Interleukin-27 and interferon-gamma are involved in regulation of autoimmune arthritis

Abstract

Inflammation underlying immune pathology and tissue damage involves an intricate interplay between multiple immunological and biochemical mediators. Cytokines represent the key immune mediators that trigger a cascade of reactions that drive processes such as angiogenesis and proteolytic damage to tissues. IL-17 has now been shown to be a pivotal cytokine in many autoimmune diseases, supplanting the traditional Th1-Th2 paradigm. Also, the dual role of proinflammatory IFN-γ has unraveled new complexities in the cytokine biology of such disorders. A major hurdle in fully understanding the effector pathways in these disorders is the lack of information regarding the temporal kinetics of the cytokines during the course of the disease, as well as the interplay among the key cytokines. Using an experimental model of arthritic inflammation, we demonstrate that the temporal expression of cytokines during the incubation phase is a critical determinant of disease susceptibility. The susceptible rats raised a vigorous IL-17 response early, followed by IFN-γ and IL-27 response in that sequence, whereas the resistant rats displayed an early and concurrent response to these three cytokines. Accordingly, treatment with exogenous IFN-γ/IL-27 successfully controlled arthritic inflammation and inhibited the defined mediators of inflammation, angiogenesis, cell survival, apoptosis, and tissue damage. Furthermore, IFN-γ enhanced IL-27 secretion, revealing a cooperative interplay between the two cytokines. Our results offer a novel immunobiochemical perspective on the pathogenesis of autoimmune arthritis and its therapeutic control.

FIGURE 1.

Cytokine mRNA expression profiles in Lewis and WKY rats following an arthritogenic challenge. Mtb-immunized Lewis and WKY rats were killed at different time points corresponding to the phases of AA in the Lewis rat. The draining LNC harvested from these rats were restimulated with Bhsp65 for 24 h (A, B, and D), whereas the SAC were restimulated with sonicated Mtb (10 μg/ml) for 6 h (C). The cytokine mRNA expression (n = 4 per group at each phase) was quantified using real-time qRT-PCR. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA served as an internal control. *, p < 0.05 when cytokine mRNA levels at the corresponding time point were compared. Pk, peak phase; Rec, recovery phase.

FIGURE 2.

Cross-regulation among cytokines. A–F, inhibition of IL-17 and its related transcription factors by IFN-γ and IL-27. LNC harvested from Mtb-immunized Lewis rats at the onset of AA were restimulated for 24 h (A–C) or 48 h (D and E) with Bhsp65 in the presence or absence of different concentrations of the indicated cytokines or anti-cytokine antibody. The cDNA from these cells was used to quantify the mRNA expression of IL-17 (A–C), whereas the culture supernatant was used to determine the level of IL-17 protein by Multiplex (D and E). LNC lysate was used to determine the level of phosphorylated STAT3 (pSTAT3) and ROR-γt using GAPDH as an internal control (F). G–I, IFN-γ-mediated enhancement of IL-27 (p28 subunit) but reduction of TGF-β1 mRNA expression. SAC were restimulated for 6 h with Mtb in the presence or absence of IL-17 and IFN-γ (10 ng/ml). The cDNA was used to quantify the mRNA expression of IL-27 (G), TGF-β1 (H), and IL-23 (I) by qRT-PCR. *, p < 0.05.

FIGURE 3.

Differential regulation of AA in Lewis rats by cytokine treatment in vivo. Separate groups of Mtb-immunized Lewis rats were treated with IL-17 (A), IFN-γ (B and D), and IL-27 (E and H) on the days indicated by arrows. The control rats were treated with PBS. LNC from IFN-γ-treated (C) and IL-27-treated (F) animals (those indicated in B and E) were harvested at the Ons phase of AA and restimulated in vitro with Bhsp65 for 24 h and then processed to determine IL-17 mRNA expression (C and F) by qRT-PCR and the level of phosphorylated STAT3 (pSTAT3) and ROR-γt (G) by Western blotting. Representative hematoxylin/eosin-stained hind paw sections of different rat groups are shown (I). B, bone; C, cartilage; JS, joint space; P, pannus. Arrows indicate mononuclear cell infiltrates. *, p < 0.05; Med, medium.

FIGURE 4.

Cytokine expression in SIC. Total SIC (A) and adherent SIC (B) from the joints of arthritic Lewis rats were restimulated with Bhsp65 (24 h) and Mtb (6 h), respectively. The cytokine mRNA expression in these cells was quantified by qRT-PCR using SYBR Green Master Mix. Results were expressed as ΔmRNA normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). ova, ovalbumin; Med, medium.

FIGURE 5.

Differential effect of cytokines on FLS. FLS in passage 2 were stimulated with IL-17, IFN-γ, and IL-27, and the effects of these cytokines on MMP activity (MMP9 and MMP2) (A), MMP9 mRNA expression (B), VEGF secretion (C), signaling molecules (D), and caspase-3/7 activity (E) are shown. pAkt, phosphorylated Akt. *, p < 0.05; Med, medium.

FIGURE 6.

Schematic model explaining the novel interactions among IL-27, IFN-γ, and IL-17. IFN-γ and IL-27 inhibit IL-17 expression in the periphery. IL-17 is the dominant cytokine in the joints and is critical for the development of joint inflammation. IL-17 induces MMP9 activity and phosphorylated Akt in FLS, leading to the development of arthritis. Our results suggest that IFN-γ- and IL-27-mediated down-regulation of MMP9 activity, VEGF secretion, and phosphorylated Akt (pAkt) in the joints leads to the down-modulation of arthritis. The model also highlights the effects of exogenously administered IL-27 and IFN-γ on inhibiting the arthritogenic events and, accordingly, the role of these two cytokines in conferring protection against AA to WKY rats. (* = p<0.05; Med, medium).