IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses

Abstract

CD4(+) T helper 2 (T(H)2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, T(H)2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal lymphopoietin, IL33 and IL25 (also known as IL17E) have been implicated in inducing T(H)2 cell-dependent inflammation at mucosal sites, but how these cytokines influence innate immune responses remains poorly defined. Here we show that IL25, a member of the IL17 cytokine family, promotes the accumulation of a lineage-negative (Lin(-)) multipotent progenitor (MPP) cell population in the gut-associated lymphoid tissue that promotes T(H)2 cytokine responses. The IL25-elicited cell population, termed MPP(type2) cells, was defined by the expression of Sca-1 (also known as Ly6a) and intermediate expression of c-Kit (c-Kit(int)), and exhibited multipotent capacity, giving rise to cells of monocyte/macrophage and granulocyte lineages both in vitro and in vivo. Progeny of MPP(type2) cells were competent antigen presenting cells, and adoptive transfer of MPP(type2) cells could promote T(H)2 cytokine responses and confer protective immunity to helminth infection in normally susceptible Il25(-/-) mice. The ability of IL25 to induce the emergence of an MPP(type2) cell population identifies a link between the IL17 cytokine family and extramedullary haematopoiesis, and suggests a previously unrecognized innate immune pathway that promotes T(H)2 cytokine responses at mucosal sites.

Figure 1. IL-25 elicits a c-kit^int-GFP^neg and c-kit^int-GFP^pos cell population in the GALT

a, b, Cell numbers from IL-25-treated IL-4/eGFP reporter mice. Numbers of total mLN cells or of T cells (CD4^pos or CD8^pos), B cells (CD19^pos) and macrophages (CD11b^pos MHC class II^pos) (a) and total numbers of non-B non-T (NBNT) c-kit^pos cells (b). c-e, Frequency of c-kit^int cells in mLNs (c), Peyer's patches (PP) (d) and cecal patch (CP) (e) was assessed by flow cytometry. f, g, Frequencies (f) and total numbers (g) of c-kit^int cells in control or Nippostrongylus brasiliensis-infected (INF) mice. Crtl = control. Plots shown are gated on live, CD4^neg CD8^neg CD11b^neg CD11c^neg and B220^neg cells or as indicated. *, P < 0.05. Error bars indicate s.e.m. Data in a-e are representative of more than five independent experiments (control, n=16; IL-25-treated, n=34). Data in f-g are representative of at least two independent experiments (Crtl, n=2; INF, n=6).

Figure 2. IL-25-elicited c-kit^int cells promote Th2 cytokine-dependent responses in vivo

a-c, CFSE-labeled CD45.2 OVA-specific CD4^pos T cells were adoptively transferred i.v. into CD45.1 congenic recipients and mice were immunized i.p. with OVA/IFA in the presence or absence of IL-25-elicited c-kit^int cells. Proliferation of donor CD45.2 cells in recipient mice receiving T cells alone (red shaded histogram) or T cells and OVA/IFA immunization ± c-kit^int cells (black histograms) was measured by flow cytometry (a). Frequency of donor CD4^pos cells/total CD4^pos T cells isolated from the peritoneum (b). IL-13 production from αCD3/αCD28-stimulated mLN cells was measured by ELISA (c). Data in a-c are representative of two independent experiments (n=7). *, P < 0.05, **, P < 0.01. d-g, Adoptive transfer of c-kit^int cells protects Trichuris-infected (INF) Il17e^-/- mice. Cytokine production by αCD3/αCD28-stimulated mLN cells (d), Trichuris-specific serum IgG1 antibody titers (e), intestinal mucin responses (f), and number of worms from Trichuris-infected mice (g) were assessed at day 20 post-infection. Scare bar, 50 μm.

Figure 3. IL-25-elicited c-kit^int cells exhibit multi-potent capacity

a, b, Frequencies of c-kit^int cells in the mLNs of IL-25-treated IL-4/eGFP reporter mice (a) and expression of HSC markers by c-kit^int-GFP^neg (blue histograms) or c-kit^int-GFP^neg (green histograms) cells from IL-25-treated IL-4/eGFP reporter mice (b). Number (italics) indicates the mean fluorescent intensity. Plots shown are gated on live, lineage^neg cells (CD3ε, CD8α, CD8β, TCRβ, TCRγδ, B220, CD19, CD11b, CD11c, Gr-1, NK1.1 and Ter119) or as indicated. Data in a and b are representative of two independent experiments (control, n=4; IL-25-treated, n=7). c, e, Flow cytometric analysis of myeloid cell and granulocyte differentiation of FACS-purified lineage^neg/lo c-kit^int-GFP^pos (c) or c-kit^int-GFP^neg (e) cells from IL-25-treated IL-4/eGFP reporter mice following in vitro culture in SCF and IL-3. d, f, Cytospin preparation of progeny derived from IL-25-elicited c-kit^int-GFP^pos cells (d) or c-kit^int-GFP^neg cells (f). Scale bar, 20 μm. Data in c-f are representative of at least three independent experiments.

Figure 4. Progeny from IL-25-elicited c-kit^int-GFP^neg cells promote Th2 cell differentiation

a, FACS-purified IL-25-elicited c-kit^int cells were cultured for 8 days in SCF and IL-3 and the resulting progeny from c-kit^int-GFP^pos (black histogram) or c-kit^int-GFP^neg (gray shaded histogram) cells were assessed for expression of IL-4/eGFP and MHC class II. Results in (a) are representative of three independent experiments. b, CFSE-dilution by OVA-specific CD4^pos CD62L^hi CD44^lo T cells following 4 day co-culture with progeny from FACS-purified IL-25-elicited c-kit^int-GFP^neg cells in the presence of OVA peptide with or without addition of mAb against MHC class II or IL-4Rα. Gray histogram indicates CFSE-dilution by OVA-specific CD4^pos T cells cultured in medium alone. c, IL-4 and IL-13 protein levels in cell-free supernatants from (b) were assayed by ELISA. Results in b-c are representative of at least two independent experiments.