Autoantibody mediated inflammation co-activates FcgR and IFNgR pathways in osteoclasts

Bone remodeling is a continuous and uninterrupted process in which old bone tissue is removed by osteoclasts (the bone-resorbing cells) while, in parallel, new bone substance is formed by osteoblasts (the bone-building cells). Normal osteoclast activity is essential for the vitality of the tissue and the stability of the bones. In contrast, osteoclasts are responsible for the destruction of bone and cartilage in inflammatory bone diseases such as rheumatoid arthritis (RA).

Rheumatoid arthritis (RA) affects approximately 1% of the Western world population, making it one of the most widespread autoimmune diseases. Inflammatory bone loss in RA is caused by altered bone homeostasis with increased osteoclast generation and activity resulting in accelerated bone resorption. The triggers that lead to progressive bone loss given the appropriate genetic predisposition are still insufficiently researched. It is described that activation of osteoclast differentiation and bone loss in RA is stimulated by IgG immune complexes like anti-citrullinated protein antibodies (ACPAs), which activate the Fc gamma receptor (FcgR) on the osteoclast surface and shift the balance toward bone resorption. Apart from this, by secreting interferon gamma (IFNg), immune cells can block osteoclast differentiation in vitro, although the role of IFNg is discussed controversially. IFNg has been described to induce osteoclast activation in vivo via Tumor necrosis factor alpha (TNFa). Despite the role of IFNg and IgG immune complexes in regulating osteoclast differentiation, no link between these two signaling pathways has been described.


In our study, we hypothesized that bone loss is RA is mutually influenced by the IgG immune complexes formed during the immune response and by the inflammatory cytokine IFNg. In our recently published study in Frontiers in Immunology we aimed to investigate when and how exactly, the FcgR and IFNgR signaling pathways interact with each other to regulate osteoclast differentiation.


Total internal reflection (TIRF) microscopy measurements revealed that FcgR3 and IFNgR are localized in close proximity to each other on the human osteoclast surface. Interestingly, the average distance of FcgR3 and IFNgR increases in the process of osteoclast differentiation, suggesting that the average distance of these receptors is dependent on the maturation state of human osteoclast. Even more, in vitro activation of both receptors indicates that FcgR3 and IFNgR activation influence the membrane localization of the other receptor and possibly shape their individual distribution during activation.

IFNgR activation promotes TRAF6 degradation in vitro, leading to reduced osteoclast formation. However, opposite functions of IFNg have been described depending on the model used. Here we demonstrated that the inhibitory effect of IFNg in vitro depends on the differentiation state of human osteoclasts. While IFNg completely inhibits the differentiation of early osteoclast precursor cells into multinucleated osteoclasts, IFNg promotes fusion of premature osteoclasts. Moreover, in gene expression analyses, IFNgR activation induces FcgR expression in early osteoclasts but not in premature osteoclasts suggesting a co-regulation of FcgR and IFNgR in human osteoclast progenitor cells.

Based on hierarchical cluster analysis of differentially phosphorylated proteins by scioPhospho protein profiling, FcgR and IFNgR activation in premature osteoclasts exhibit distinct clusters compared to non-stimulated controls. Moreover, Volcano plot analyses of the stimulated osteoclasts compared with untreated osteoclasts showed strong induction of protein phosphorylation in IFNg-treated cells but little in ACPA-treated cells. Surprisingly, combined activation of both receptors showed a similar proteomic pattern as the non-stimulated control cells, suggesting a negative balancing mechanism within the two activated pathways. Functional gene ontology (GO) annotation showed an increased phosphoproteome for cellular metabolic (ROS) processes, MAPK signaling, cytoskeleton organization, cell differentiation and activation in IFNg-treated mature osteoclasts that are all in line with the observed stimulatory effect of IFNg on mature osteoclasts. The GO annotation for phosphorylated proteins that were downregulated by IFNg treatment could be clustered to the biological process (BP) of cell differentiation, cell cycle, cell signaling, cell motility, transmembrane transport and migration. In conclusion, the phosphoproteome data show that FcgR or IFNgR activation on multinucleated osteoclasts trigger distinct downstream signaling events while co-stimulated mature osteoclasts clustered close to the control cells. This indicates that FcgR3 activation could attenuate IFNgR induced effects in mature osteoclasts.


In summary, we were able to show for the first time that FcgR3 and IFNgR interact directly with each other during osteoclast differentiation. More specifically, we now know that the IFNgR is dependent on the expression of FcgR3 for efficient signaling; on the other hand, targeted activation of FcgR3 inhibits the inhibitory effect of IFNgR on osteoclast differentiation. Taken together, our data show that FcgR and IFNgR signaling pathways influence osteoclast activity depending on the differentiation state, which may reflect the bone erosion state of rheumatoid arthritis. Our data can be an interesting starting point for the development of new innovative bone building agents and antiresorptive drugs for the treatment of osteoporosis.


These findings are described in the article titled “Inflammatory activation of FcgR and IFNgR pathways co-influence the differentiation and activity of osteoclasts” published in Frontiers in Immunology (2022; doi: 10.3389/fimmu.2022.958974). This work was performed by Bettina Groetsch, Elisabeth Schachtschabel, Philipp Tripal, Benjamin Schmid, Ana-Suncana Smith, Georg Schett and Aline Bozec.

Protein O-GlcNAcylation modulates osteoclast formation

Mechanobiological changes during estrogen deficiency leading to bone loss



Osteoporosis, which predominantly occurs in females, affects 200 million women worldwide and is characterised by bone loss and microarchitectural degradation, which leads to fractures, pain and immobility. Conventional treatments for osteoporosis target bone loss by inhibiting osteoclast activity, but these only prevent osteoporotic fractures in 50% of sufferers. An advanced understanding of the underlying mechanisms and treatments for osteoporosis is required to avert the projected trend, whereby the worldwide economic burden of treatment will reach $132 billion by 2050. Osteoblasts and osteocytes are known to regulate the differentiation, function and survival of osteoclasts, through expression of paracrine factors, which can be altered following mechanical loading and are affected by the presence of estrogen. Studies have recently revealed changes in osteoblast and osteocyte mechanobiology during estrogen deficiency, but, how these changes effect osteoclastogenesis is not fully understood. The first study of this thesis sought to assess changes in osteoblast-induced osteoclastogenesis under combined estrogen deficiency and mechanical loading, using a combination of conditioned media and co-culture experiments. MC3T3-E1 cells were cultured with pre-menopausal levels of estrogen which was then withdrawn from the media and the osteoblast like-cells were exposed to oscillatory fluid flow. These studies found that conditioned media from estrogen-treated MC3T3-E1 cells inhibited the differentiation of RAW264.7 cells and inhibited podosome belt formation. Estrogen deficient osteoblasts down-regulated osteoprotegerin (OPG) expression in response to fluid flow when compared to estrogen-treated osteoblasts, leading to a significant increase in the ratio of RANKL/OPG gene expression. Subsequently, an increase in osteoclast number and an up-regulation in nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and Cathepsin K expression was observed in RAW264.7 cells. In addition, estrogen deficient osteoblasts co-cultured with RAW264.7 cells resulted in an increase in osteoclastogenesis and matrix degradation compared to co-cultures with estrogen-treated osteoblasts. Additionally, it was found that Rho-ROCK inhibition in osteoblasts, exacerbated the increase in osteoblast-induced osteoclastogenesis that arose under estrogen deficiency. It is proposed that the reduction in OPG production by mechanically stimulated osteoblasts during estrogen deficiency may leave osteoclast differentiation and matrix degradation activity unchecked and, thereby, play an important role in bone loss during osteoporosis. The second study sought to establish whether mechanically stimulated osteocytes induce osteoclastogenesis and bone resorption during estrogen deficiency. This study built upon the post-menopausal model and co-culture methods established in the first study. The findings revealed that under postmenopausal conditions RANKL/OPG gene expression is increased in mechanically stimulated osteocytes (OCY454 cells) compared to estrogen-treated osteocytes and a significant increase in osteocyte-induced osteoclast formation (by bone marrow macrophage cells) occurs leading to increased resorption. Estrogen deficient osteocytes also up-regulated sclerostin expression following mechanical loading. Interestingly, Wnt antagonists WIF1 and FRZB were down-regulated in estrogen deficient osteocytes following loading. A second aim of this study was to investigate whether a neutralising antibody against sclerostin (Scl-Ab) could revert osteocyte-mediated osteoclastogenesis and resorption by attenuating RANKL/OPG gene expression. It was demonstrated that administration of the Scl-Ab reduced pro-osteoclastogenic signalling (RANKL/OPG) between osteocytes and osteoclasts, which led to reduced resorption. Osteocytes are housed within lacunae and their dendrites extend through canaliculae to enable the formation of gap junctions with neighbouring osteocytes and osteoblasts. The lacunar-canalicular network and vascular pores facilitate movement of interstitial fluid, which confers mechanical stimulation on osteocytes and facilitates transport of nutrients and signalling molecules to and from the osteocyte. There is some evidence that this microporous network is altered during estrogen deficiency, which might alter fluid flow through the network and the ensuing responses of osteoblasts and osteocytes. While previous studies have investigated early-stage estrogen deficiency, the long term effects of estrogen deficiency and the time-sequence of changes in cortical bone microporosity are not known. For this reason, the final study of this thesis sought to determine the temporal changes that occur in the cortical vasculature and the lacunar-canalicular network during short and longer term estrogen deficiency. This study quantified microporosity and lacunar occupancy in an ovariectomised rat model of osteoporosis by micro-CT analysis, backscatter electron imaging and histological analysis. It was shown that initial increases in canalicular diameter and vascular porosity arose during short term estrogen deficiency (week 4 OVX) compared to aged-matched controls, but that these were reduced along with a decrease in lacunar diameter and lacunar occupancy in long term estrogen deficiency (week 14 OVX). These changes could be explained by perilacunar remodelling, micropetrosis or a mechanobiological adaptive response, such as increases in bone mass and bone mineralisation that may occur to alter the mechanical environment in an attempt to restore tissue homeostasis. This study also demonstrated that administration of Scl-Ab to ovariectomised rats resulted in a significant decrease in osteoclast number and prevented the occurrence of empty lacunae in long term estrogen deficiency (week 14 OVX) compared to untreated week 14 OVX animals. Together, the studies in this thesis found that the inhibitory effects mechanically loading has on pro-osteoclastogenic signalling in osteocytes and osteoblasts is attenuated during estrogen deficiency. Using cell culture and mechanobiological techniques, the changes in paracrine factor expression in estrogen deficient osteoblasts and osteocytes and their implications in terms of osteoclastogenesis and resorption were elucidated. Additionally, the temporal changes in cortical microporosity that occur were established. Collectively, the research presented in this thesis provides an important, but previously unrecognised, insight into the mechanobiological changes that occur during estrogen deficiency leading to bone loss. The information gained from this work may inform future mechanobiological treatments for osteoporosis.


Full text of D. Allison’s thesis is available here