre-coated slides for 30 min in the presence of Brefeldin A and monensin. Slides were then stained with a goat anti-rat IL-22BP pAb followed by a donkey anti-goat IgG-Alexa fluor 568. Controls were performed using the secondary antibody alone. Data are representative of two independent experiments.

Figure 4

Figure 4. A subset of intestinal DC expresses high levels of IL-22BP in the steady state

(A) Constitutively migrating intestinal lymph DCs (ilDCs) subsets were obtained by lymph collection after thoracic duct cannulation of mesenteric lymphadenectomized rat, and cell sorting. IL-22BP gene expression was analyzed by RT-qPCR for the three subsets of ilDCs and compared to intestinal tissues and its CD4+ DC spleen counterpart (n=3 for ilDCs; n=2 for tissues). (B) The 3 subpopulations of rat MLN-DCs were isolated by cell sorting and analyzed for IL-22BP expression by RT-qPCR (n=3). (C) Total DCs were depleted from total MLN cells by FACS-depletion of CD103+ cells. IL-22BP gene expression was analyzed by RT-qPCR (n=3). (D) Mouse CD8+ and CD11b+ CD103+ DC subsets and macrophages were isolated from intestinal lamina propria by cell sorting. IL-22BP gene expression was analyzed by RT-qPCR (n=3 for DC and small intestine tissue; n=2 for macrophages). LP,lamina propria; SI, Small Intestine; Mø, Macrophages. (E) Mouse MLN CD103+ DCs were separated into CD11b+ and CD11b− by cell sorting and analyzed for IL-22BP expression by RT-qPCR (n=3). (F) IL-22BP gene expression was analyzed by RT-qPCR in small intestine and colon of wt (n=3) and Flt3L−/− (n=3) mice. In panels A, B, C (rat), E and F (mouse) bars represent mean ± SEM ratio of IL-22BP gene to HPRT expression as determined by the 2-ΔΔCt method of relative quantification. In panel D (mouse), bars represent mean ± SEM ratio of IL-22BP gene to GAPDH expression as determined by the 2−ΔΔCt method of relative quantification.. * p<0.05.

Figure 5

Figure 5. Rat CD4+ DCs express a counterpart of the human short isoform of IL-22BP

Spleen CD4+ cDC were isolated by cell sorting. IL-22BP gene expression was analyzed by RT-PCR using primers allowing amplification of whole il22ra2 mRNA.

Figure 6

Figure 6. IL-22BP is expressed in human monocyte-derived dendritic cells

Human monocytes from peripheral blood of healthy donors were differentiated into DCs in complete medium with GM-CSF and IL-4 for 6 days. (A) On day 6, IL-22BP gene expression was analyzed by RT-qPCR. Each point represents the ratio of IL-22BP gene to HPRT expression, as determined by the 2−ΔΔCt method of relative quantification, for an individual healthy donor (n=14). (B) Cells were collected at the indicated times during MDDC differentiation and IL-22BP gene expression analyzed by RT-qPCR. Each point represents the ratio of IL-22BP gene to HPRT expression as determined by the 2−ΔΔCt method of relative quantification. Data are representative of two independent experiments. (C) Monocytes and MDDC were let to adhere on poly-L-Lysine pre-coated slides for 30 min in the presence of Brefeldin A and monensin, and then stained with a mouse IgG1 anti-human IL-22BP as primary antibody followed by goat anti-mouse IgG1-Alexa fluor 568. Controls were performed using the secondary antibody alone. Data are representative of four independent experiments. (D) IL-22BP gene expression was analyzed by RT-PCR using primers allowing amplification of whole il22ra2 mRNA. Data are representative of three independent experiments. *** p<0.001.

Figure 7

Figure 7. Retinoic acid is a potent inducer of IL-22BP expression by DCs

Human monocytes from peripheral blood of healthy donors were differentiated into DCs in complete medium with GM-CSF and IL-4 for 6 days. When indicated, ligands were added at day 4 of culture. On day 6, IL-22BP gene expression was analyzed by RT-qPCR. Bars represent mean ± SEM ratio of IL-22BP gene to HPRT expression as determined by the 2−ΔΔCt method of relative quantification from 7 independent experiments ** p<0.01; * p<0.05.

Figure 8

Figure 8. AM580-induced differentiation of MDDC enhances IL-22BP expression

(A) MDDC were differentiated in the absence or presence of AM580 added at day 0 or day 4 of culture. IL-22BP gene expression was analyzed by RT-qPCR. Bars represent mean ± SEM of fold change compared to GM-CSF/IL-4 derived MDDC, of IL-22BP gene to HPRT, expression as determined by the 2−ΔΔCt method of relative quantification (n=4). (B) Cells were harvested at the indicated times during MDDC differentiation in the presence or the absence of AM580 added at day 0. IL-22BP gene expression was analyzed by RT-qPCR. Each point represents mean ± SEM of fold change compared to GM-CSF/IL-4 derived MDDC, of IL-22BP gene to HPRT expression as determined by the 2−ΔΔCt method of relative quantification (n=4). (C) MDDC and AM580-diferentiated MDCC were stained for IL-22BP as described in figure 6. (D) MDDC were differentiated in the absence or presence of AM580 added at day 0 or day 4 of culture. Cell surface markers were analyzed by flow cytometry. Red histograms, isotype control staining; blue histrograms, antibody staining. Data are representative of at least four independent experiments. (E) MDDC were differentiated in the absence or presence of RA and specific inhibitors of RA nuclear receptors added at d0. IL-22BP gene expression was analyzed by RT-qPCR. Bars represent mean ± SEM of fold change compared to GM-CSF/IL-4 derived MDDC, of IL-22BP gene to HPRT, expression as determined by the 2−ΔΔCt method of relative quantification (n=4). (F) MDDC were differentiated in the presence or not of retinal and/or DEAB, a selective inhibitor of RALDH2. IL-22BP gene expression was analyzed by RT-qPCR. Bars represent mean ± SEM of fold change compared to GM-CSF/IL-4 derived MDDC, of IL-22BP gene to HPRT, expression as determined by the 2−ΔΔCt method of relative quantification (n=3). (G) RALDH2 gene expression was analyzed by RT-qPCR in rat CD172+ CD4+ spDCs and CD172high ilDCs. Bars represent mean ± SEM of IL-22BP gene to HPRT, expression as determined by the 2−ΔΔCt method of relative quantification (n=3). * p<0.05.

Figure 9

Figure 9. IL-22BP is down-regulated when DCs undergo maturation

(A) Rat spleen CD4+ DC were isolated by cell sorting and cultured for the indicated times in complete medium, in the absence or the presence of the TLR9 ligand CpG ODN2006. IL-22BP gene expression was analyzed by RT-qPCR (n=3). (B) LPS or LPS + IFNγ was added at day 6 on human MDDC. IL-22BP gene expression was analyzed by RT-qPCR after 24h (n=2). (C) Rat spleen CD4+ cDC were isolated by cell sorting and cultured for 24 hours in the absence or the presence of AM580. IL-22BP gene expression was analyzed by RT-qPCR (n=3). Data are presented as mean ± SEM of ratios of IL-22BP gene to HPRT expression as determined by the 2−ΔΔCt method of relative quantification (Rat, A and C; Human, B).

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