Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation

Abstract

Interleukin (IL) 23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 affects memory T cell and inflammatory macrophage function through engagement of a novel receptor (IL-23R) on these cells. Recent analysis of the contribution of IL-12 and IL-23 to central nervous system autoimmune inflammation demonstrated that IL-23 rather than IL-12 was the essential cytokine. Using gene-targeted mice lacking only IL-12 (p35-/-) or IL-23 (p19-/-), we show that the specific absence of IL-23 is protective, whereas loss of IL-12 exacerbates collagen-induced arthritis. IL-23 gene-targeted mice did not develop clinical signs of disease and were completely resistant to the development of joint and bone pathology. Resistance correlated with an absence of IL-17-producing CD4+ T cells despite normal induction of collagen-specific, interferon-gamma-producing T helper 1 cells. In contrast, IL-12-deficient p35-/- mice developed more IL-17-producing CD4+ T cells, as well as elevated mRNA expression of proinflammatory tumor necrosis factor, IL-1beta, IL-6, and IL-17 in affected tissues of diseased mice. The data presented here indicate that IL-23 is an essential promoter of end-stage joint autoimmune inflammation, whereas IL-12 paradoxically mediates protection from autoimmune inflammation.

Figure 1.

IL-23, and not IL-12, is required for induction of CIA. (A) Cumulative incidence and the mean clinical score of mice that developed clinical disease are plotted against days after primary immunization with CII/CFA. (B) Disease incidence (number of diseased mice/total mice in group), disease severity (median maximum disease score attained and range of clinical scores), and mean day of onset of clinical disease are shown for mice of all genotypes. ^a, Only mice that developed clinical signs of disease were analyzed. N/A, not applicable. (C) Histopathology of decalcified paws at day 42 after primary CII immunization from WT, p35^−/−, and p19^−/− mice. Hematoxylin and eosin staining of tarsal joints from both WT and p35^−/− mice frequently showed severe pathology with articular cartilage erosion (▴), synovial inflammation (S), and pannus (P) formation. p19^−/− joints showed no pathology, characterized by smooth, intact articular cartilage (▵) and no cellular infiltrate in the joint space (J). Original magnification, 100.

Figure 2.

Type II collagen (CII)–specific T and B cell responses in IL-23– and IL-12–deficient mice. (A) Normal CII-specific T cell proliferation for both IL-23– and IL-12–deficient mice from DLN cells taken day 10 after CII/CFA immunization (n = 3–4/group) were cultured individually in triplicate. Data show the mean proliferative response of DLN cells for each genotype ± SEM. (B) DLN cells (day 10 after CII immunization) from individual mice were cultured and assayed as described in Materials and Methods. Data represent the mean response of 3–4 mice ± SEM. (C) Divergent humoral responses by p19^−/− mice versus p35^−/− and p40^−/− mice. Sera levels of CII-specific total Ig and IgG₁ at day 42 after primary CII immunization (n = 5/group) were determined by ELISA. Differences in Ig levels between relevant groups are indicated by p-values as determined by the unpaired Student's t test. Data represent the mean ± SEM.

Figure 3.

Reduced IFN-γ, but elevated inflammatory cytokine and chemokine expression by p35^−/− mice. Cytokine and chemokine mRNA levels in hind paws day 42 after primary CII/CFA immunization were determined by quantitative real-time PCR and expressed in units relative to ubiquitin mRNA levels. (A) mRNA expression levels for IFN-γ and the IFN-γ–inducible chemokines IP-10 and Mig. (B) mRNA expression of genes encoding proinflammatory factors. RNA from 3 to 5 individual paw samples for each genotype were pooled for analysis, except for IFN-γ and IL-17, where the data represent the mean ± SEM; n = 3. The abundance of mRNA encoding IFN-γ and IL-17 were very low and, hence, were confirmed using expression data from individual mice. (Naive) Paws from unimmunized mice. WT samples, both inflamed and naive, consist of pooled RNA from both C57BL/6 and B6 × 129 F2 mice.

Figure 4.

IL-23 promotes the production of IL-17, which is regulated by the IL-12 pathway. Intracellular detection of IFN-γ and IL-17 protein by CD4⁺ T cells from DLN cells at day 10 after CFA immunization. Cells were either analyzed immediately after isolation (ex vivo) or after 5 d of culture in the presence of rIL-12 or rIL-23, both at 10 ng/ml. All plots are gated on live CD4⁺ T cells and are representative of four separate experiments.

Figure 5.

A model proposing the respective roles of IL-12/IFN-γ and IL-23 in T cell-priming events versus end-stage autoimmune inflammation. The precise mechanism through which IL-12/IFN-γ may down-regulate the magnitude of the inflammatory response (i.e., reducing IL-17 production) remains to be defined. It is recognized that IFN-γ may have dual roles during late-stage inflammation (i.e., antiinflammatory actions through termination of activated T cell responses [references 9, 29] while also stimulating macrophage function [reference 5]). ^a, The IL-23R is present on CD4⁺ CD45RB^lo “memory” T cells (30), but this cell phenotype may also include activated CD4⁺ T cells that are not strictly memory T cells.