Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category

Abstract

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23-IL-17 and IL-12-T helper cell (Th)1 pathways in the pathogenesis of EAU. IL-23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-gamma. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-gamma-producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

Figure 1.

IL-23 is essential for EAU induction by up-regulating proinflammatory cytokine. (A) EAU scores were evaluated by histopathology of eyes from WT and the different IL-12 family cytokine KO mice 21 d after immunization. (B) DTH response in mice challenged 48 h before the end of the experiment. These data are representative of three experiments. (C) Representative histopathology of ocular damage in WT and p35 KO mice. Bar, 0.25 mm. (D) LNs were harvested from WT and the different IL-12 family KO mice 21 d after immunization. Cytokine secretion was measured in 48-h IRBP-stimulated (30 μg/ml) culture supernatants. ***, P < 0.001 versus P35 KO; +, P < 0.05; ++, P < 0.01 versus WT. Cytokines were measured in pooled supernatants, so although data represent a group average of five mice, no error bars could be generated.

Figure 2.

Anti–IL-23 treatment prevents, but does not reverse, EAU. B10RIII mice were immunized with IRBP peptide (161–180) as indicated. Groups of five mice were treated i.p. with antibodies against p19, p40, or with isotype from the priming (starting day −1) or from the effector phase of EAU induction (starting day 7) every other day, as indicated in Materials and methods. (A) EAU score evaluated in eyes by histopathology 17 d after immunization. (B) DTH responses of mice challenged 48 h earlier. (C) LN proliferation to IRBP peptide 161–180. (D) Cytokine secretion from LN cells stimulated with IRBP peptide 161–180 for 48 h. The data are from a representative experiment of two, with five mice per group. **, P < 0.01; ***, P < 0.001 versus the respective isotype control.

Figure 3.

Enhanced EAU in IL-12p35 KO and in IFN-γ KO mice is associated with increased systemic and local IL-17 responses. WT and GKO mice on B10.RIII background (A–C) and WT and P35 KO mice on C57BL/6 background (D–F) were immunized with IRBP. On day 21 after immunization, eyes were collected for intracellular cytokine staining of eye-infiltrating inflammatory cells (A and D) or for histopathology (data pooled from two experiments with five mice per group; B and E). *, P < 0.05 versus WT. For FACS analysis, cells isolated from eyes were incubated either with PMA plus ionomycin and brefeldin A (PMA/Io) or with brefeldin A only (unstimulated). Shown are cells gated on CD4. Cytokine secretion from LN cells was measured by ELISA after 48 h of culture with IRBP.

Figure 4.

Anti–IL-17 treatment prevents and reverses EAU. B10RIII mice were immunized with IRBP as indicated. Groups of five mice were treated i.p. with antibodies against IL-17 or with isotype from the time of priming (starting day −1) or from the effector phase of EAU induction (starting day 7) every other day. (A) EAU score evaluated in eyes by histopathology 17 d after immunization. (B) Ag-specific DTH response. (C and D) LN cells were explanted into culture, and IRBP-specific proliferation and cytokine production were measured by multiplex ELISA or by single-plex ELISA (IL-22). Data show a representative experiment of two. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the related isotype control.

Figure 5.

Th17 effector cells induce EAU in the absence of an IFN-γ response. Short-term Th17 lines were generated from IFN-γ KO mice immunized with p161–180 under Th17 conditions, as described in Materials and methods. (A) Intracellular IL-17 expression after a second stimulation with p161–180. IFN-γ expression is confirmed to be negative. (B) Supernatant from the stimulated cultures was tested for cytokine secretion by ELISA. (C and D) Pathogenicity of Th17 cells. Cells were collected from culture after the second cycle of stimulation, and 5 × 10⁶/mouse were infused into WT or GKO recipients (five mice per group). On day 10 after transfer, eyes were collected for histopathology and disease was scored. Inset shows the inflammatory infiltrate, similar in WT and IFN–γ KO. Bar, 0.25 mm (0.02 mm in inset). A representative experiment of two is shown.

Figure 6.

Severe EAU induced with a T cell line that is stably polarized to the Th1 phenotype. (A and B) After 48 h of stimulation with p161–180, the indicated number of cells was infused into naive B10RIII mice. Shown are average scores (A) and representative histopathology. (B) Inset shows the typical inflammatory infiltrate. Bar, 0.25 mm (0.02 mm in inset). (C) ELISA analysis of cytokines in supernatants of the stimulated cells. (D and E) Intracellular cytokine staining after stimulation with Ag for 24 h in neutral conditions (D) or for 5 d under Th17-inducing conditions (Ag plus IL-23 or Ag plus IL-23 plus anti–IFN-γ; E). (F) “Parking” of Th1 cells in allotype-congenic recipient mice. After stimulation with p161–180 in the presence of irradiated APCs, the T cell line was infused into naive Thy1.1 x Thy1.2 heterozygous recipients (2 × 10⁶/mouse). Note heterozygous Thy1.1/2⁺ recipient cells versus Thy1.2⁺ donor cells by FACS analysis. Thy1.2 single-positive cells were sorted out after 90 h, stimulated with IRBP peptide 161–180 for 24 h (with PMA-ionomycin and brefeldin A added during the last 4 h), and stained for intracellular IL-17 and IFN-γ. Representative experiment of two with five mice per group.

Figure 7.

Host IL-17 does not seem to play a role in pathogenesis of EAU induced with the Th1 cell line. Two million freshly activated Th1 line cells were infused i.v. into naive B10.RIII Thy1.1 homozygous mice. EAU scores were assessed by fundoscopy. (A) Eyes of recipient mice were removed for isolation of infiltrating cells 10 d after cell transfer. Cells isolated from eyes and incubated with PMA plus ionomycin and brefeldin A (PMA/Io) or with brefeldin A only (unstimulated) were stained for intracellular IFN-γ versus IL-17 and were analyzed by FACS. (B) Recipient mice were treated with neutralizing antibodies to IL-17, IFN-γ, or TNF-α, or with isotype control (0.25 mg/mouse/day, starting day 0). 10 d after transfer, EAU was scored by fundoscopy and confirmed by histopathology. Data show histopathology scores of a representative experiment of three. +, P < 0.05; ++, P < 0.01 versus isotype control; **, P < 0.01 and ***, P < 0.001 versus anti–IL-17.

Figure 8.

IFN-γ KO (GKO) mice fail to develop EAU after infusion of uveitogenic DCs despite the presence of a Th17 response. Flt3L-elicited splenic DCs obtained from B10.RIII WT mice were matured and pulsed with IRBP p161–180 in vitro for 4 h and injected into syngeneic WT recipients (n = 8) or GKO recipients (n = 10). EAU (histopathology) and immunological responses were evaluated on day 18 after uveitogenic DC transfer. Data show a representative experiment of three. ***, P < 0.001 versus WT.

Figure 9.

IL-17 KO mice develop EAU and maintain production of proinflammatory cytokines. IL–17 KO mice were immunized with a uveitogenic regimen of IRBP. (A and B) On day 21 after immunization, eyes were collected for EAU evaluation by histopathology (A) and specific DTH responses were recorded as the difference between IRBP- and PBS-injected ears (B). (C and D) LN cells were explanted into culture, and IRBP-specific proliferation (C) and cytokine production (D) were evaluated. The data show averaged responses from 17 animals in three experiments. (E) On day 21 after immunization, eyes were collected and eye-infiltrating cells were extracted and stained for intracellular IFN–γ and IL-17. (F) Ocular extracts prepared as described in Materials and methods were assayed for cytokine levels. (G) Histopathology of EAU in IL-17 KO and WT mice. Bar, 0.25 mm. Data show a representative experiment of three. **, P < 0.01; ***, P < 0.001 versus WT.

Figure 10.

Schematic representation of the patterns of effector T cell dominance in the different EAU models. Conditions of initial exposure to Ag that may determine effector dominance are the quality/quantity of TLR signals and the type/variety of cells participating as APCs.