Irisin is a peptide produced by proteolytic cleavage of fibronectin type III domain-containing protein 5 (FNDC5), a membrane-bound protein highly expressed in the skeletal muscle. FNDC5 expression increases in response to acute bouts of exercise by regulation of PGC-1α, leading to a burst of circulating irisin (Jedrychowski et al., 2015). Initially, irisin was described as a hormone that induces thermogenesis in adipose tissue (Boström et al., 2012), but more recent studies have shown that it has a potent ability to modulate bone turnover. These effects support the tenet that irisin may be a key mediator of muscle-bone crosstalk during exercise. Initial studies demonstrated that irisin enhanced cortical bone formation and prevented unloading-induced bone loss in vivo and stimulated osteoblasts in vitro (Colaianni et al., 2014; Colaianni et al., 2015; Colaianni et al., 2017). Conversely, genetic deletion of Fndc5 was separately shown to block resorption-driven bone loss and maintain osteocyte function following ovariectomy; irisin treatment in vitro also prevented osteocyte apoptosis and stimulated sclerostin and RANKL release, key promoters of osteoclastogenesis, through the α_Vβ₅ integrin receptor (Kim et al., 2018). This study addresses the hypothesis that irisin also directly stimulates osteoclast differentiation and function in vitro and in vivo.

First, we used continuous treatment with a range of irisin doses (0, 2, 5, 10, and 20 ng/mL) for 7 days in an in vitro osteoclast differentiation assay using primary marrow hematopoietic progenitors. These doses were based on previous studies establishing the physiologic range of circulating irisin during and after exercise (Jedrychowski et al., 2015). We found a qualitative enhancement of both osteoclast number and size (Figure 1a), and significant increases in osteoclast number across this dose range (2, 5, and 10 ng/mL: p<0.0001, 20 ng/mL: p=0.044; Figure 1b, Source data 1). Based on these results, we selected 10 ng/mL irisin to compare continuous (7 days) and transient treatment for the first 4 or 24 hr of culture, and for all further experiments with resorption and gene expression. Both transient treatments led to enhanced osteoclast numbers versus controls (4 hr: p=0.0218, 24 hr: p=0.0008), but continuous irisin resulted in the greatest increase (p<0.0001) and was higher than both 4 hr (p=0.0002) and 24 hr only treatments (p=0.0152; Figure 1c, Source data 1). The stimulatory effect of continuous 10 ng/mL irisin was then further confirmed in primary hematopoietic cells from both sexes of C57BL/6J mice (p=0.0003) and in the RAW 264.7 macrophage cell line (p=0.0428; Figure 1d, Source data 1). RAW-derived osteoclasts appeared morphologically similar to primary cells and mirrored observations of qualitatively larger cells with irisin treatment (Figure 1e).

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As integrins are present on the osteoclast membrane and known to play a role in differentiation (Duong et al., 2000; Yavropoulou and Yovos, 2008), and earlier work identified integrin α_Vβ₅ as a receptor for irisin on osteocytes (Kim et al., 2018), we examined the expression of both subunits in osteoclast cultures and found increased relative mRNA expression above controls with irisin treatment (α_V: p=0.552, β₅: p=0.031; Figure 1f, Source data 1). Blocking integrin α_Vβ₅ with a neutralizing antibody (AVB5-AB) resulted in no differences in osteoclast number per well with irisin treatment (10 ng/mL) versus untreated controls (p=0.79) compared to significant increases with an IgG antibody control (p=0.012) or no-antibody conditions (p=0.0295), indicating this integrin acts as the receptor for irisin on osteoclasts (Figure 1g, Source data 1).

Next, we asked whether irisin-induced osteoclastogenesis led to enhanced bone resorption. Osteoclast differentiation cultures were performed on a variety of native and synthetic substrates with and without continuous 10 ng/mL irisin. TRAP-positive osteoclasts were observed in situ on dentin slices after 7 days (Figure 2a, left), and subsequent toluidine blue staining revealed a qualitative increase in resorption pit area on the surface with irisin treatment (Figure 2a, right). Irisin significantly increased osteoclast numbers on dentin (p=0.013), as well as total resorption area (p=0.045). However, when normalized by osteoclast number, resorption was not significantly different (p>0.99), indicating a dominant effect of cell number in increasing total resorption (Figure 2b, Source data 1). Irisin enhancement of total resorption was further confirmed via the Corning OsteoAssay, with a similar significant increase in total resorption area (p=0.048; Figure 2c, Source data 1). Release of carboxy-terminal collagen crosslinks (CTX) from osteoclast cultures on collagen substrates was measured to assess the effect of transient irisin treatment on resorption at early stages in culture and was significantly increased with both continuous irisin treatment for 3 days (p=0.0153) and for the first 24 hr only (p<0.0001), with this transient treatment also resulting in significantly higher resorption than continuous treatment (p=0.0041; Figure 2d, Source data 1).

Figure 2

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To investigate the key signaling pathways influenced by the action of irisin on the osteoclast, we performed an unbiased analysis of RNA sequencing (RNAseq) data from in vitro osteoclast cultures after 7 days of continuous 10 ng/mL irisin treatment, which demonstrated a qualitatively differential gene expression pattern compared to untreated controls, as typified by hierarchical clustering, volcano plot, and principal component analysis (Figure 3a). Irisin treatment significantly increased the expression of resorption markers Adamts5 (p=0.0381) and Loxl2 (p=0.009), and markers for secreted clastokines known to stimulate osteoblasts: Postn (p=0.0002), Igfbp5 (p=0.03), Tgfb2 (p=0.0073), and Sparc (p=0.0365). Significant decreases in the expression of macrophage markers Mst1r (p=0.0416) and Itgax (p<0.0001) and the lymphocyte markers Cd72 (p=0.0086), Slamf8 (p=0.0052), and H2-aa (p=0.0017) indicated a preferential shift of the hematopoietic progenitor lineage toward osteoclast differentiation (Source data 1). RT-qPCR further showed that irisin significantly increased the expression of key differentiation markers, namely, Rank, the receptor for RANKL (p=0.019), c-src (p=0.008), Fam102a (p=0.039), Nrf2 (p=0.0008), and the osteoclast fusion markers Dcstamp (p=0.0039) and Atp6vod2 (p=0.012). Other early markers of osteoclast differentiation such as Cfos (p=0.41), Itgb3 (p=0.09), Nfatc (p=0.11), Rela (p=0.12), and Rgs12 (p=0.09) were slightly but not significantly increased. Significant increases were confirmed for both key resorption markers: Acp5 (p=0.012) and Loxl2 (p=0.035), and clastokine markers: Sparc (p=0.009) and Wnt10a (p=0.047; Figure 3b, Source data 1).

Figure 3

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Next, we turned to a genetic model of forced expression of Fndc5 in C57BL/6J mice using the McK muscle-specific promoter. RT-qPCR analysis of the whole tibia demonstrated a significant increase in Fndc5 expression at both 4.5 months (p=0.000082) and 13 months (p=0.0041), indicating promoter activity in the bone marrow in addition to the muscle (Figure 4a, Source data 1). Markedly lower cortical bone area was observed by µCT at younger ages in Fndc5-transgenic (TG) mice compared to wild type (WT) (2 and 4.5 months), with significant decreases in trabecular bone volume fraction at 2 (p<0.0001) and 4.5 months (p=0.0003) and in cortical thickness at 2 months (p<0.0001; Figure 4b, Source data 1). Dynamic histomorphometry revealed smaller bones and similar decreases in overall bone volume fraction and trabecular thickness that persisted from 2 to 13 months (Figure 4c, Source data 1). Significant decreases in both the number of osteoblasts per bone perimeter (p=0.05) and bone formation rate (p=0.0046) were observed only at 2 months, and not at 13 months. However, in vitro osteoblast differentiation from bone marrow mesenchymal cells harvested at 2–3 months did not differ by genotype (Figure 4—figure supplement 1). Osteoclast numbers per bone perimeter were slightly increased in Fndc5-transgenic mice versus wild type across 2–13 months but did not reach statistical significance (Figure 4c). However, primary bone marrow progenitors isolated from Fndc5-transgenic mice at 2–3 months demonstrated markedly greater osteoclastogenic potential compared to the wild type, yielding significantly higher numbers of osteoclasts that were qualitatively larger than controls during in vitro differentiation (p<0.0001; Figure 4d, Source data 1).

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This study demonstrates that irisin plays an important role in regulating bone remodeling not only by stimulating osteoblasts and osteocytes as previously described, but by also directly acting on osteoclasts to promote differentiation and resorption. This stimulatory effect was observed across multiple experiments with primary murine progenitors and the RAW 264.7 macrophage cell line and occurred with either intermittent or continuous irisin exposure across a range of physiologic concentrations previously reported in humans (Jedrychowski et al., 2015). Analogous to its action on osteocytes, we confirmed the expression of integrin subunits α_V and β₅ on osteoclasts and identified it as a likely receptor for irisin, particularly since blocking this receptor complex with a neutralizing antibody completely suppressed the stimulatory effect of irisin on osteoclastogenesis (Figure 1). Furthermore, we found that both recruitment and differentiation of more osteoclast progenitors with irisin treatment appeared to be driving factors of enhanced bone resorption, based on in situ studies on native dentin as well as synthetic calcium phosphate and collagen substrates (Figure 2). Using unbiased RNAseq analysis and qRT-PCR of irisin-treated osteoclasts, we noted that some markers of early osteoclast differentiation, nuclear fusion markers, and enzymes related to bone resorption were upregulated, matching the functional effects observed in vitro. In addition, several osteoclast-secretory factors known to stimulate osteoblasts were significantly upregulated, suggesting that the direct actions of irisin on this cell type may have further impact on cell signaling to enhance coupled remodeling (Figure 3).

To confirm the capacity of irisin to impact osteoclast function in vivo, we employed a genetic strategy with chronically forced expression of Fndc5 using the muscle-specific Mck promoter. In that mouse model, we demonstrated that high levels of irisin were expressed in the whole bone marrow at both ages, and this was associated with low bone mass. The relative contribution of osteoclastogenesis to the skeletal phenotype cannot be resolved in our genetic model. While early decreases in osteoblast numbers and bone formation rate were observed by histomorphometry at 2 months of age in the transgenic mouse, these metrics became equivalent to controls at 13 months, whereas a trend for increased osteoclast numbers was noted at 13 months. The in vitro differentiation cultures from the Fndc5-transgenic marrow progenitors from 2- to 3-month-old mice provide further supportive evidence for the action of irisin on the osteoclast. Bone marrow mesenchymal progenitors from Fndc5-transgenic mice at 2–3 months of age were not different from the wild type in regard to the pace or quantitation of osteoblast differentiation or mineralization. On the other hand, in vitro osteoclastogenesis from transgenic hematopoietic progenitors was markedly enhanced compared to control mice (Figure 4). Additionally, we have found that continuous exogenous treatment with 10 ng/mL irisin used to stimulate in vitro osteoclast differentiation from wild type progenitors had no observable effect on osteoblast differentiation or mineralization (Figure 1—figure supplement 1). Taken together, our results demonstrate a clear action of irisin on the osteoclast, but further suggest that in this forced expression model, irisin may act at early stages to reduce bone mass by suppressing osteoblast-mediated bone formation, while the later and persistent effects on bone turnover may be more focused on the osteoclast.

While our observations of irisin’s effects on osteoblastogensis differ from other published studies (Colaianni et al., 2014), it is important to note that both dose and duration of irisin exposure may be critical determinants of the skeletal response and hence be responsible for phenotypic differences. For example, from our gene expression studies, it is clear that the activity of the Mck promoter for Fndc5 is very high at 2 months but waned with age, albeit still maintaining relatively increased levels (Figure 4a). This exposure difference may impact skeletal cell responsiveness as noted by an earlier study demonstrating suppressed osteoclastogenesis in vitro with supraphysiologic levels of irisin (Marino et al., 2014). Similarly, we found that 20 ng/mL irisin had little effect on osteoclast number in vitro (Figure 1b), and initial experiments with doses of irisin equal or exceeding 100 ng/mL actually showed reduced osteoclast numbers (data not shown). Our previous work demonstrated that irisin concentrations in humans are approximately 2–4 ng/mL but increase to 10 ng/mL or more with exercise, a dose range we employed for the in vitro studies (Jedrychowski et al., 2015). Thus, both dosing and timing relative to developmental age may be determinants of irisin’s function as a dynamic signaling molecule in the skeleton, and its actions on specific bone cell types. It should be noted that the relatively narrow dose range for stimulating osteoclastogenesis could have physiologic implications since exercise induces rapid changes in serum calcium (Ng et al., 2018). If irisin targets both osteoclasts and osteocytes, it may serve as another counter-regulatory hormone to maintain serum calcium during the early phases of exercise much like parathyroid hormone (Ng et al., 2018).

Taken together, our studies provide more evidence that irisin mediates muscle-bone crosstalk by regulating bone remodeling. While further studies are necessary to fully elucidate irisin’s mechanism of action on the osteoclast and to resolve contrasting cellular effects observed between cell types and experimental models, it remains clear that this myokine is a potent regulator of bone remodeling that is able to both act directly on all key cell types of the bone remodeling unit, and potentially modulate signaling between them.

All data generated or analysed during this study are included in the manuscript figures and Source data 1.

1. 1. Arlot ME
   2. Burt-Pichat B
   3. Roux JP
   4. Vashishth D
   5. Bouxsein ML
   6. Delmas PD

   (2008) Microarchitecture influences microdamage accumulation in human vertebral trabecular bone

   Journal of Bone and Mineral Research 23:1613–1618.

   https://doi.org/10.1359/jbmr.080517

   • PubMed
   • Google Scholar
2. 1. Bharti AC
   2. Takada Y
   3. Aggarwal BB

   (2004) Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

   The Journal of Immunology 172:5940–5947.

   https://doi.org/10.4049/jimmunol.172.10.5940

   • PubMed
   • Google Scholar
3. 1. Boström P
   2. Wu J
   3. Jedrychowski MP
   4. Korde A
   5. Ye L
   6. Lo JC
   7. Rasbach KA
   8. Boström EA
   9. Choi JH
   10. Long JZ
   11. Kajimura S
   12. Zingaretti MC
   13. Vind BF
   14. Tu H
   15. Cinti S
   16. Højlund K
   17. Gygi SP
   18. Spiegelman BM

   (2012) A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis

   Nature 481:463–468.

   https://doi.org/10.1038/nature10777

   • PubMed
   • Google Scholar
4. 1. Colaianni G
   2. Cuscito C
   3. Mongelli T
   4. Oranger A
   5. Mori G
   6. Brunetti G
   7. Colucci S
   8. Cinti S
   9. Grano M

   (2014) Irisin enhances osteoblast differentiation in vitro

   International Journal of Endocrinology 2014:1–8.

   https://doi.org/10.1155/2014/902186

   • Google Scholar
5. 1. Colaianni G
   2. Cuscito C
   3. Mongelli T
   4. Pignataro P
   5. Buccoliero C
   6. Liu P
   7. Lu P
   8. Sartini L
   9. Di Comite M
   10. Mori G
   11. Di Benedetto A
   12. Brunetti G
   13. Yuen T
   14. Sun L
   15. Reseland JE
   16. Colucci S
   17. New MI
   18. Zaidi M
   19. Cinti S
   20. Grano M

   (2015) The myokine irisin increases cortical bone mass

   PNAS 112:12157–12162.

   https://doi.org/10.1073/pnas.1516622112

   • Google Scholar
6. 1. Colaianni G
   2. Mongelli T
   3. Cuscito C
   4. Pignataro P
   5. Lippo L
   6. Spiro G
   7. Notarnicola A
   8. Severi I
   9. Passeri G
   10. Mori G
   11. Brunetti G
   12. Moretti B
   13. Tarantino U
   14. Colucci SC
   15. Reseland JE
   16. Vettor R
   17. Cinti S
   18. Grano M

   (2017) Irisin prevents and restores bone loss and muscle atrophy in hind-limb suspended mice

   Scientific Reports 7:2811.

   https://doi.org/10.1038/s41598-017-02557-8

   • PubMed
   • Google Scholar
7. 1. Cornwell M
   2. Vangala M
   3. Taing L
   4. Herbert Z
   5. Köster J
   6. Li B
   7. Sun H
   8. Li T
   9. Zhang J
   10. Qiu X
   11. Pun M
   12. Jeselsohn R
   13. Brown M
   14. Liu XS
   15. Long HW

   (2018) VIPER: visualization pipeline for RNA-seq, a snakemake workflow for efficient and complete RNA-seq analysis

   BMC Bioinformatics 19:135.

   https://doi.org/10.1186/s12859-018-2139-9

   • PubMed
   • Google Scholar
8. 1. Dempster DW
   2. Compston JE
   3. Drezner MK
   4. Glorieux FH
   5. Kanis JA
   6. Malluche H
   7. Meunier PJ
   8. Ott SM
   9. Recker RR
   10. Parfitt AM

   (2013) Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR histomorphometry nomenclature committee

   Journal of Bone and Mineral Research 28:2–17.

   https://doi.org/10.1002/jbmr.1805

   • PubMed
   • Google Scholar
9. 1. Dobin A
   2. Davis CA
   3. Schlesinger F
   4. Drenkow J
   5. Zaleski C
   6. Jha S
   7. Batut P
   8. Chaisson M
   9. Gingeras TR

   (2013) STAR: ultrafast universal RNA-seq aligner

   Bioinformatics 29:15–21.

   https://doi.org/10.1093/bioinformatics/bts635

   • PubMed
   • Google Scholar
10. 1. Duong LT
    2. Lakkakorpi P
    3. Nakamura I
    4. Rodan GA

    (2000) Integrins and signaling in osteoclast function

    Matrix Biology 19:97–105.

    https://doi.org/10.1016/S0945-053X(00)00051-2

    • PubMed
    • Google Scholar
11. 1. Hildebrand T
    2. Laib A
    3. Müller R
    4. Dequeker J
    5. Rüegsegger P

    (1999) Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus

    Journal of Bone and Mineral Research 14:1167–1174.

    https://doi.org/10.1359/jbmr.1999.14.7.1167

    • PubMed
    • Google Scholar
12. 1. Hildebrand T
    2. Rüegsegger P

    (1997) A new method for the model‐independent assessment of thickness in three‐dimensional images

    Journal of Microscopy 185:67–75.

    https://doi.org/10.1046/j.1365-2818.1997.1340694.x

    • Google Scholar
13. 1. Jedrychowski MP
    2. Wrann CD
    3. Paulo JA
    4. Gerber KK
    5. Szpyt J
    6. Robinson MM
    7. Nair KS
    8. Gygi SP
    9. Spiegelman BM

    (2015) Detection and quantitation of circulating human irisin by tandem mass spectrometry

    Cell Metabolism 22:734–740.

    https://doi.org/10.1016/j.cmet.2015.08.001

    • PubMed
    • Google Scholar
14. 1. Kim H
    2. Wrann CD
    3. Jedrychowski M
    4. Vidoni S
    5. Kitase Y
    6. Nagano K
    7. Zhou C
    8. Chou J
    9. Parkman VA
    10. Novick SJ
    11. Strutzenberg TS
    12. Pascal BD
    13. Le PT
    14. Brooks DJ
    15. Roche AM
    16. Gerber KK
    17. Mattheis L
    18. Chen W
    19. Tu H
    20. Bouxsein ML
    21. Griffin PR
    22. Baron R
    23. Rosen CJ
    24. Bonewald LF
    25. Spiegelman BM

    (2018) Irisin mediates effects on bone and fat via αv integrin receptors

    Cell 175:1756–1768.

    https://doi.org/10.1016/j.cell.2018.10.025

    • PubMed
    • Google Scholar
15. 1. Lin J
    2. Wu H
    3. Tarr PT
    4. Zhang CY
    5. Wu Z
    6. Boss O
    7. Michael LF
    8. Puigserver P
    9. Isotani E
    10. Olson EN
    11. Lowell BB
    12. Bassel-Duby R
    13. Spiegelman BM

    (2002) Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres

    Nature 418:797–801.

    https://doi.org/10.1038/nature00904

    • PubMed
    • Google Scholar
16. 1. Love MI
    2. Huber W
    3. Anders S

    (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

    Genome Biology 15:550.

    https://doi.org/10.1186/s13059-014-0550-8

    • PubMed
    • Google Scholar
17. 1. Marino S
    2. Logan JG
    3. Mellis D
    4. Capulli M

    (2014) Generation and culture of osteoclasts

    BoneKEy Reports 3:570.

    https://doi.org/10.1038/bonekey.2014.65

    • PubMed
    • Google Scholar
18. 1. Meinel L
    2. Fajardo R
    3. Hofmann S
    4. Langer R
    5. Chen J
    6. Snyder B
    7. Vunjak-Novakovic G
    8. Kaplan D

    (2005) Silk implants for the healing of critical size bone defects

    Bone 37:688–698.

    https://doi.org/10.1016/j.bone.2005.06.010

    • PubMed
    • Google Scholar
19. 1. Ng AYH
    2. Tu C
    3. Shen S
    4. Xu D
    5. Oursler MJ
    6. Qu J
    7. Yang S

    (2018) Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics

    JBMR Plus 2:328–340.

    https://doi.org/10.1002/jbm4.10058

    • Google Scholar
20. 1. Rajagopalan S
    2. Lu L
    3. Yaszemski MJ
    4. Robb RA

    (2005) Optimal segmentation of microcomputed tomographic images of porous tissue-engineering scaffolds

    Journal of Biomedical Materials Research Part A 75A:877–887.

    https://doi.org/10.1002/jbm.a.30498

    • Google Scholar
21. Conference

    1. Ridler TC

    (1978) Picture thresholding using an iterative selection method

    IEEE Transactions on Systems, Man, and Cybernetics. pp. 630–632.

    https://doi.org/10.1109/TSMC.1978.4310039

    • Google Scholar
22. 1. Ritchie ME
    2. Phipson B
    3. Wu D
    4. Hu Y
    5. Law CW
    6. Shi W
    7. Smyth GK

    (2015) Limma powers differential expression analyses for RNA-sequencing and microarray studies

    Nucleic Acids Research 43:e47.

    https://doi.org/10.1093/nar/gkv007

    • PubMed
    • Google Scholar
23. 1. Vesprey A
    2. Yang W

    (2016) Pit assay to measure the bone resorptive activity of bone Marrow-derived osteoclasts

    Bio-Protocol 6:1836.

    https://doi.org/10.21769/BioProtoc.1836

    • Google Scholar
24. 1. Yavropoulou MP
    2. Yovos JG

    (2008)

    Osteoclastogenesis - Current knowledge and future perspectives

    Journal of Musculoskeletal & Neuronal Interactions 8:204–216.

    • PubMed
    • Google Scholar

Acceptance summary:

The reviewers determined that is a well-designed study of the effects of Irisin on osteoclastogenesis. Using a combination of in vitro, RNAseq, and in vivo studies (transgenic mouse model over-expressing Fndc5, an upstream regulator of Irisin in response to exercise), this work provides strong evidence that, in addition to its known effects on osteoblasts and osteocytes, irisin directly regulates osteoclasts and that the hematopoietic progenitors were biased towards osteoclast differentiation.

Decision letter after peer review:

Thank you for submitting your article "Irisin directly stimulates osteoclastogenesis and bone resorption in vitro and in vivo" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kathryn Cheah as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Warren Grayson (Reviewer #2); Sundeep Khosla (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

This is a well-designed study of the effects of Irisin on osteoclastogenesis that reports the effects of dosing and timing of Irisin on osteoclast number, morphology, and integrin α_Vβ₅ expression in vitro. Using a combination of in vitro, RNAseq, and in vivo studies (transgenic mouse model over-expressing Fndc5, an upstream regulator of Irisin in response to exercise), the authors provide strong evidence that, in addition to its known effects on osteoblasts and osteocytes, irisin directly regulates osteoclasts. The authors also found that the hematopoietic progenitors were biased towards osteoclast differentiation along with an increase in some resorption markers and clastokines. The reviewers noted that the lack of mechanistic insights is a major limitation of the study (as also acknowledged by the authors).

Essential revisions:

1) The authors should show validation of the transgenic mouse in terms of the quantitative changes in Fndc5 or Irisin expression. This is important given that the in vivo data contradicts to the previously published results that reported Irisin increases bone mass and cortical bone thickness. In this study, the authors have observed a decrease in osteoblasts and an increase in osteoclasts and concomitant decrease in mineralized surface (MS/BS).

2) Another concern is related to the bone histomorphometry (Figure 4B). There is a clear reduction in BV/TV, which seems to be driven more by a reduction in the MS/BS and osteoblast numbers (p values close to 0.05) than by increased osteoclast numbers. The increase in osteoclast progenitors in the FNDC5 Tg mice is consistent with the authors' hypothesis. The authors should address this in the Discussion – perhaps the most likely explanation is that there may be a more robust increase in bone resorption and osteoclast numbers earlier in life (the mice were analyzed at 4.5 months). If data on younger mice are available, these should be provided; if not, this issue should at least be discussed.

Summary:

This is a well-designed study of the effects of Irisin on osteoclastogenesis that reports the effects of dosing and timing of Irisin on osteoclast number, morphology, and integrin α_Vβ₅ expression in vitro. Using a combination of in vitro, RNAseq, and in vivo studies (transgenic mouse model over-expressing Fndc5, an upstream regulator of Irisin in response to exercise), the authors provide strong evidence that, in addition to its known effects on osteoblasts and osteocytes, irisin directly regulates osteoclasts. The authors also found that the hematopoietic progenitors were biased towards osteoclast differentiation along with an increase in some resorption markers and clastokines. The reviewers noted that the lack of mechanistic insights is a major limitation of the study (as also acknowledged by the authors).

The authors thank the editors and reviewers for their time in considering this manuscript. In response to the summary, we acknowledge the lack of in-depth mechanistic insights in this study but note the novelty of these findings in regard to previously uncharacterized effects of irisin on a key cell type in bone homeostasis, the osteoclast. We therefore feel that the study stands on its own while also setting the foundation for more detailed mechanistic studies that are currently in planning and underway. Below, we address each of the specific critiques in turn, with rationale and reference to corresponding changes in the revised manuscript.

Essential revisions:

1) The authors should show validation of the transgenic mouse in terms of the quantitative changes in Fndc5 or Irisin expression.

We have now revised Figure 4 to more clearly demonstrate the validation of the transgenic forced expression of FNDC5 by showing quantitative increases in whole-bone mRNA expression of FNDC5 in tibia at both 4.5 and 13 months of age (Figure 4A). To establish the characterization of this transgenic model more completely we have further included data at earlier and later timepoints where available, at 2 and 13 months for µCT (Figure 4B) and at 13 months for histomorphometry (Figure 4C) (see Results and Discussion, fifth paragraph).

This is important given that the in vivo data contradicts to the previously published results that reported Irisin increases bone mass and cortical bone thickness. In this study, the authors have observed a decrease in osteoblasts and an increase in osteoclasts and concomitant decrease in mineralized surface (MS/BS).

To more fully explore the respective roles of osteoblasts and osteoclasts in this model we have included the additional histomorphometry data at 13 months of age (Figure 4C) as well as in vitro osteoblast cultures carried out from both wild type bone marrow progenitors with exogenous irisin treatment (Figure 1—figure supplement 1) and compared wild type and transgenic bone marrow progenitors (Figure 1—figure supplement 1). We have endeavored to address contradictions with previously published results throughout the Discussion (Results and Discussion, eighth paragraph).

2) Another concern is related to the bone histomorphometry (Figure 4B). There is a clear reduction in BV/TV, which seems to be driven more by a reduction in the MS/BS and osteoblast numbers (p values close to 0.05) than by increased osteoclast numbers. The increase in osteoclast progenitors in the FNDC5 Tg mice is consistent with the authors' hypothesis. The authors should address this in the Discussion – perhaps the most likely explanation is that there may be a more robust increase in bone resorption and osteoclast numbers earlier in life (the mice were analyzed at 4.5 months). If data on younger mice are available, these should be provided; if not, this issue should at least be discussed.

Thank you for that important point about the lower bone mass. We have now included µCT data for an earlier time point at 2 months, which does indeed also show a reduction in trabecular and cortical bone, and smaller bone area in transgenic mice. We have added the histomorphometry data at this earlier time point to gain insight into osteoblasts and osteoclasts relative to bone changes. In sum for the revised manuscript we included µCT at three time points (2, 4.5 and 13 months) and dynamic histomorphometry at two time points (2 months and 13 months). The original submission noted that histomorphometry was done at 4.5 months; that was in error, it was performed on 2-month-old mice and corresponds with the µCT at that time point. As noted, we have now also included in vitro osteoblast differentiation cultures of bone marrow mesenchymal progenitors from 2-3-month wild type and transgenic mice and found no increase in osteoblast differentiation or mineralization (Figure 4—figure supplement 1). Similarly, in vitro osteoblast differentiation of wild type bone marrow progenitors showed no effect of the continuous exogenous treatment with 10 ng/mL irisin that was shown to stimulate osteoclast differentiation (Figure 1—figure supplement 1).

These in vitro osteoblast findings have been included in the Discussion to inform interpretation on the respective roles on osteoblasts and osteoclasts in the transgenic model. In the transgenic model itself, the decrease in osteoblast numbers and bone formation at 2 months did not persist at 13 months, when it showed a slight but nonsignificant increase possibly related to the increase in osteoclast number (Figure 4C). At the earlier time points, osteoclast numbers did not differ by genotype, whereas by 13 months, there were more osteoclasts per bone surface in the transgenic. While the level of irisin exposure with exogenous treatment in vitro and forced FNDC5 expression in vivo are not directly comparable, these data together suggest that irisin influences both osteoblast and osteoclast differentiation throughout skeletal development. A more complete discussion of these data and the insights they may provide into the respective roles of osteoblasts and osteoclasts in response to irisin have been worked into the Discussion (Results and Discussion, eighth paragraph).

Author details

1. Eben G Estell

   Maine Medical Center Research Institute, Scarborough, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   eestell@mmc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8319-6886

2. Phuong T Le

   Maine Medical Center Research Institute, Scarborough, United States

   Contribution

   Conceptualization, Supervision, Validation, Methodology

   Competing interests

   No competing interests declared

3. Yosta Vegting

   Maine Medical Center Research Institute, Scarborough, United States

   Contribution

   Conceptualization, Validation, Methodology

   Competing interests

   No competing interests declared

4. Hyeonwoo Kim

   Dana Farber Cancer Institute, Boston, United States

   Contribution

   Conceptualization, Resources, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Christiane Wrann

   1. Dana Farber Cancer Institute, Boston, United States
   2. Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
   3. Department of Medicine, Harvard Medical School, Boston, United States
   4. Department of Cell Biology, Harvard University Medical School, Boston, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

6. Mary L Bouxsein

   Beth Israel Deaconess Department of Orthopedic Surgery, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

7. Kenichi Nagano

   Harvard School of Dental Medicine, Boston, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

8. Roland Baron

   Harvard School of Dental Medicine, Boston, United States

   Contribution

   Conceptualization, Resources, Supervision, Writing - review and editing

   Competing interests

   No competing interests declared

9. Bruce M Spiegelman

   Dana Farber Cancer Institute, Boston, United States

   Contribution

   Conceptualization, Resources, Writing - review and editing

   Competing interests

   No competing interests declared

10. Clifford J Rosen

    Maine Medical Center Research Institute, Scarborough, United States

    Contribution

    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Project administration, Writing - review and editing

    For correspondence

    rosenc@mmc.org

    Competing interests

    Senior editor, eLife

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