Enhanced Immunoprotective Effects by Anti-IL-17 Antibody Translates to Improved Skeletal Parameters Under Estrogen Deficiency Compared With Anti-RANKL and Anti-TNF-α Antibodies

Abstract

Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF-kB ligand (RANKL), tumor necrosis factor a (TNF-a), and interleukin 17 (IL-17), all of which augment osteoclastogenesis. RANKL and TNF-a are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF-a. IL-17 is also an important mediator of bone loss, and an antibody against IL-17 is undergoing phase II clinical trial for rheumatoid arthritis. Although there are a few studies showing suppression of Th17 cell differentiation and induction of regulatory T cells (Tregs) by infliximab, the effect of denosumab remains poorly understood. In this study, we investigated the effects of anti-TNF-a, anti-RANKL, or anti-IL-17 antibody administration to estrogen-deficient mice on CD4 þ T-cell proliferation, CD28 loss, Th17/Treg balance and B lymphopoesis, and finally, the translation of these immunomodulatory effects on skeletal parameters. Adult Balb/c mice were treated with anti-RANKL/-TNF-a/-IL-17 subcutaneously, twice a week, postovariectomy (Ovx) for 4 weeks. Animals were then autopsied; bone marrow cells were collected for FACS and RNA analysis and serum collected for ELISA. Bones were dissected for static and dynamic histomorphometry studies. We observed that although anti-RANKL and anti-TNF-a therapies had no effect on Ovx-induced CD4 þ T-cell proliferation and B lymphopoesis, anti-IL-17 effectively suppressed both events with concomitant reversal of CD28 loss. Anti-IL-17 antibody reduced proinflammatory cytokine production and induced Tregs. All three antibodies restored trabecular microarchitecture with comparable efficacy; however, cortical bone parameters, bone biomechanical properties, and histomorphometry were best preserved by anti-IL-17 antibody, likely attributable to its inhibitory effect on osteoblast apoptosis and increased number of bone lining cells and Wnt10b expression. Based on the superior immunoprotective effects of anti-IL-17, which appears to translate to a better skeletal preservation, we propose beginning clinical trials using a humanized antibody against IL-17 for treatment of postmenopausal osteoporosis.

Interleukin 27 (IL-27) Alleviates Bone Loss in Estrogen-deficient Conditions by Induction of Early Growth Response-2 Gene

“…One of the mechanisms by which IL-27 exerts its anti-inflammatory action is by inducing IL-10 production from FoxP3-negative Tr1 cells with aid from transcription factor-like Egr-2, which is essential for the differentiation of Tr1 cells (15 Th17 cells are considered to be osteoclastogenic T helper subsets as they augment the synthesis and secretion of RANKL, which induces osteoclastogenesis (10,24). IL-27 cytokine has been reported to prevent Th17 development by inhibition of the ROR␥t transcription factor that is essential for Th17 development (33).…”

“…CD4 ϩ T cells, macrophages, and B cells were isolated using columns, Macs separator, and Micro beads from Miltenyi Biotech (Singapore) by positive selection. All the steps were performed using the manufacturer's instruction and as per as protocols in our previously published papers (10,24,35). After isolation the cells were collected in 1 ml of TRIzol (Invitrogen) for RNA isolation.…”

“…6B). Transcript level of FoxP3, which is a negative regulator of Th17 development (24), was very low in T cells harvested from Ovx animals ( Fig. 6D).…”

“…Not much difference was observed between sham and the sham group treated with IL-27 cytokine. Transcription factorlike ROR-␥t is crucial for Th17 differentiation (24), and its transcript level was determined in CD4 ϩ T cells. As expected, the expression of ROR-␥t was robustly enhanced in T cells harvested from Ovx animals, whereas treatment with IL-27 significantly reversed this effect and brought it back to the sham control and sham group treated with IL-27 ( Fig.…”

“…ϩ T Cells on Osteoblast Differentiation-Pro-inflammatory cytokines like IL-17 and TNF-␣ induce osteoblast apoptosis (24). Thus, we determined the effect of IL-27 on IL-17-mediated osteoblast apoptosis by annexin-propidium iodide staining.…”