Interleukin-34 orchestrates bone formation through its binding to Bone Morphogenic Proteins

doi:10.21203/rs.3.rs-4847401/v1

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Interleukin-34 orchestrates bone formation through its binding to Bone Morphogenic Proteins

https://doi.org/10.21203/rs.3.rs-4847401/v1

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During growth, the contribution of IL34, a ligand of MCSFR, have not been established. The aim of this work was therefore to establish these implications using two models of IL34 invalidation generated in zebrafish and mouse. Significant growth delay and hypo-mineralization of skeletal elements were observed in both models, as well as craniofacial dysmorphoses in mice. With regard to bone cells, an unexpected increase in the number of osteoclasts and an accumulation of pre-osteoblasts were observed. In vitro analyses complemented by protein binding and molecular docking studies established that IL34 interacts directly with certain Bone Morphogenetic Proteins, modulating their various activities such as the stimulation of osteoblast differentiation. A new mechanism of action for IL34 has thus been characterized, opening up new therapeutic perspectives.

Biological sciences/Physiology/Bone

Biological sciences/Cancer/Bone cancer

Health sciences/Diseases/Cancer/Bone cancer

Interleukin-34 (IL34) is a soluble cytokine discovered in 2008 by its ability to bind to c-FMS/MCSFR/CSF1R/CD115 receptor ¹. This work has rekindled interest in the MCSFR signaling pathway and in the roles of the twin cytokines MCSF/IL34 in the differentiation and activation of myeloid cell lineage, such as macrophages, Langerhans cells, microglia cells and osteoclasts ^2–5. IL34 binding to MCSFR can occur as a homodimer or heterodimer with MCSF/CSF1, depending on the relative amounts of the two cytokines ⁶. The twin cytokines induce similar patterns of phosphorylation of MCSFR but with variable intensity and kinetic, raising the question of their functional redundancy and specific functions. Their functional redundancy is confirmed by the greater severity of the bone phenotype associated with MCSFR versus MCSF invalidation in mice ⁷. Additional receptors of IL34 have been identified and include Protein-Tyrosine Phosphatase β/ζ receptor (PTPβ/ζ) ⁸, Triggering Receptor Expressed on Myeloid cells-2 (TREM2) ⁹ and syndecan-1 ¹⁰. PTPβ/ζ is mainly expressed by neuronal progenitors and glial cells and known as pleiotrophin/heparin-binding growth-associated molecule receptor ¹¹. TREM2 is a lipid-binding receptor ¹², carried by myeloid lineage cells, whose differentiation and migratory capacities it modulates ¹³. Finally, IL34 binds to Syndecan-1 (CD138) and this binding modulates IL34-mediated activation of MCSFR ¹⁰. The diversity of IL34 receptors and co-ligands suggests that this cytokine plays an important role in the differentiation and activation of myeloid, neural and glial cells. To analyze these functions, IL34 was invalidated in zebrafish and mouse and the phenotypes of these mutants was fully deciphered during growth.

IL34 invalidation was genetically achieved in zebrafish and mouse using respectively CrispR/Cas9 technology on one-cell stage embryos (Fig. 1a; Fig.S1a) and conventional homologous recombination in embryonic stem cells (Fig. 1b; Fig.S2). Two zebrafish loss of function lines were generated for il34, corresponding to a 23bp deletion and a 50bp deletion with a 6bp insertion in exon 3 (Fig. 1a; Fig.S1b-e). In both zebrafish lines, individuals homozygous for the mutations presented a severe growth delay (Fig. 1a). Von Kossa and alcian blue staining of zebrafish embryo craniofacial skeleton at 5 days post fertilization revealed poorer skeletal mineralization in the null mutants but no evident dysmorphosis (Fig. 1a). The IL34 invalidated mouse line was obtained by CRE-recombinase activation on genetically modified Il34 gene (Fig. 1b; Fig.S2) with LoxP sites in introns 2 and 5 enabling to remove exons 3 to 5 while maintaining a lacZ reporter sequence located in the 5’ part of intron 2 (Il34^LacZ allele in Fig.S2). The functionality of the generated Il34^LacZ allele was validated in the skin (Fig.S3), a well-known site of IL34 expression (for instance ^14,15), with in mice homozygous for this allele (thereafter called Il34^−/− mice), the expected absence of IL34 expression (Fig.S3a) associated to a significant reduction of CD207⁺ Langerhans cells (Fig.S3b-d). The LacZ reporter was also functional as attested by the ß-galactosidase staining on skin section of mice heterozygous for the Il34^LacZ allele (Fig.S3e). IL34 invalidated mice were phenotypically altered. Indeed, 15 days-old IL34 invalidated mice exhibited a severe growth delay with all skeleton elements affected and the presence of dysmorphoses specifically in the craniofacial skeleton associated with hydrocephaly (Fig. 1b). MicroCT morphometric analysis of the craniofacial skeleton and the tibia evidenced significant reductions in the skull growth in all planes (sagittal, vertical and transversal) and of the long bone growth in the length and width dimensions (red vs black values in Fig. 1c; Fig.S4a). Interestingly, the use of a murine IL34 blocking antibody (Sheff.5) during the first post-natal week in WT mouse pups (protocol described in Fig.S4b) similarly induced skull growth alterations in all plane but to a lower extent (brown vs black in Fig. 1c). MicroCT scans were also used to determine bone structure parameters and bone mineral density in different anatomical sites, namely the mandibular, the vertebral, the cranial and the tibial bones. No significant difference in the trabecular thickness (Tb.Th), the trabecular space (Tr. Sp) or the percentage of bone volume (BV/TV) was observed between Il34^+/+ and Il34^−/− mice whichever bone considered (Fig.S4 and Fig. 2c, red vs black). On the contrary, the trabecular number (Tb.N) was significantly increased only for the vertebral bone (Fig.S4). Injections of the Sheff.5 blocking antibody had no impact on the bone structure parameters (Fig.S5). Regarding the bone mineral density, a significant reduction was observed in the cranial and the tibial bones of Il34^−/− mice comparatively to Il34^+/+ mice (Fig. 2a, b and d). The Sheff.5 antibody transitory treatment was insufficient to induce a similar reduction in WT mice (Fig. 2a, Fig.S5). Histological analyses on tibia sections performed at the level of the proximal epiphysis (Safranin-O staining Fig. 2e; Masson’s trichrome staining Fig.S6) revealed an important reduction of the growth plate hypertrophic chondrocytes area (Il34^+/+ 0.277 ± 0.021 mm² and Il34^−/− 0.146 ± 0.094 mm²). Tartrate resistant acid phosphatase (TRAP) and Osterix (Osx/SP7) dual staining carried out by histoenzymology and immunohistochemistry respectively (Fig. 2f and Fig.S7 for higher magnification) outlined an increase of both staining corresponding to osteoclastic and pre-osteoblastic cells in the null mutant comparatively to the wild-type (Il34^+/+) littermate. The quantitative analysis showed an increase of 120% for the numbers of osteoclasts and pre-osteoblasts (Fig. 2f). Interestingly, the RUNX2 immunohistochemistry staining, which enables identification of cells of the osteoblastic lineage (Fig.S8), showed no difference in the number of stained cells between Il34^−/− and Il34^+/+ mice, suggesting a slowdown of the osteoblast differentiation process with an accumulation of Osterix-positive pre-osteoblasts in the null mutant without reduction of the total number of cells committed in this process.

In order to identify the part of the Il34^−/− mouse skeleton phenotype linked to the increased number of osteoclastic cells, a RANKL blocking antibody (IK22.5) was injected during the first postnatal week (protocol described in Fig.S4B) to totally block the osteoclastogenesis. Such blockade had no consequence on the morphometric parameters in the null mutant (green vs red in Fig. 1c) but impacted the trabecular parameters, the BV/TV and the BMD with significant differences for cranial and tibial bones (green vs red in Fig.S4c and Fig. 2c-d). Histological analyses on tibia sections performed at the level of the proximal epiphysis enabled visualization in the null mutant treated with the IK22.5 blocking antibody of a massive reduction of the TRAP positive cells (Fig. 2f and Fig.S7) associated with an apparent normalization (from 0.146 ± 0.094 mm² to 0.250 ± 0.033 mm²) of the growth plate hypertrophic chondrocytes area (Safranin-O staining Fig. 2e) whereas no impact was noticed on the number of Osterix-positive (Fig. 2F and Fig.S7) and RUNX2-positive (Fig.S8) cells.

The direct impact of IL34 on the osteoblastic differentiation suggested by these last results was addressed in vitro with the model of human mesenchymal stem cell differentiation into osteoblasts induced by a standard osteoblastic differentiation medium (composition described in the materials and methods). This differentiation, quantified by the phosphocalcic mineral deposition (alizarin red staining) and the expression levels of differentiation markers (RUNX2, ALP and OCN), was accelerated by addition of the bone morphogenic protein 2 (BMP2) as shown in Fig. 3a-b, Fig.S9 and Fig.S10. The addition of IL34 alone to the differentiation medium had no impact on the rate of osteoblastic differentiation, but interestingly it was able to potentiate the effect of BMP2 when added in combination with an optimal IL34/BMP2 concentration ratio (ng/mL) of two (Fig. 3a-b and Fig.S9) corresponding to an equal amount in molarity of the two cytokines (Fig.S11a). Such an IL34 potentiation effect was effective on the canonical BMP signaling pathway with an increased and earlier phosphorylation of the SMAD1/5 proteins observed in the presence of BMP2 (Fig. 3c, Fig. 4a), BMP4 and BMP7 (Fig.S11), whereas IL34 has no similar impact on the phosphorylation of the SMAD2 protein induced by the TGFβ (Fig. 4a, Fig.S12). Interestingly, the potentiation effect of IL34 was blocked by the use of a specific human-IL34 blocking antibody named BT34 (Fig. 4b, Fig.S12), and the blocking of BMP2 pro-differentiation signaling with its natural inhibitor NOGGIN was annihilated by the presence of IL34 (Fig. 4c, Fig.S12). The potentiation effect of IL34 was moreover rapid (Fig. 4d, Fig.S12) and as previously mentioned sensitive to the ratio between the two cytokines (Fig. 4e-g, Fig.S12). This observation supported the existence of a physical and strong interaction between IL34 and some proteins of the BMP family. To complete the evidence on a physical interaction between IL34 and members of the BMP protein family, the potential impact of BMP2 addition on the IL34-induced osteoclastogenesis was evaluated in vitro. Results showed that BMP2 dose-dependently blocked IL34-induced osteoclastogenesis while no impact was observed on MCSF-induced osteoclastogenesis (Fig. 3d-e). Furthermore, the phosphorylation of MCSFR in response to IL34 was inhibited in presence of BMP2 and this inhibition was reversed by addition of NOGGIN supporting the existence of a functional physical link between IL34 and some members of the BMP protein family (Fig. 4h, Fig.S12). In order to definitively establish this physical interaction, surface plasmon resonance experiments were performed and demonstrated effective binding of IL34 to BMP2, BMP4 and BMP7 with KD values of 3.63E-07 M, 4.26E-07 M and 9.22E-07 M respectively (Fig. 5a and Fig.S13).

A molecular modelling approach, corresponding to a protein-protein docking study, established that IL34 binding to BMP2 occurred at the “Knuckle” sites of the BMP2 dimers known to correspond to the binding sites of BMP type 1 receptors and did not impinge on the “Wrist” sites that correspond to the binding sites of BMP type 2 receptors (Fig. 5b-d; Fig.S14). The amino acids of BMP2 and IL34 involved in binding were identified (Fig. 5e and Fig.S15), with BMP2 involving a pocket (formed by F305, W310, W313, Y385 and M388) in which the phenylalanine in position 85 for BMPR1A (F85) or the arginine in position 48 for IL34 (R48) are positioned during their respective interactions (Fig. 5f). It is important to note that the amino acids involved are phylogenetically highly conserved in both BMP2 and IL34 and that, in addition, the amino acids of BMP2 implicated are also found conserved in several members of the BMP family (Fig.S16). It was therefore possible to model the binding to IL34 of certain BMPs for which crystallographic structures were available, such as BMP3, BMP6 and BMP7 (Fig.S17). The existence of direct physical links between BMP proteins and IL34 having been established, the question of the consequences of these links on the binding of BMPs to their receptors on the one hand and the binding of IL34 to M-CSFR on the other was raised. Binding of BMP2 to the type 1 BMP receptor is hindered by IL34, which binds to the same site as shown above. It should be noted that this “Knuckle” site is also the binding site for co-receptor proteins of the RGM family (Fig.S14c) and that it is partially masked by NOGGIN binding (Fig.S14d). With regard to the binding of BMP2 to type 2 BMP receptors, modelling shows that binding of the ACVR2A receptor, for example, is entirely possible on a BMP2 dimer with two IL34 binders (Fig. 6a), the “Wrist” sites not being masked by the presence of IL34. These different possibilities for binding type 1 and 2 receptors and IL34 to a BMP2 dimer are shown in 3D in Movie S1. Concerning the binding of IL34 to MCSFR, the binding of IL34 to BMP2 occurs at a site that overlaps with the binding site of MCSFR to IL34 (Fig. 6b), preventing the simultaneous binding of MCSFR and BMP2 to IL34.

IL34, one of the latest cytokines identified ¹⁶, has been shown to bind to a variety of receptors with consequences for the differentiation and activation of myeloid, neural and glial cells (For review ^4,5). Surprisingly, the implications of this cytokine during development and growth had not been addressed in detail, unlike those of its receptor MCSFR (for review ¹⁷), although its ability to stimulate differentiation of osteoclasts, cells important for skeletal growth, had been established via binding and activation of this receptor (18–20). The primary aim of the work presented was therefore to determine these implications by generating two in vivo models of IL34 invalidation, one in zebrafish and the other in mice, and to characterize the associated skeletal phenotypes. Both models showed significant growth retardation, with reductions in cartilage mineralization in zebrafish and bone mineralization in mice. In mice invalidated for IL34, obtained with an expected frequency (Mendelian inheritance) but demonstrating a reduced life expectancy (3 weeks); significant craniofacial dysmorphoses were observed with the presence of hydrocephalus. Such defects in craniofacial development are consistent with the previously established implications of IL34 in neural and microglial cells differentiation and activation ^14,21–27. This model should therefore provide a useful additional tool for deciphering the precise functions of IL34 during normal and pathological development of the central nervous system.

Histological study of bone tissue from IL34-invalidated mice revealed a marked increase in the number of osteoclasts in the growth plate, in contrast to the phenotype envisaged for the loss of a factor known to stimulate osteoclastogenesis ^19,28,29. As this increase in osteoclast numbers was associated with an accumulation of pre-osteoblasts (OSX-positive) and a reduction in the hypertrophic zone of the growth plate, the question of a role for IL34 in the differentiation of osteoblasts and chondroblasts was raised. To check whether these two points were simply not secondary to the increase in osteoclastogenesis, the consequences of inhibiting RANKL (a factor essential to osteoclastogenesis) during the first week of life in Il34^−/− mice were analyzed. No impact was observed on the accumulation of OSX-positive cells, while normalization of the size of the hypertrophic zone was observed. These results suggest that IL34 may directly regulate osteoblastic differentiation and probably indirectly that of chondroblasts via osteoclasts, bearing in mind that in inflammatory situations, both mature osteoblasts and hypertrophic chondrocytes can become important sources of pro-osteoclastic IL34 ^30–32. Interestingly, a relationship has already been observed between the level of osteoclastic activity and the size of the hypertrophic zone of the growth plate, and vice versa. Thus, a decrease in the hypertrophic zone goes hand in hand with an increase in osteoclastic activity ^33,34 and an increase in this zone with a decrease in osteoclastic activity ^35–39. It should then be noted that the disruption of one or other of the elements in this relationship, over and above the repercussions on the other, induces growth retardation in all cases, as has been reported in patients with disorders of osteoclastogenesis (for example, in patients suffering from juvenile osteoporosis ⁴⁰ or osteopetrosis ⁴¹) as well as in those with chondrodysplasia (for review ⁴²). The increase in osteoclasts observed in Il34^−/− mice, appropriately associated with a reduction in the hypertrophic zone of the growth plate, could therefore explain the growth retardation. Establishing the origin of the increase in osteoclasts in relation to the absence of IL34 was not immediately obvious. Osterix expression marks pre-osteoblasts, which are known as an important source of RANKL during growth ^43,44. The accumulation of OSX-positive cells at the subchondral level in Il34^−/− mice could therefore explain the increased number of osteoclasts, taking into account that an analysis of the number CD11b-positive cells (osteoclast precursors) in the bone marrow and spleen of Il34^−/− mice reveled a marked increase (Fig. S18). The question then arose as to the origin of the accumulation of these OSX-positive pre-osteoblasts in Il34^−/− mice, given that the total number of cells committed to osteoblastic lineage according to RUNX2 labeling did not appear to be affected. An impact of IL34 absence on osteoblastogenesis was therefore strongly suspected.

Members of the TGFβ-BMP family, in particular BMP2, are major stimulators of osteoblastogenesis (for review ⁴⁵). The co-addition of IL34 with BMP2 in the culture medium of mesenchymal stem cells undergoing osteoblastic differentiation has shown, for certain ratios, a potentiation of the effect of BMP2 on this differentiation. Protein binding studies showed that IL34 could bind directly to BMP2, and 3D modeling identified the amino acids involved in this binding in the sequences of IL34 and BMP2. With regard to the BMP family, the amino acids involved in IL34 binding were found to be highly conserved, and the veracity of the direct binding of BMP4 and BMP7 to IL34 was established, suggesting that IL34 may potentiate the effects of several family members. IL34 binds to the "Knuckle" site of BMP2, which is also the binding site for type 1 receptors to BMPs, without obscuring the "Wrist" binding site for type 2 receptors. Protein binding studies have also shown that IL34 can directly bind type 2 receptors to BMPs (Fig. S19), enabling it to occupy the "Knuckle" site of a BMP and transform it into a "Wrist"-like site. A biphasic mechanism of action associated with IL34 binding to BMP dimers can then be proposed (Fig. 6c-d), corresponding to the progressive modification of the ratio between type 1 and 2 BMP receptors. Thus, in the absence of IL34, basal activity is observed with a receptor ratio of 2/2. Then with an amount of IL34 equivalent to that of BMP2, maximum activity is observed corresponding to a receptor ratio of 1/3. Finally, with an excess of IL34, zero activity is observed with a receptor ratio of 0/4. Interestingly, several studies have reported that in an inflammatory context, BMP2 could inhibit IL34 expression ^46–48, suggesting the possible existence of a feedback loop of IL34 potentiation of BMP2 activity. The ratio of IL34 to BMP2 has also been shown to impact IL34 binding to the MCSFR so the osteoclastogenesis in bone. IL34 thus appears to play a key role in bone formation, modulating both osteoclastogenesis via its direct binding to the MCSFR and osteoblastogenesis via its binding to BMPs.

In a more general context, IL34's ability to directly control MCSF receptor activation and indirectly BMP receptors activation defines it as a major player in the development, growth, homeostasis and function of most organs. Further studies will obviously be needed to determine which members of the BMP family are IL34 partners in each organ, in normal physiology and pathological situations (for review ⁴⁹) including cancers for which IL34 is already presented as a therapeutic target of major interest ^50–52.

In vivo experiments

All zebrafish (Danio rerio) used for this project were located in the aquaria at the Bateson Centre, at the University of Sheffield (UK). Zebrafish were present in tanks at a density of no more than four zebrafish per liter, with 14 hours light and 10 hours dark cycle, at a temperature of 28°C. All experimental procedures were carried out in accordance with the UK Home Office Project License PPL70/8178 and personal license IO6008638. All transgenic mice (Mus Musculus) used for this project were housed under pathogen-free conditions at the Experimental Therapy Unit at the Faculty of Medicine of the University of Nantes, France (Agreement D44015 and DUO 6781). All protocols applied in the present study were first validated by the French ethical committee of the “Pays de la Loire” (CEEA-PdL-06) and authorized by the French ministry of agriculture and fisheries (authorization # 18415-201901101823350 v2).

Generation of IL34 mutant zebrafish

The zebrafish Il34 gene (ENSDARG00000091003.2 or ZDB-GENE-050419-150) contains seven exons as human and mouse genes (Figure 1). IL34 mutant zebrafish was generated using the CRISPR-Cas9 technology as previously described ^53,54. Exon 3 was targeted using the sequence shown in Supplementary Figure 1A and the corresponding 20 bp spacer region was placed into a guide RNA template for in vitro transcription. The gRNA was then transcribed using the MEGAshortscript T7 kit (Life Technologies, UK) and microinjected with Cas9 protein (NEB, UK) into the yolk of zebrafish embryos the one cell stage. F0 adult fish were crossed with wild-type fish to identify founder with germline transmission. Primers used for genotyping were (Fw 5’-TCA GCC AAT AAA TAT CAG ATC CA-3’ and Rv 5’-CGT CTC CTG GTT GCA TTT-3’) which amplify a 300bp fragment of the wild type sequence of Zebrafish IL34 exon3 covering the chosen CRISPR target sequence. Obtained fragments of shorter sizes were sequenced to identify mutations induced in the different founders. Two mutations corresponding to a 23bp deletion (mutant #1 in Figure 1) and a 50bp deletion combined to a 6bp insertion (mutant #2 in Figure 1) were obtained. Phenotypes of Zebrafishes homozygous for each of these mutations (Il34^-/- from F3 or following generations) were compared to ensure for link to Il34 deficiency and not from potential background mutations. Genotyping was performed on DNA extracted from the caudal fins by PCR using same primers as those used to identify founders. Fragments of 300bp, 277bp and 256bp were amplified respectively for Il34 exon3 WT, mutant #1 and mutant #2 sequences.

Van Kossa and Acian Blue staining of zebrafish skeleton

For Von Kossa staining, samples were fixed in 4% PFA for 2 hours at room temperature, rinsed in water containing 0.01% tween 20, and left to incubate in a solution of silver nitrate under a 60 W light bulb for 1 hour. After rinsing with water containing 0.01% tween 20, samples were fixed in 2.5% sodium-thiosulfate for 10 minutes, rinsed and again fixed in 4% PFA for 30 minutes at room temperature. Preservation was done in glycerol and samples were kept at room temperature in dark until images were taken.

For Alcian Blue Staining, samples were fixed overnight in 4% PFA at 4ºC. After several washes in a phosphate buffer solution containing 0.1% tween 20 (PBS-T) and dehydration using methanol, samples were transferred into Alcian blue staining solution (0.1% Alcian Blue, 70% ethanol, 1% concentrated hydrochloric acid) and left to stain overnight at room temperature. Samples where then rinsed in PBS-T and bleached in 30% hydrogen peroxide for 10 minutes at 37ºC. A 30% saturated borate solution was then used to eliminate all residues of bleaching solution before putting the samples into a trypsin digestion solution for 30 minutes at 37ºC until brains and eyes appeared translucent. A rehydration was performed and samples were put in glycerol for preservation until images were taken.

Zebrafish were imaged for both stains using the SMZ1500 stereomicroscope, with a DS-Fi1 camera (both Nikon, Japan), at 20 X magnification and Nikon Elements software.

Generation of Il34 mutant mouse

The Il34 mutant mouse was generated at the Mouse Clinical Institute (IGBMC, Illkirch, France; Project IR00004258 / K4258) by classical embryonic stem cells (ES) injection in blastocyst stage embryo. Three JM8.N4 ES cell clones carrying the targeted Il34^{tm1a(EUCOMM)Wtsi} allele were purchased at the European Conditional Mouse Mutagenesis Consortium (EUCOMM) and the clone EPD0146_4_F02 (embryonic stem line JM8.N4; C57BL/6) that was confirmed by PCR and Sanger sequencing (Supplementary Figure 2) as being correctly targeted was used to generate the Il34 conditional mutant mouse line. Breeding with ERT2-Cre mice (B6.Cg‐Tg(UBC‐cre/ERT2)1Ejb/J, JR#8085, Jackson Laboratory, Bar Harbor, Maine, USA) enabled to (Tamoxifen dependently) delete exons 3–5 of Il34 and the neomycin-resistance cassette generating the Il34^+/LacZ mice (Supplementary Figure 2). Breeding with CAG-FLPe mice (C57BL/6-Tg(CAG-flpe)16Ito, RBRC10707, RIKEN BRC, Tsukuba, Ibaraki 305-0074, Japan) allowed to delete the whole LacZ–NeoR cassette and generate mice carrying a loxP-flanked Il34 allele (Il34^+/f). Homozygous Il34^LacZ/LacZ mice (called Il34^-/- in the manuscript) were used for analysis. Mice were genotyped by PCR (Supplementary Figure 2) with the primers Il34-S2: 5’-GTC AGT ATC GGC GGA ATT-3’, Il34-S3: 5’-GTT TGG CCG ATG CTG GCA AAG G-3’ and Il34-AS2: 5’-CTG TCT TAT GAA GAT GGC ATG CC-3’. Il34-S2 and Il34-AS2 primers enable to amplify a 440bp fragment in presence of Il34^LacZ allele, and Il34-S3 and Il34-AS2 primers fragments of 240bp and 290bp respectively in presence of wild type (WT) and Il34^f alleles (Supplementary Figure 2).

Alizarin Red and Alcian Blue double staining of mouse skeleton

The whole-mount skeletal staining protocol used is derived from the protocol of Rigueur and Lyons ⁵⁵. Briefly, after euthanasia, all skin, internal organs, adipose tissue and as much as possible muscle were removed before fixation in a PBS 1X pH 7.4 solution containing 2% of paraformaldehyde and 0.2% glutaraldehyde. Skeletons were then dehydrated in ethanol and placed in acetone for permeabilization. Cartilage staining was then realized by submerging the skeletons in the Alcian blue stain (Alcian blue 8GX 0.03 % (w/v), 80 % EtOH, 20 % glacial acetic acid). After washes in 70 % and 95 % ethanol, a pre-clear of the tissue was realized in a 1 % KOH solution. Bone staining was then carried out in Alizarin red stain (Alizarin red 0.005 % (w/v) in 1 % (w/v) KOH). The Alizarin red solution was then replaced with a v/v mix of glycerol and 1 % KOH to remove the excess red color. Skeleton were transferred to 100 % glycerol for long-term storage and imaging.

New-born mice treatment with blocking antibodies

The protocol used to treat newborn mice with blocking antibodies was previously described ³⁸. Briefly, newborn C57BL/6 mice from naïve and transgenic IL34^+/LacZ mothers received four subcutaneous injections (25 mg/kg of body weight) of respectively Sheff-5 (rat anti-mouse IL34 blocking IgG1 antibody, Diaclone, Besançon, France) and IK22-5 rat anti- mouse RANKL blocking IgG2a antibody ⁵⁶ or isotopic corresponding control every 2 days beginning at day 1 after birth (Supplementary Figure 4b). The mice were finally sacrificed at postnatal day 15 for phenotyping.

Micro-CT analysis

A Skyscan 1076 micro-CT scanner (Skyscan, Kontich, Belgium) was used to analyze and compare between the different groups of mice the bone morphometric, structural and mineral parameters at different anatomical sites namely the tibia, the mandible, the vertebra and the cranium. All samples were scanned using the same parameters (pixel size 9 μm, 50 kV, 0.5 mm Aluminum filter, 20 minutes of scanning). The scanner reconstruction was carried out using the NRecon software and the analyses were performed using CTAn, CTVox, and DataViewer software (Skyscan). In order to obtain the different measurements, the IMAGE-J software (National Institutes of Health, Bethesda, MD, USA) was used. In this way, the acquisition of the image in CTVox was systematically calibrated with a phantom of 5 mm (known size) and all measurements were finally sized using the analysis scale in the IMAGE-J software.

Bone morphometric parameters including tibia total length and width were sized using specific reference marks (Figure 1c and Supplementary Figure 4a), and for the cranium measurements were made using the method previously described ⁵⁷. Briefly, seven measurements regarding the sagittal, vertical and transversal planes of craniofacial growth were made (Figure 1c and Supplementary Figure 4a).

Bone mineral and structural parameters including the bone mineral density (BMD), the percentage of bone volume (BV/TV), the trabecula thickness (Tb.Th), the trabecula separation (Tb.Sp) and the trabecula number (Tb.N) were analyzed for each bone at different anatomical sites using a volume of interest (VOI) measuring 2.0 mm x 1.1mm × 1.1 mm. The VOI was sectioned using the Data Viewer software, and analyzed using the CTAn software. The different points chosen for the analysis are presented in Supplementary Figure 4. To facilitate the identification of changes in the different structures, a “color density range” was used in the CTAn software that made it possible to adjust the correspondence of color and brightness values using image gray scales. For tibia and head images, a brightness level of -32 and a contrast level of 6 from the color density range of the CTAn software were systematically used.

Histology, histo-enzymology and immunohistochemistry

Histology, histo-enzymology and immunohistochemistry were performed on 3 µm thickness paraffin embedded sections of the different samples prepared as previously described ⁵⁸. Masson's trichrome and Safranin-O stains were performed following classical protocols and tartrate-resistant acid phosphatase (TRAP) histo-enzymology was carried out as previously described ⁵⁹. Immunohistochemistry was performed by using the protocol as previously described ⁶⁰ and the following antibodies: rabbit monoclonal anti-RUNX2 (Abcam, ref#ab192256, 1/1000), rabbit polyclonal anti- osterix (OSX) (Abcam ref#ab22552, 1/1000), anti-CD207 (eBioscience, ref# 14-2073-80, 1/100).

LacZ staining

Sections (12μm) of IL34^+/LacZ mice epidermis embedded in OCT were cut using Cryostat Leica CM3050S. Slices were fixed with PFA 1% 5min, rinsed with PBS 1x and incubated in Xgal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) solution overnight at 37°C. Sections were rinsed with PBS 1X, left to dry and mounted with EUKITT® medium.

In vitro experiments

Reagents

Recombinant human Macrophage-Colony Stimulating Factor (M-CSF), human interleukin-34 (IL-34), human M-CSF receptor (M-CSFR/CD115), human TGF-β1, human bone morphogenetic protein 2 (BMP-2), human bone morphogenetic protein 4 (BMP-4), human bone morphogenetic protein 7 (BMP-7), human Noggin, Activin RIIA receptor (ActRIIA), human Activin RIIB receptor (ActRIIB), human TRANCE (RANKL) and antibody anti-human M-CSFR, Anti-Phosho-M-CSFR (Y723) were obtained from R&D Systems (Abingdon, UK). Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”. WO/2016/097420 A1, 2016). Antibodies directed against human Smad1 (D59D7), human Smad2 (D43B4), anti Phospho-Smad1/5 (ser463/465) (41D10), anti-phospho-Smad2 (Ser465/467) (138D), β-Actin (8H10D10) and HRP-conjugated secondary antibodies were purchased from Cell Signalling (Ozyme, Saint Quentin Yvelines, France). AlphaLISA® SureFire® Ultra Total SMAD1 and p-SMAD1 (Ser463/465) Assay kits were purchase from PerkinElmer (Villebon-sur-Yvette, France).

Cell cultures

The cell lines used in the present study were purchased from the American Tissue Cell Collection (ATCC, Molsheim, France). HEK293 (HEK) transfected with the pCDNA3 empty plasmid or the pCDNA3 plasmid containing the M-CSFR gene as described by Segaliny et al., ⁶. Human Mesenchymal Stem Cells-Bone Marrow (HMSC-BM) (CLS catalog number 300665) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Lonza, Levallois-Perret, France) supplemented with 10% fetal bovine serum (FBS; Hyclone Perbio, Bezons, France) and 2 mmol/L of L-glutamine.

Human osteoclast differentiation

CD14⁺ monocytes were isolated from peripheral blood of 3 healthy donors CD14⁺ cells were initially isolated from human peripheral blood donors provided by the French blood bank institute (Etablissement Français du Sang, Nantes, France, authorization number: NTS 2000-24), by using MACS microbeads (MiltenyiBiotec, Bergisch Gladbach, Germany) as previously described ⁶¹. For osteoclast differentiation, CD14⁺cells were cultured in alpha-MEM (Lonza) supplemented with 10% human serum (Invitrogen, France) and in the presence of human M-CSF (25 ng/mL) or human IL34 (100 ng/mL) +/- human BMP2 (40 or 100 ng/mL) for 3 days. Then cells were treated with same molecules in the presence of human RANKL (100 ng/mL) for 11 days. Medium was renewed every 3 days. After 11 days of treatment, osteoclasts were analyzed by Acid Phosphatase (TRAP) staining kits (Sigma Aldrich, Saint-Quentin Fallavier, France). TRAP+ multinucleated cells with 3 nuclei and more were considered as osteoclasts and were manually enumerated.

Human osteoblastic differentiation

Human Mesenchymal Stem Cells-Bone Marrow (HMSC-BM) (CLS catalog number 300665) were purchased from CLS (Germany). Osteoblast differentiation assays were performed as previously described ^60,62. Briefly, HMSC-BM were cultured in DMEM was supplemented 10% of FBS, vitamin D3 (10⁻⁸M; Sigma) and dexamethasone (10⁻⁷M; Sigma). After 3 days, ascorbic acid (50 ng/mL; Sigma) and b-glycerophosphate (10 mM; Sigma) were added to allow mineralization detected by alizarin red-S staining for three weeks. Images were captured using a stereomicroscope (Nikon), and mineralized surfaces were quantified using Image J software. Mineralization process was carried out in the presence or absence of human cytokine IL-34 (25 ng/mL), BMP2 (10 ng/mL) or combination of both molecules for 3 weeks. RNA samples were collected at days 3, 4, 14 and 21 after the induction of differentiation.

Flow cytometry

FACS analysis of CD11b monocytic bone marrow and spleen cells were performed as previously described ⁶³. Briefly, after red blood cell lysis (Sigma-Aldrich), bone marrow and spleen cells were labelled with anti- CD11b (clone M1/70; BD Bioscience, Le Pont de Claix, France). Data were acquired using a FACS Canto-II (BD Biosciences).

Western blot

The cells were collected in a RIPA buffer (10 mM Tris pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS containing a cocktail of protease and phosphatase inhibitors Halt™ (Thermo Fisher, Waltham, MA, USA). The protein concentration was determined using a BCA (bicinchoninic acid) method by BC Assay Protein Quantitation Kit (Interchim, Montluçon, France). 50 μg of protein extracts were prepared in a Laëmmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue) and then separated by SDS-polyacrylamide gel electrophoresis. After electrophoretic transfer, the immobilon-P membranes (Millipore, Molsheim, France) were blotted with the antibodies referenced in the “Reagents” section. The membranes were then probed with secondary antibodies coupled with horseradish peroxidase. Antibody binding was visualized with an enhanced chemiluminescence (ECL) kit Clarity™ Western ECL Substrate (Bio-Rad, Marnes-la-Coquette, France). The luminescence was detected with a ChemiDoc MP Imaging System (Bio-Rad). Blots images and semi-quantitative analysis were done using ImageJ software (USA).

SMAD1/5 signaling measured by Alpha SureFire® Technology

Direct quantification analysis of cell signaling was performed by using Alpha SureFire® Technology from PerkinElmer in a Victor® Nivo™ multimode microplate reader (ALSU-PSM1; PerkinElmer, Villebon-sur-Yvette, France).

RNA isolation and real-time PCR

Total RNA was extracted using NucleoSpin® RNA Plus (Macherey-Nagel, Duren, Germany). 1μg of total RNA was used for first strand cDNA synthesis using the OneScript® RT Mix (Ozyme). Real-time PCR was performed on 20 ng of reverse transcribed total RNA (cDNA), 300 nM of primers (QuantiTect Primer® Assays, Qiagen) and PowerUp™ SYBR™ Master Mix from Applied Biosystems™ (Thermo Fisher) in a CFX96 Touch Deep Well Real-Time PCR Detection system from Bio-Rad. Thermal cycle conditions were perform by following manufacture protocol. The analysis was performed with CFX Manager Software (Bio-Rad) using human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Hypoxanthine Phosphoribosyl transferase 1 (HPRT1) and TATA box binding protein (TBP) as invariant controls (QuantiTect Primer® Assays, Qiagen). Oligonucleotides were designed with Primer-Blast software (NCBI) and purchased from Eurogentec (Eurogentec, Angers, France). The 2^−ΔΔCt (cycle threshold) method was used to calculate expression levels. List of primers and gene name symbols with corresponding full names are indicated in Tables S1 and S2 below.

Surface plasmon resonance (SPR) assays

All SPR experiments were performed on a T200 apparatus (Cytiva) at 25 °C in PBS pH7.4 containing 0.05% of surfactant P20. Human recombinant BMP2, BMP4 and BMP7 proteins were immobilized (1500- 2300 RU) at pH 4.5 on CM5-S sensor chip by amine coupling following the manufacturer’s instructions (Cytiva, Velizy-Villacoublay, France). IL34 kinetics were measured using one cycle titration, for these five increasing concentrations of recombinant human IL-34 (12.5, 25, 50, 100, 200 nM) were injected during 60s at 100 µL/min on coated BMPs. The last injection was followed by a 600s dissociation time in running buffer. The KD values were evaluated using a bivalent fitting model (T200 Evaluation software 3.2.1, Cytiva). All sensorgrams were corrected by subtracting the low signal from the control reference surface (without any immobilized protein) and blank buffer injections before fitting. For KD evaluation of IL34 on human recombinant receptors BMPRIIA, Act RIIA and Act RIIB, these receptors were captured on immobilized anti-human Fc (Cytiva), four increasing concentrations of IL34 (18.75, 37.5, 75, 150, 300 nM) were injected. The KD values were evaluated by using a steady-state fitting model. The binding responses of IL-34 (50 nM) alone, Nogging (50 nM) alone and a mixing of IL-34 and Noggin were measured by 180s injection on different coated BMP proteins (BMP-2, BMP-4, BMP-7) at a flow rate of 30 µL/min followed by a dissociation time of 400s in running buffer.

Protein-Protein docking and analysis

Structures of M-CSFR, BMPR1 and IL-34 were extracted from their bound crystallographic forms (1REW for BMPR1A + BMP2 ⁶⁴, 4WRL for M-CSF:M-CSFR1 ⁶⁵ and 4DKD for IL-34:M-CSFR1 ⁶⁶). Docking experiments were performed using either BMP-2 fixed and the partner protein mobile, or the reverse, as previously published ⁶⁴. ClusPro analysis ⁶⁷ was performed in balanced mode, only the first 10 binding modes clusters were considered for analysis, the best modes were selected by visual inspection. Interface analysis was performed using the PISA web server ⁶⁸. Visualization and superimposition of docking poses and crystallographic structures were done using PyMOL (The PyMOL Molecular Graphics System, Version 2.5 Schrödinger, LLC; Schrödinger, LLC 2015).

Statistical analysis

All experiments were repeated at least three times in independent experiments. The differences between the experimental conditions were assessed with Student’s t test or a one-way ANOVA followed by the Mann–Whitney test or Kruskal-Wallis test (in the case of more than two independent samples of equal or different sample size). The results are given as a mean ± SD. Results were considered significant at p-values of ≤0.05, p-values of ≤0.01 and p-values of ≤0.001. GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA) and Real Statistics Resource Pack Software (Release 8.91), copyright (2013-2023) Charles Zaiontz (www.real-statistics.com) were used for statistical analyses.

Conflict of interests:

“Authors declare that they have no competing interests.”

Funding:

Il-34 ^−/− mice were generated at the “Institut Clinique de la Souris” (Illkirch-Graffenstaden, France) and their development was supported by an INSERM grant (Project IR00004258 / K4258). The work was supported by a maturation grant funding program of the Ouest Valorisation Technology Transfer Office (SATT Ouest Valorisation, Nantes, France; ref. DV2439) and by Agence Nationale de la Recherche (ANR-20-CE14-0037).

Supplementary Information

“All data are available in the main text or the supplementary materials.”

Funding

acquisition: FL, CB-W, DH

Author contributions:

Conceptualization: JM-G, JWV-F, ST, FL, DH

Acknowledgments:

The authors wish to thank members from the Therapeutic Experimental Unit (Nantes, France) for their technical assistance.

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Interleukin-34 orchestrates bone formation through its binding to Bone Morphogenic Proteins

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