Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ

Abstract

Introduction: The potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals.

Methods: We used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice.

Results: Percentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly.

Discussion: ZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw.

Keywords: IFN-γ; NK cells; antibody-dependent cellular cytotoxicity (ADCC); cytotoxicity; denosumab; humanized-BLT mice; osteonecrosis of the jaw; zoledronic acid.

Figure 1

Surface expression of human and mouse CD45 on peripheral blood, bone marrow, splenocytes and bone marrow-derived osteoclasts of hu-BLT mice; comparison of immune cell percentages in human, WT mice and hu-BLT mice PBMCs. Hu-BLT mice acquired humanized hemato-lymphatic system by co-transplantation of human fetal liver and thymus fragments under the renal capsule of NOG/Scid γc-/- immune deficient NSG mice followed by the intravenous injection of autologous CD34+ hematopoietic cells. Eight weeks after tissue implantation, mice were euthanized and tissues were harvested to obtain single cell suspension. PBMCs, bone marrow (BM), and spleen of hu-BLT mice were analyzed for human and mice CD45+ immune cells using flow cytometric analysis (A). Hu-BLT mice BM isolated monocytes were used to generate osteoclasts (OCs) for 21 days as described in Materials and Methods section. On day 21, hu-BLT OCs were washed with 1 X PBS before the pictures were taken by Leica DMI 6000B inverted microscope. Hu-BLT OCs were then treated with Fast Garnet GBC and sodium nitrate (1:1), incubated at 37 degree C for one hour, cells were rinsed and treated with hematoxylin for two mins before pictures were taken by Leica DMI 6000B inverted microscope. Pre- and Post-TRAP-stained pictures are shown in the figure. Hu-BLT OCs were analyzed for human and mice CD45+ immune cells using flow cytometric analysis (B). Immune cell composition was determined in human PBMCs, hu-BLT PBMCs and B6 female WT mice PBMCs using flow cytometric analysis (C).

Figure 2

IFN-γ secretion and NK-cell mediated cytotoxicity of PBMCs, BM, and splenocytes of NACL and ZOL-injected hu-BLT mice. Hu-BLT mice were administered with either 0.9% NACL or ZOL (500 µg/kg) via IV as described in Materials and Methods section. Four weeks after injections, mice were euthanized and tissues were harvested to obtain single cell suspension. PBMCs (A), BM cells (B), and splenocytes (C) of hu-BLT mice were cultured (2 × 10⁶ cells/2ml) with IL-2 (1000 U/ml) for three days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISA. PBMCs (A), BM cells (B), and splenocytes (C) were used as effector cells in standard 4-hour ⁵¹Cr release assay against human OSCSCs tumors. Lytic units (LU) 30/10⁶ cells were determined using inverse number of effector cells required to lyse 30% of OSCSCs × 100. LUs per 1% NK cells were determined using CD16+CD56+ percentages obtained by flow cytometric analysis (n=2) (A–C). **(p value 0.001-0.01), *(p value 0.01-0.05).

Figure 3

Human CD45+ immune cell percentages, IFN-γ secretion and NK-cell mediated cytotoxicity in oral gingival cells of NACL or ZOL-injected and tooth extracted hu-BLT mice. Hu-BLT mice were administered with either 0.9% NACL or ZOL (500 µg/kg) via IV followed by maxillary left first molar extraction as described in Materials and Methods section. Two weeks (n=3) (A) and four weeks (n=2) (B) after injections, mice were euthanized and oral gingival tissues were harvested to obtain single cell suspension. Surface expression of CD45+CD3+, CD45+CD16+CD56+, and CD45+CD3+gdT+ in oral gingival cells were determined using flow cytometric analysis as described in the Materials and Methods. Oral gingival cells mice were cultured (2 × 10⁶ cells/2ml) with IL-2 (1000 U/ml) for three days, after which the supernatants were harvested and the levels of IFN-γ was determined using specific ELISA. IFN-γ secretion was determined per human CD45+ cells using human CD45+ percentages obtained by flow cytometric analysis. IFN-γ per one NK cell were determined using CD16+CD56+ percentages obtained by flow cytometric analysis. Oral gingival cells were used as effector cells in standard 4-hour ⁵¹Cr release assay against human OSCSCs tumors. Lytic units (LU) 30/10⁶ cells were determined using inverse number of effector cells required to lyse 30% of OSCSCs × 100. LU per 1% NK cells were determined using CD16+CD56+ percentages obtained by flow cytometric analysis (A, B). **(p value 0.001-0.01), *(p value 0.01-0.05) ns (no significance).

Figure 4

IFN-γ secretion in PBMCs, BM, spleen and pancreas of NACL or ZOL-injected and tooth-extracted hu-BLT mice. Hu-BLT mice were administered with either 0.9% NACL or ZOL (500 µg/kg) via IV followed by maxillary left first molar extraction as described in Materials and Methods section. Two (A) or four weeks (B) after tooth extraction, mice were euthanized and tissues were harvested to obtain single cell suspension. PBMCs (n=4), BM (n=4), splenocytes (n=4), and pancreas (n=3) of hu-BLT mice were cultured (2 × 10⁶ cells/2ml) with IL-2 (1000 U/ml) for three days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISA. IFN-γ secretion per human CD45+ cell was determined using CD45 percentages obtained by flow cytometric analysis. IFN-γ per one NK cell was determined using CD16+CD56+ percentages obtained by flow cytometric analysis (A, B). ****(p value<0.0001), ***(p value<0.001), **(p value 0.001-0.01), *(p value 0.01-0.05), ns (no significance).

Figure 5

NK cell-mediated cytotoxicity in PBMCs, BM, spleen and pancreas of NACL or ZOL-injected and tooth-extracted hu-BLT mice. Hu-BLT mice were administered with either 0.9% NACL or ZOL (500 µg/kg) via IV followed by maxillary left first molar extraction as described in Materials and Methods section. Two (A) or four weeks (B) after tooth extraction, mice were euthanized and tissues were harvested to obtain single cell suspension. PBMCs (n=4), BM (n=4), splenocytes (n=4), and pancreas (n=3) of hu-BLT mice were cultured (2 × 10⁶ cells/2ml) with IL-2 (1000 U/ml) for three days, after which the cells were used as effector cells in standard 4-hour ⁵¹Cr release assay against human OSCSCs tumors. Lytic units (LU) 30/10⁶ cells were determined using inverse number of effector cells required to lyse 30% of OSCSCs × 100. LUs per 1% NK cell were determined using CD16+CD56+ percentages obtained by flow cytometric analysis (A, B). ***(p value<0.001), **(p value 0.001-0.01), *(p value 0.01-0.05), ns (no significance).

Figure 6

IFN-γ secretion and NK-cell mediated cytotoxicity in NACL and denosumab-injected hu-BLT mice. Hu-BLT mice were administered with either 0.9% NACL or denosumab (120 mg/mice) via IV as described in Materials and Methods section. Four weeks after injections, mice were euthanized and tissues were harvested to obtain single cell suspension. PBMCs (n=2), BM cells (n=2), splenocytes (n=2), pancreas (n=2), and gingiva (n=3) of hu-BLT mice were cultured (2 × 10⁶ cells/2ml) with IL-2 (1000 U/ml) for three days, after which the supernatants were harvested and the levels of IFN-γ were determined using specific ELISA (A). PBMCs (n=2), BM cells (n=2), and splenocytes (n=2) were used as effector cells in standard 4-hour ⁵¹Cr release assay against human OSCSCs tumors. Lytic units (LU) 30/10⁶ cells were determined using inverse number of effector cells required to lyse 30% of OSCSCs × 100 (B). LUs per 1% NK cells were determined using CD16+CD56+ percentages obtained by flow cytometric analysis (n=2) (C). ***(p value 0.0001-0.001), **(p value 0.001-0.01), *(p value 0.01-0.05), ns (no significance).

Figure 7

Higher levels of NK cell-mediated ADCC was seen against OCs treated with a combination of denosumab and RANKL. Human OCs were generated as described in the Materials and Methods. OCs were left untreated or treated with denosumab (20 μg/ml) or RANKL (25 ng/ml) or a combination of denosumab (20 μg/ml) and RANKL (25 ng/ml) for 30 minutes. The unbounded antibodies were washed away, and the surface expression levels were analyzed after cells were stained with goat anti-human FITC using flow cytometry. IgG2 isotype antibodies were used as controls. One of three representative experiments is shown in the figure (A). OCs were generated as described in the Materials and Methods. Purified NK cells (1×10⁶ cells/ml) from healthy individuals were treated with IL-2 (1000 U/ml) for 18 hours and were as effectors in chromium release assay. OCs were labeled with ⁵¹Cr for an hour after which cells were washed to remove unbound ⁵¹Cr. ⁵¹Cr-labeled OCs were then left untreated or treated with denosumab (20 μg/ml) or RANKL (25 ng/ml) or a combination of denosumab (20 μg/ml) and RANKL (25 ng/ml) for 30 minutes. The unbounded antibodies were washed away, and the cytotoxicity against the OCs was determined using a standard 4-6 hour ⁵¹Cr release assay. The lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of OCs × 100 (n=3) (B). *(p value 0.01-0.05).