ORIGINAL ARTICLE JBMR CCL20/CCR6 Signaling Regulates Bone Mass Accrual in Mice Michele Doucet, Swaathi Jayaraman, Emily Swenson, Brittany Tusing, Kristy L Weber,Ã and Scott L Kominsky Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA ABSTRACT CCL20 is a member of the macrophage inflammatory protein family and is reported to signal monogamously through the receptor CCR6. Although studies have identified the genomic locations of both Ccl20 and Ccr6 as regions important for bone quality, the role of CCL20/CCR6 signaling in regulating bone mass is unknown. By micro–computed tomography (mCT) and histomorphometric analysis, we show that global loss of Ccr6 in mice significantly decreases trabecular bone mass coincident with reduced osteoblast numbers. Notably, CCL20 and CCR6 were co-expressed in osteoblast progenitors and levels increased during osteoblast differentiation, indicating the potential of CCL20/CCR6 signaling to influence osteoblasts through both autocrine and paracrine actions. With respect to autocrine effects, CCR6 was found to act as a functional G protein–coupled receptor in osteoblasts and although its loss did not appear to affect the number or proliferation rate of osteoblast progenitors, differentiation was significantly inhibited as evidenced by delays in osteoblast marker gene expression, alkaline phosphatase activity, and mineralization. In addition, CCL20 promoted osteoblast survival concordant with activation of the PI3K-AKT pathway. Beyond these potential autocrine effects, osteoblast-derived CCL20 stimulated the recruitment of macrophages and T cells, known facilitators of osteoblast differentiation and survival. Finally, we generated mice harboring a global deletion of Ccl20 and found that Ccl20-/- mice exhibit a reduction in bone mass similar to that observed in Ccr6-/- mice, confirming that this phenomenon is regulated by CCL20 rather than alternate CCR6 ligands. Collectively, these data indicate that CCL20/CCR6 signaling may play an important role in regulating bone mass accrual, potentially by modulating osteoblast maturation, survival, and the recruitment of osteoblast-supporting cells. © 2016 American Society for Bone and Mineral Research. KEY WORDS: OSTEOBLAST; BONE MODELING AND REMODELING; CELL/TISSUE SIGNALING; CYTOKINES Introduction at specific organ sites and the recruitment of immune cells (eg, dendritic cells, T cells, B cells) in response to inflammatory signals, – CL20 (MIP-3a) is a member of the macrophage inflamma- respectively.(5 7) Indeed, Ccr6-/- mice show impaired localization of tory protein (MIP) family, which was discovered in 1988 and leukocytes under nonpathological conditions and impairment of C – consists of six chemokines of the CC type. Amongst CC both innate and adaptive immune responses.(8 10) chemokines, including those of the MIP family, CCL20 has Consistent with its role in regulating chemotaxis of immune some distinctive characteristics. Although most chemokines are effectors, studies indicate that CCL20 may be involved in various promiscuous in their ligand-receptor interactions, CCL20 inflammatory disorders. In addition to its potential involvement participates in a monogamous ligand-receptor relationship, in psoriasis, inflammatory bowel disease, and asthma, CCL20 has wherein it signals through a single receptor, CCR6, which in turn been reported to play a role in various cancers.(11,12) Exploring its serves solely as the receptor for CCL20.(1) Further, although potential involvement in renal cell carcinoma (RCC), we showing conservation among species,(2,3) the gene structure of observed high levels of CCL20 in patient samples of RCC Ccl20 differs from that of the CC chemokine family and is located bone metastasis, which is classically associated with excessive in a genomic region apart from that in which most CC bone resorption. Correspondingly, inflammatory chemokines of chemokine genes are clustered. Expression of CCL20 has been the MIP family have been identified as potential mediators of reported on epithelial surfaces of the liver, lung, and intestine, as pathological bone loss where MIP-1a and MIP-1d have been well as in bone marrow and secondary lymphoid organs.(4) shown to directly enhance osteoclast development.(13,14) Evidence suggests that CCL20 functions in both homeostatic Interestingly, genome-wide linkage analyses have identified and inflammatory capacities, regulating the residence of leukocytes the genomic locations of both Ccl20 and Ccr6 (2q33-37 and Received in original form October 30, 2015; revised form February 3, 2016; accepted February 13, 2016. Accepted manuscript online Month 00, 2016. Address correspondence to: Scott L Kominsky, PhD, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 232, Baltimore, MD 21205, USA. E-mail: [email protected] ÃPresent address: Department of Orthopaedic Surgery, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA Additional Supporting Information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. xx, No. xx, Month 2016, pp 1–10 DOI: 10.1002/jbmr.2813 © 2016 American Society for Bone and Mineral Research 1 6q27, respectively) as regions important for bone quality,(15–17) recommendations of the American Society of Bone and Mineral indicating a potential role for CCL20 in the regulation of bone Research.(20,21) For histomorphometric analysis, 8-week-old mass. Although the expression of CCL20 and CCR6 in human Ccr6-/- and WT littermate mice received injections of 1% bone cells (eg, osteoblast, osteoclast, chondrocyte) has been (wt/vol) calcein at 8 and 3 days before euthanization. Femurs reported,(18,19) the role of CCL20/CCR6 signaling in bone remains were embedded in polymethyl methacrylate for sectioning and unknown. analyzed using a semiautomatic method (Osteoplan II, Kontron, Bone disorders (eg, osteoporosis, rheumatoid arthritis, bone Poway, CA, USA). metastasis) affect millions of men and women worldwide. Advances in the prevention and treatment of these disorders Cell isolation and culture are dependent on our ability to identify key factors regulating Bone marrow was isolated from female Ccr6-/-, Ccl20-/-, and WT bone formation and remodeling. Here, we investigate the effect littermate mice as previously described.(14) T cells were isolated of CCL20/CCR6 signaling on bone mass by examining the þ þ from total bone marrow using the CD4 CD25 T Cell Isolation skeletal phenotype of Ccr6-/- and Ccl20-/- mice. We demonstrate, Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manu- for the first time to our knowledge, that loss of CCL20/CCR6 facturer’s instructions. To generate bone marrow macrophages signaling results in reduced trabecular bone volume, coincident (BMM), marrow cells were washed and resuspended in with a decrease in osteoblast numbers, potentially stemming complete media (aMEM þ 10% fetal bovine serum [FBS] þ 1% from deficiencies in osteoblast maturation, survival, and the penicillin/streptomycin) containing 5 ng/mL macrophage recruitment of osteoblast-supporting cells. Taken together, colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, these data indicate that CCL20/CCR6 signaling may represent a MN, USA) and incubated in a 100-mm dish overnight. Non- novel pathway through which bone accrual and maintenance adherent cells were then collected, strained through 70-mm nylon are regulated. mesh, and treated with 50 ng/mL M-CSF for 3 days to generate M-CSF-dependent BMM. To generate BMSC, marrow cells were Materials and Methods washed and resuspended in complete media and incubated overnight. Adherent cells were collected as BMSC, and cell purity Animals was confirmed >95% negative for nonspecific esterase (a marker Animal experiments were carried out in accordance with for cells of the monocyte/macrophage lineage). approved protocols and guidelines of the Johns Hopkins Animal To determine the number of BMSC contained within the bone Care and Use Committee. Ccr6-/- (B6.129P2-Ccr6tm1Dgen/J) mice, marrow, marrow was plated at clonal density, incubated in backcrossed at least five generations to C57BL/6 mice, were complete medium for 10 to 14 days, and colony formation was purchased from The Jackson Laboratory (Bar Harbor, ME, USA) assessed by methylene blue dye staining. To determine the and subsequently crossed with C57BL/6 mice (The Jackson osteogenic potential of BMSC within the bone marrow, marrow Laboratory) to generate the Ccr6-/- and WT littermate control mice was plated at clonal density, incubated in osteogenic medium À used in all experiments. To generate Ccl20-/- mice, embryonic (complete media containing dexamethasone [10 8 M], ascorbic stem (ES) cells containing a Ccl20 mutant allele (Ccl20tm11a)were acid [50 mg/mL], and b-glycerolphosphate [5mM]) for up to obtained from the European Conditional Mouse Mutagenesis 18 days, and alkaline phosphatase positive colony formation was Program (Neuherberg, Germany). Within Ccl20tm11a, the second assessed by alkaline phosphatase staining. For osteoclast exon is flanked by loxP sites and when excised results in a differentiation, BMM were cultured in complete media contain- frameshift causing loss of CCL20 mRNA expression (Supplemental ing M-CSF (20 ng/mL) and receptor activator of NF-kB ligand Fig. S1). Ccl20tm11a ES cells (C57BL/6N) were injected into mouse (RANKL; 5 ng/mL; R&D Systems) for a period of 7 days. Fifty blastocysts (C57BL/6J) at the Johns Hopkins University