The newly discovered cytokine IL-34 is expressed in gingival fibroblasts, shows enhanced expression by pro-inflammatory cytokines, and stimulates osteoclast differentiation

Abstract

Background: Interleukin-34 (IL-34) is a recently discovered cytokine functionally overlapping macrophage colony stimulating factor (M-CSF), a mediator of inflammation and osteoclastogenesis in bone-degenerative diseases such as rheumatoid arthritis. The objective of this study was to assess the expression of IL-34 in human gingival fibroblasts and investigate if the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin-1Β (IL-1β) modulate its expression, and moreover if IL-34 could contribute to recruitment of bone-resorbing osteoclasts.

Methods: IL-34 expression was evaluated in gingival fibroblasts by real time PCR following stimulation by TNF-α, IL-1β, and treatment with inhibitors of intracellular pathways. The formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining of bone marrow macrophages treated with IL-34 or M-CSF in addition to receptor activator of nuclear factor kappa-B ligand (RANKL).

Results: IL-34 was expressed in gingival fibroblasts. The expression was enhanced by TNF-α and IL-1β, regulated by the transcription factor nuclear factor kappa B (NF-κΒ) and activation of c-Jun N-terminal kinase (JNK). Further, IL-34 supports RANKL-induced osteoclastogensis of bone marrow macrophages, independently of M-CSF.

Summary: In conclusion, this study shows for the first time IL-34 expression in human gingival fibroblasts, stimulated by TNF-α and IL-1β, key mediators of periodontal inflammation. Furthermore, IL-34 can be substituted for M-CSF in RANKL-induced osteoclastogenesis. IL-34 may contribute to inflammation and osteoclastogenesis in bone-degenerative diseases such as periodontitis.

Figure 1. Time-dependent increase of IL-34, M-CSF, and IL-6 expression in gingival fibroblasts by TNF-α and IL-1β.

(A) TNF-α (50 ng/ml) and IL-1β (100 pg/ml) stimulate IL-34, (B) M-CSF, and (C) IL-6 expression in a time-dependent manner. Data expressed as means ± SEM. Data represent three individual experiments.

Figure 2. Dose-dependent increase of IL-34, M-CSF, and IL-6 expression in gingival fibroblasts by TNF-α and IL-1β.

(A) TNF-α and (B) IL-1β increase IL-34 expression in a dose-dependent manner. (C) TNF-α and (D) IL-1β increase M-CSF expression in a dose-dependent manner. (E) TNF-α and (F) IL-1β increase IL-6 expression in a dose-dependent manner. Analysis performed at 48 h of incubation. Data expressed as means ± SEM. Data represent three individual experiments.

Figure 3. Involvement of NF-κΒ and JNK pathways in TNF-α and IL-1β stimulated IL-34 and M-CSF in gingival fibroblasts.

(A) Down regulation of TNF-α stimulated IL-34 by IKKβ inhibitor V (IKKV) and JNK inhibitor and down regulation of (B) IL-1β stimulated IL-34 by IKKβ inhibitor V (IKKV). (C) Down regulation of TNF-α and (B) IL-1β stimulated M-CSF by IKKβ inhibitor V (IKKV). Analysis performed at 48 h of incubation. Data expressed as means ± SEM. Data represent three individual experiments.

Figure 4. IL-34 substitutes M-CSF in RANKL induced osteoclastogenesis.

(A) IL-34, together with RANKL, induces osteoclastogenesis of bone marrow macrophages in a concentration-dependent manner, to comparable levels of M-CSF/RANKL. (B) Representative photos (20X) of RANKL (4 ng/ml)/IL-34 (100 ng/ml) versus RANKL (4 ng/ml)/M-CSF (25 ng/ml) at day 4. Data expressed as means ± SEM.