Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells

Abstract

IL-23, an IL-12 family member, has been implicated in the development of Th17 cells and the progression of autoimmune diseases. However, due to the lack of availability of sensitive Ab reagents specific for the IL-23 receptor (IL-23R), it has been difficult to characterize the cell types that express the IL-23R and are responsive to IL-23 in vivo. To address the role of IL-23 in vivo, we have generated a novel "knock-in" mouse in which we have replaced the intracellular domain of the IL-23R with the GFP. We show that in addition to Th17 cells, a subset of myeloid cells express IL-23R and respond to IL-23 by producing IL-17 and IL-22. Our studies further demonstrate that IL-23R expression is crucial for generation of encephalitogenic Th17 cells, but its expression on the innate immune system is dispensible in the development of experimental autoimmune encephalomyelitis.

Figure 1

Generation of IL-23R reporter mice. A, IL-23 genomic locus and targeting strategy. An IRES-EGFP cassette was introduced right after the EXON8 of the endogenous IL-23R gene. B, Southern blot was used to identify the properly targeted ES cells. DNA purified from ES cells was digested by NheI and probed with a 1.1 kb specific probe generated by PCR. The hybridized 15.7 kb fragment corresponds to the wild type allele, and the hybridized 6.8 kb fragment corresponds to the targeted allele.

Figure 2

IL-23R expressing cells are enriched in the lamina propria (LP) of naive mice. Cells from lymph nodes, spleen and LP were prepared from naive WT and IL-23R-GFP.KI mice, stained and analyzed by flow cytometry. A, Lymph node cells were stained for CD3, TCRγδ, CD11c, and CD11b. B, IL-23R(GFP) expression was also analyzed in LP cells. LP cells were stained for CD45 and TCRγδ (B left panel). The expression of CD3, CD4, and TCRγδ was analyzed in the CD45⁺IL-23R/GFP⁺ compartment of LP cells (B right panel).

Figure 3

IL-23R-/- mice have a defective T_H17 response and are resistant to EAE. A, EAE was induced in WT and IL-23R-/- mice by immunization with MOG_35-55 emulsified in CFA. The course of EAE in these mice were monitored and shown as mean clinical score. B, IL-23R-GFP.KI mice were immunized with MOG_35-55/CFA. On day 16 at the peak of disease, mononuclear cells were isolated from the CNS and stimulated with PMA/ionomycin. Intracellular cytokine staining for IFN-γ and IL-17 was performed. The histograms represent the percentages of cytokine positive cells in the CD4⁺IL-23R/GFP^-and CD4⁺IL-23R/GFP⁺ gates, respectively. C, WT and IL-23R-/- mice were immunized with MOG_35-55/CFA. On day 8, spleens (right panel) and LNs (left panel) were collected and cultured with MOG_35-55 alone or in the presence of rIL-23 for 6-8 days. Cells were re-stimulated with PMA/Ionomycin before intracellular cytokine staining for IL-17 and IFN-γ was performed. Quadrants represent intracellular cytokine cytokine staining in the CD4⁺ T cell gate. D, CD4⁺CD44⁺ memory T cells from WT or IL-23R-/- mice were activated with anti-CD3 in the presence rIL-23 (30 ng/ml) with irradiated syngenic APCs. Four days later cells were re-stimulated with PMA/Ionomycin and intracellular cytokine staining was performed for IL-17 and IFN-γ. E, For passive transfer of EAE, MOG_35-55 specific T cells from immunized WT or IL-23R-/- mice were cultured in the presence of MOG_35-55 with rIL-23 (30 ng/ml). On day 8 CD4⁺ T cells were reactivated with plate bound anti-CD3 and anti-CD28 for 48 hrs. Equal number of MOG_35-55 specific CD4⁺ T cells either from WT or IL-23-/- mice were transferred into WT recipient. The course of the EAE was monitored and is shown as mean clinical score.

Figure 4

IL-23R expression on APCs is dispensable for the development of EAE. A, Single cell suspensions were prepared from Lymph nodes (LNs) from naive either RAG2 -/- or IL-23R-GFP.KI RAG2 -/- mice and IL-23R(GFP) expression was analyzed on total LNs' cells. B, IL-23R-GFP.KI mice were immunized with MOG_35-55/CFA. On day 17, IL-23R(GFP) expression was analyzed on myeloid cell populations as indicated in histogram. C, MOG specific T_H17 cells were i.v transferred into RAG2-/- and RAG2-/-IL-23R-/- mice. The course of EAE in these mice were monitored and shown as mean clinical score. D, Naïve T cells derived from MOG specific mice were i.v transferred into RAG2-/- or IL-23R-/- RAG2-/- mice. 24 h later, the mice were immunized with MOG_35-55 emulsified in CFA. The course of EAE in these mice were monitored and shown as mean clinical score.