RANKL-induced M1 macrophages are involved in bone formation

Abstract

The activation of M1 macrophages can be achieved by stimulating them with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, M1 can be found under physiological conditions without any pathological stimuli. This study aimed to understand the involvement of RANKL-induced M1 macrophages in bone formation compared with pathologically induced macrophages. Fischer rats were used to investigate macrophage distribution in normal and injured femoral condyles in vivo. Bone marrow-derived macrophages (BMDMs) were activated with LPS+IFN-γ and RANKL to achieve M1 activation in vitro. Gene expression related to inflammation, osteoclastogenesis, angiogenesis, and migration was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS). Tissue macrophages showed distinct expression patterns at different bone regions. RANKL was found in close proximity to inducible nitric oxide synthase-positive (iNOS+) cells in vivo, suggesting an association between RANKL expression and iNOS+ cells, especially in trabecular bone. RANKL-induced macrophages showed a different cytokine secretion profile compared with pathologically induced macrophages. Both osteoclasts and M1 macrophages peaked on day 7 during bone healing. RANKL could trigger M1-like macrophages with properties that were different from those of LPS+IFN-γ-induced macrophages. These RANKL-activated M1 macrophages were actively involved in bone formation.

Immunology: White blood cells crucial for bone healing

White blood cells induced by physiological rather than pathological stimuli have a crucial role in bone healing. Yinghong Zhou and colleagues from the Queensland University of Technology in Brisbane, Australia, analyzed the distribution of white blood cells known as macrophages in rat femurs. They found the anti-inflammatory M2 type of macrophages in the bone marrow and the pro-inflammatory M1 macrophages at the bone surface. Alongside the M1 macrophages, they also found high levels of a bone-remodeling protein called RANKL, which is essential for bone remodeling. They then treated macrophages in cell culture with either RANKL or proteins associated with bacterial infection, and found that only the RANKL-treated M1 macrophages were actively involved in bone repair, a finding that could be useful in treating bone disorders.

References

[1] Lavin Y, Mortha A, Rahman A et al. Regulation of macrophage development and function in peripheral tissues. Nat Rev Immunol, 2015, 15: 731-744

[2] Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol, 2008, 8: 958-969

[3] Ley K. The second touch hypothesis: T cell activation, homing and polarization. F1000Res, 2014, 3: 37

[4] Stein M, Keshav S, Harris N et al. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med, 1992, 176: 287-292

[5] Doyle AG, Herbein G, Montaner LJ et al. Interleukin-13 alters the activation state of murine macrophages in vitro comparison with interleukin-4 and interferon-gamma. Eur J Immunol, 1994, 24: 1441-1445

[6] Cherry JD, Olschowka JA, O'Banion MK. Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation, 2014, 11: 98

[7] Hotamisligil GS. Endoplasmic reticulum stress and atherosclerosis. Nat Med, 2010, 16: 396-399

[8] Oh J, Riek AE, Weng S et al. Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J Biol Chem, 2012, 287: 11629-11641

[9] Karaiskos C, Hudspith BN, Elliott T et al. Defective macrophage function in crohn's disease: role of alternatively activated macrophages in inflammation. Gut, 2011, 140: S324-S325

[10] Sindrilaru A, Peters T, Wieschalka S et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest, 2011, 121: 985-997

[11] Fujisaka S, Usui I, Bukhari A et al. Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice. Diabetes, 2009, 58: 2574-2582

[12] Lacey DL, Timms E, Tan HL et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell, 1998, 93: 165-176

[13] Kong YY, Yoshida H, Sarosi I et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature, 1999, 397: 315-323

[14] Zheng H, Yu X, Collin-Osdoby P et al. RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption. J Biol Chem, 2006, 281: 15809-15820

[15] Jablonski KA, Amici SA, Webb LM et al. Novel markers to delineate murine M1 and M2 macrophages. PLoS ONE, 2015, 10: e0145342

[16] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods, 2001, 25: 402-408

[17] Chun L, Yoon J, Song Y et al. The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses. J Biomed Mater Res, 1999, 48: 899-903

[18] Weisser SB, McLarren KW, Kuroda E et al. Generation and characterization of murine alternatively activated macrophages. Methods Mol Biol, 2013, 946: 225-239

[19] Xuan W, Qu Q, Zheng B et al. The chemotaxis of M1 and M2 macrophages is regulated by different chemokines. J Leukoc Biol, 2015, 97: 61-69

[20] Davies LC, Jenkins SJ, Allen JE et al. Tissue-resident macrophages. Nat Immunol, 2013, 14: 986-995

[21] Edin S, Wikberg ML, Dahlin AM et al. The distribution of macrophages with a M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PLoS ONE, 2012, 7: e47045

[22] Alexander KA, Chang MK, Maylin ER et al. Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model. J Bone Miner Res, 2011, 26: 1517-1532

[23] Wu AC, Raggatt LJ, Alexander KA et al. Unraveling macrophage contributions to bone repair. BoneKEy Rep, 2013, 2: 373

[24] Schlundt C, El Khassawna T, Serra A et al. Macrophages in bone fracture healing: their essential role in endochondral ossification. Bone 2015; pii: S8756-3282(15)00392-0.

[25] Price JS, Oyajobi BO, Russell RG. The cell biology of bone growth. Eur J Clin Nutr, 1994, 48 Suppl 1 S131-S149

[26] Mackaness GB. Cellular resistance to infection. J Exp Med, 1962, 116: 381-406

[27] Martinez FO. Regulators of macrophage activation. Eur J Immunol, 2011, 41: 1531-1534

[28] Kim JB, Han AR, Park EY et al. Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol Pharm Bull, 2007, 30: 2345-2351

[29] Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy, 2005, 4: 281-286

[30] Kondo S, Kohsaka S, Okabe S. Long-term changes of spine dynamics and microglia after transient peripheral immune response triggered by LPS in vivo. Mol Brain, 2011, 4: 27

[31] Wada T, Nakashima T, Hiroshi N et al. RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med, 2006, 12: 17-25

[32] Xing L, Schwarz EM, Boyce BF. Osteoclast precursors, RANKL/RANK, and immunology. Immunol Rev, 2005, 208: 19-29

[33] Loi F, Córdova LA, Zhang R et al. The effects of immunomodulation by macrophage subsets on osteogenesis in vitro. Stem Cell Res Ther, 2016, 7: 15

[34] Schell H, Lienau J, Epari DR et al. Osteoclastic activity begins early and increases over the course of bone healing. Bone, 2006, 38: 547-554

[35] Bastian O, Pillay J, Alblas J et al. Systemic inflammation and fracture healing. J Leukoc Biol, 2011, 89: 669-673

[36] Spiller KL, Nassiri S, Witherel CE et al. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds. Biomaterials, 2015, 37: 194-207