TNF-Polarized Macrophages Produce Insulin-like 6 Peptide to Stimulate Bone Formation in Rheumatoid Arthritis in Mice

Abstract

The risk of osteoporosis is increased in rheumatoid arthritis (RA). Anti-tumor necrosis factor (TNF) therapy has markedly improved the outcomes of RA patients but does not improve osteoporosis in some reports. This could be a combined result of disease severity and other therapeutic agents, such as glucocorticoids that accelerate osteoporosis progression. We evaluated the effects of anti-TNF therapy on osteoporosis in an animal model of RA and explored the possible mechanisms involved. Six-week-old TNF transgenic (TNF-Tg) mice with early stage erosive arthritis were treated with TNF antibody (Ab) or control immunoglobulin (IgG) weekly for 4 weeks. We found that TNF Ab completely blocked the development of erosive arthritis in TNF-Tg mice, but only slightly increased vertebral bone mass, associated with reduction in parameters of both bone resorption and formation. Similarly, TNF Ab slightly increased trabecular bone mass in tibias of 8-month-old TNF-Tg mice with advanced erosive arthritis. Interestingly, TNFα increased osteoblast differentiation from mouse bone marrow stromal cells (BMSCs) containing large number of macrophages but not from pure mesenchymal progenitor cells (MPCs). TNFα-polarized macrophages (TPMs) did not express iNos and Arginase 1, typical markers of inflammatory and resident macrophages. Interestingly, TPMs stimulated osteoblast differentiation, unlike resident and inflammatory macrophages polarized by IL-4 and interferon-λ, respectively. RNA-seq analysis indicated that TPMs produced several anabolic factors, including Jagged1 and insulin like 6 (INSL6). Importantly, inhibition of either Jagged1 or INSL6 blocked TNFα-induced osteoblast differentiation. Furthermore, INSL6 Ab significantly decreased the expansion of TNF-induced MPCs in BMSCs, and anti-TNF Ab reduced INSL6 expression by macrophages in vitro and in TNF-Tg mice in vivo. We conclude that TPMs produce INSL6 to stimulate bone formation and anti-TNF Ab blocks not only enhanced bone resorption but also the anabolic effect of TPMs on bone, limiting its effect to increase bone mass in this model of RA.

Regulation of TNF-Induced Osteoclast Differentiation

“…Thus, it is widely accepted that targeted depletion of M1 and boosting activities of M2 macrophages are emerging as an attractive combined therapeutic strategy for autoimmune diseases [ 141 , 142 , 143 ]. However, a recent report shows that TNF-polarized macrophages (TPMs) produce insulin-like 6 peptide (INSL6) and Jagged1 to stimulate bone formation, slowing down bone loss caused by enhanced bone resorption in RA [ 144 ], which explains why anti-TNF therapy has a limited effect in improving the lost bone in patients with RA [ 145 , 146 , 147 ].…”

“…The hallmarks of M1 activation are iNOS expression and high levels of IL-12 with low levels of IL-10 production [ 102 ]. A recent report shows that TNF-polarized macrophages do not produce iNOS, although they express the major M1 surface markers [ 144 ]. RANKL can stimulate the expression of iNOS, which acts as an autocrine negative feedback mechanism to restrain RANKL-mediated osteoclastogenesis [ 154 ].…”

“…Many factors, including disease severity or activity and glucocorticoid treatment, which accelerates bone loss, could influence the effect of anti-TNF agents on bone mass in RA patients [ 146 , 187 , 194 , 195 ], and explain the discrepancies in these reported outcomes. In 6-week-old TNF-Tg mice with early-stage erosive arthritis, 4 weeks of TNF antibody treatment completely blocked the development of erosive arthritis, but only slightly increased vertebral bone mass, associated with a reduction in parameters of both bone resorption and formation [ 144 ]. Similarly, TNF antibody slightly increased trabecular bone mass in tibiae of 8-month-old TNF-Tg mice with advanced erosive arthritis [ 144 ].…”

“…In 6-week-old TNF-Tg mice with early-stage erosive arthritis, 4 weeks of TNF antibody treatment completely blocked the development of erosive arthritis, but only slightly increased vertebral bone mass, associated with a reduction in parameters of both bone resorption and formation [ 144 ]. Similarly, TNF antibody slightly increased trabecular bone mass in tibiae of 8-month-old TNF-Tg mice with advanced erosive arthritis [ 144 ]. Interestingly, TNFα increased osteoblast differentiation from bone marrow stromal cells containing a large number of macrophages, but not from pure mesenchymal progenitor cells (MPCs) [ 144 ].…”

“…Similarly, TNF antibody slightly increased trabecular bone mass in tibiae of 8-month-old TNF-Tg mice with advanced erosive arthritis [ 144 ]. Interestingly, TNFα increased osteoblast differentiation from bone marrow stromal cells containing a large number of macrophages, but not from pure mesenchymal progenitor cells (MPCs) [ 144 ]. Further analysis indicated that TNFα-polarized macrophages (TPMs) produced several anabolic factors, including Jagged1 and insulin-like 6 (INSL6).…”