Effects of IL-34 and anti-IL-34 neutralizing mAb on alveolar bone loss in a ligature-induced model of periodontitis

Abstract

Macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) are ligands for the colony-stimulating factor-1 receptor (CSF-1r) expressed on the surface of monocyte/macrophage lineage cells. The importance of coordinated signaling between M-CSF/receptor activator of the nuclear factor kappa-Β ligand (RANKL) in physiological and pathological bone remodeling and alveolar bone loss in response to oral bacterial colonization is well established. However, our knowledge about the IL-34/RANKL signaling in periodontal bone loss remains limited. Recently published cohort studies have demonstrated that the expression patterns of IL-34 are dramatically elevated in gingival crevicular fluid collected from patients with periodontitis. Therefore, the present study aims to evaluate the effects of IL-34 on osteoclastogenesis in vitro and in experimental ligature-mediated model of periodontitis using male mice. Our initial in vitro study demonstrated increased RANKL-induced osteoclastogenesis of IL-34-primed osteoclast precursors (OCPs) compared to M-CSF-primed OCPs. Using an experimental model of ligature-mediated periodontitis, we further demonstrated elevated expression of IL-34 in periodontal lesions. In contrast, M-CSF levels were dramatically reduced in these periodontal lesions. Furthermore, local injections of mouse recombinant IL-34 protein significantly elevated cathepsin K activity, increased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and promoted alveolar bone loss in periodontitis lesions. In contrast, anti-IL-34 neutralizing monoclonal antibody significantly reduced the level of alveolar bone loss and the number of TRAP-positive osteoclasts in periodontitis lesions. No beneficial effects of locally injected anti-M-CSF neutralizing antibody were observed in periodontal lesions. This study illustrates the role of IL-34 in promoting alveolar bone loss in periodontal lesions and proposes the potential of anti-IL34 monoclonal antibody (mAb)-based therapeutic regimens to suppress alveolar bone loss in periodontitis lesions.

Keywords: IL‐34; M‐CSF; ligature‐induced periodontitis; monoclonal antibody; osteoclast precursors; osteoclastogenesis.

Figure 1.. Effects of recombinant IL-34 and M-CSF proteins on RANKL-induced osteoclastogenesis in vitro.

Expression profile of the osteoclast fusion and function genes, e.g. Oc-stamp (A), Dc-stamp (B), CatK (C), and Acp5/Trap (D), was quantified in IL-34- and M-CSF-proliferated bone marrow derived macrophages (BMDMs) (n=6) after 7 days culture in the presence or absence of recombinant RANKL. The difference in gene expression is shown as the fold change after normalization against GAPDH. Microscopic evaluation (E) of the TRAP staining and quantification of the number of TRAP+ multinucleated cells (F) in the rM-CSF- and rIL-34-stimulated OCPs exposed to RANKL in vitro (n=6). Representative images of pit formation (G) and pit area (H) quantified by Image J (n=5). *p<0.05; **p<0.01; ***p<0.001.

Figure 2.. Expression of M-CSF and IL-34 in healthy control and periodontal lesions.

Histological examination for M-CSF (A) and IL-34 (B) expression patterns at lower and higher magnification that emerged in decalcified sections of control (no ligature placed) and ligature-mediated periodontitis. C: Intensity quantification of M-CSF and IL-34 observed from microscopic images. Ligature -: no ligature placed; Ligature +: ligature attached. A.U. – arbitrary units. Scale bar = 100μm. Note experimental periodontitis was induced in C57BL/6 mice, an M-CSF-producing strain. The levels of M-CSF and IL-34 were evaluated using histological samples collected from the same mouse samples. N= 5 samples/condition. ***p<0.001, **p<0.01.

Figure 3.. Local gingival injection of mouse recombinant IL-34 protein exacerbates l alveolar bone resorption in experimental periodontitis.

In situ molecular imaging of cathepsin K (CatK) activity, a resorptive bone marker (A), and the mean fluorescent intensity (FLI) of cathepsin K activity with respect to total flux (B) observed in periodontal lesions induced by ligation alone or ligation plus local gingival injection of recombinant IL-34 mouse protein (1 ng/1μl/site). To further verify the observed difference in bone resorption between mice treated with ligation plus local gingival injection of IL-34 protein and those treated with ligation alone, the 3D bone assessment was performed using Micro-computed tomography (C) to determine periodontal bone loss (D). Total periodontal bone loss was calculated by subtracting the sum of the cemento-enamel junction (CEJ) from the alveolar bone crest (ABC) distance of the baseline no-ligature site from that of the ligature site. Finally, TRAP+ osteoclasts were identified (E) and quantified (F) in histological sections from the treated mice. N=6 male mice/condition; Ligature -: no ligature placed; Ligature +: ligature attached. FLI – Fluorescent Intensity; Note experimental periodontitis was induced in C57BL/6 mice, an M-CSF-producing strain. *p<0.05; **p<0.01; ***p<0.001.

Figure 4.. Neutralizing anti-IL-34 monoclonal antibody (mAb), but not anti-M-CSF mAb, suppressed periodontal bone loss in ligatured experimental mice.

A: Concentration of Acp5/TRAP, a bone resorption marker, in the gingival crevicular fluid collected from the ligature-induced periodontal lesions of anti-IL-34 and anti-M-CSF mAb-treated mice. The Acp5/TRAP was measured using an ELISA assay. B & C: Micro-computed tomography of ligature-induced bone resorption (B) and total periodontal bone loss (C) observed in mice that received a local periodontal injection of IgG control Ab, anti-IL-34 mAb, or anti-M-CSF mAb (10 μg/site). No ligature placed mice received a local injection of IgG control Ab. Ligature -: no ligature placed; Ligature +: ligature attached. Finally, TRAP+ osteoclasts were identified (E) and quantified (F) in histological sections from the treated mice. N= 6 male mice/group. Note that experimental periodontitis was induced in wild type mice, an M-CSF-producing strain. Scale bar – 100μm. *p<0.05; **p<0.01; ***p<0.001.