Cutting Edge: Parathyroid Hormone Facilitates Macrophage Efferocytosis in Bone Marrow via Proresolving Mediators Resolvin D1 and Resolvin D2

A primary function of bone is to house marrow, the major hematopoietic organ in mammals. Bone as an organ is composed of various cell types, including those traditionally considered “bone” cells (osteoblasts, osteoclasts, osteocytes), hematopoietic cells (hematopoietic stem cells, progenitor cells, megakaryocytes), immune cells (lymphocytes, macrophages, dendritic cells), and stromal cells (mesenchymal stem cells). Osteoblasts are “repair” cells responsible for development, secretion, and extracellular matrix mineralization. Mesenchymal stem cells are recruited to bone surfaces where they differentiate into osteoblasts. Once committed to their lineage, osteoblasts are destined to embed into the mineralized matrix as osteocytes, become flattened lining cells, or apoptose. Studies have evaluated the trajectory toward these fates, but none has detailed the aftermath of apoptotic osteoblasts. Inefficient apoptotic cell clearance leads to a local inflammatory environment (1). Hence, there are likely mechanisms for the efficient removal of apoptotic osteoblasts, and a likely facilitator is the resident macrophage. The term “osteomac” has been attributed to macrophages credited with stimulating bone formation through mechanisms that link to their phagocytic capacity (2). Macrophages facilitate apoptotic cell clearance in a process termed efferocytosis, which converts the dead and dying cell toxic environment to an anti-inflammatory environment.

With the identification of resolution-phase mediators, recent efforts have focused on the resolution phase, an active process orchestrated by special mediators that direct cellular and biochemical pathways to facilitate return to homeostasis (3). Such specialized proresolving mediators (SPMs) of inflammation include lipoxins (LXs), resolvins (Rvs), protectins, and maresins (4). Among other actions, these mediators are potent stimulators of macrophage efferocytosis, but little is known about their role in bone remodeling. The purpose of this investigation was to determine the LM profiles of two lymphoid organs—bone marrow and spleen—assess their modulation by parathyroid hormone (PTH), and determine their ability to facilitate macrophage efferocytosis of apoptotic osteoblasts. The implications identify a new feedback process in bone that has potential impact in normal bone remodeling, as well as wound healing in bone.

Male C57BL/6 mice (4–5 wk; The Jackson Laboratory, Bar Harbor, ME) were injected once with recombinant human PTH 1-34 (50 μg/kg; Bachem, Torrance, CA) or vehicle (saline) 2 h prior to sacrifice. Spleens and long bones were frozen in liquid nitrogen and then placed in ice-cold methanol containing deuterated internal standards (d₈-5S-hydroxyeicosatetraenoic acid, d₄-leukotriene B₄, d₄-PGE₂, and d₅-LXA₄; 500 pg each) and homogenized using a PTFE dounce (Kimble Chase). Proteins were precipitated (4°C), solid-phase extracted using Biotage RapidTrace+ (5), and analyzed using liquid chromatography-UV-tandem mass spectrometry, QTrap 5500 (AB SCIEX, Framingham, MA) equipped with an Agilent HP1100 binary pump (Santa Clara, CA). An Agilent Eclipse Plus C18 column (100 mm × 4.6 mm × 1.8 μm) maintained at 50°C was used with a gradient of methanol/water/acetic acid of 55:45:0.01 (v/v/v) to 100:0:0.01 at 0.4 ml/min flow rate. Multiple reaction monitoring with signature ion fragments for each molecule was used, with six diagnostic ions used for identification, and quantification was achieved using calibration curves (5). Principal component analysis was performed using SIMCA 13.0.3 software (Umetrics, Umea, Sweden) following mean centering and unit variance scaling of LMs.

PBMCs were isolated from human venous blood by density gradient centrifugation (6). Blood was obtained from healthy human volunteers under a Partners Human Research Committee–approved protocol. Cells were cultured in RPMI 1640 with 10% FBS, Pen/Strep, and GM-CSF (10 ng/ml; R&D Systems) 7 d prior to efferocytosis assays. Macrophages differentiated from PBMCs were plated overnight (96-well plates; 50,000/well) and then incubated with 1 pM–100 nM of RvD1 or RvD2 15 min prior to the addition of apoptotic human osteoblasts (5:1 osteoblasts/macrophages). Human fetal osteoblasts (hFOBs; American Type Culture Collection, Manassas, VA), cultured in DMEM F12 media (10% FBS 1% Pen/Strep, glutamine; Invitrogen, Life Technologies, Grand Island, NY), were labeled with CFSE (3 μM; Invitrogen) 15 min prior to the induction of apoptosis via UV radiation. Cells were exposed to UV light for 30 min, returned to 37°C for 2 h, harvested, and enumerated via trypan blue dye exclusion. More than 90% of cells were positive for trypan blue, reflecting cell death. hFOBs were resuspended in PBS and incubated with macrophage cultures for 4 h, followed by incubation with trypan blue to quench extracellular fluorescence and exclude cell-associated nonphagocytosed cells (i.e., not internalized), prior to rinsing and scanning at 487–517 nM on a microplate reader (SpectraMax M3; Molecular Devices, Sunnyvale, CA). Results are expressed as the fold increase in CFSE above control, as a reflection of cell engulfment.

Marrow was flushed from 4-wk-old C57BL/6 mice, expanded with 30 ng/ml M-CSF (eBioscience) in α-MEM media (10% FBS Pen/Strep, glutamine) for 5–7 d to enrich for macrophages, plated at 10,000 cells/well in eight-well chambers, attached overnight, stained for 15 min with 2 μM Green BODIPY in PBS, returned to complete medium, and incubated for 1 h to finalize dye cross-linking. MC3T3-E1 cells (previously stained as above with Red CMTPX; Invitrogen) were induced to undergo apoptosis for 48 h with 50 mM etoposide (Sigma, St. Louis, MO), and 40,000 cells/well were coincubated with macrophages for 3 h. Cells were washed, fixed with ice-cold methanol, washed, and overlaid with VECTASHIELD (Vector Laboratories, Burlingame, CA) to visualize DAPI nuclei. Z-stack images were performed on a Leica Inverted SP5X Confocal Microscope System using Leica Application Suite software (Leica, Wetzlar, Germany) and compiled into a three-dimensional video format using Imaris (Bitplane Scientific Software, South Windsor, CT). Videography was performed using the DeltaVision RT Live Cell Imaging System with SoftWoRx 3.5.1 software (Applied Precision, Issaquah, WA).

LM profiles from spleen and bone were obtained and compared using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS)–based LM metabololipidomics. Specific mediators from the arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) bioactive metabolomes were identified, including RvD1 and RvD2 from DHA, RvE1 and RvE3 from EPA, and LXA₄ and LXB₄ from the AA bioactive metabolomes (Fig. 1). These mediators were identified with matching retention times, tandem mass spectrometry fragmentation patterns, and least six characteristic and diagnostic ions, as illustrated for RvD1 and RvD2 (Supplemental Fig. 1). Quantification of individual LMs demonstrated elevated AA-, EPA-, and DHA-derived proresolving mediator levels in bone marrow from unchallenged mice compared with spleens. RvD2, RvE1, and LXB₄, among others, were significantly elevated in bone marrow. Principal component analysis demonstrated that LM profiles obtained with mouse bone marrow clustered separately from those obtained with mouse spleens (Fig. 1A) (e.g., with Protectin D1 with spleen) and RvD2 with bone marrow (Fig. 1B). These results expand the recent identification of LM biosynthesis in murine spleen (7) and bone marrow (8) with the identification of novel SPMs that, with the present results, include RvD2.

The spleen and bone marrow serve as stem and leukocyte cell reservoirs; an important difference is that the marrow is enclosed by a skeletal boundary and, hence, hematopoietic and leukocytic cells are juxtaposed with osteoblasts, osteoclasts, and a mineralized extracellular matrix. That PGs were increased in bone marrow versus spleen was not surprising because years of work have shown the diverse actions of PGs in bone (9). Unique to this study were findings that members of the DHA bioactive metabolome (i.e., the Rvs) were higher in marrow. This suggests that bone is poised to facilitate resolution of inflammation. Indeed, injurious and infectious insults to the marrow are more deleterious than to the spleen; hence, protective mechanisms for resolving inflammation in a timely manner would be essential for survival.

In the current study, PTH administration led to a rapid (2-h) and selective increase in proresolving LM levels in marrow, including RvD1 and RvD2, giving characteristic LM profiles, as demonstrated by principal component analysis (Figs. 1C, 1D, 2, Supplemental Fig. 1), but no significant difference in the spleen (data not shown). This regulation included D-series and E-series Rvs. The D-series Rvs facilitate macrophage efferocytosis of apoptotic neutrophils and reduce the resolution interval postinflammatory challenge (5). Of note, statistically significant differences were not observed for LM levels 5 d after PTH administration (data not shown).

One of the prime actions of SPMs is to facilitate macrophage efferocytosis (1, 3). Efferocytosis studies most commonly involve macrophage clearance of apoptotic neutrophils and result in macrophage production of anti-inflammatory factors, such as TGFβ (10). If dying cells are not cleared, their intracellular contents are expelled, creating an unfavorable environment (11). In the bone marrow, the fate of osteoblasts that undergo apoptosis is unclear. Experiments performed in which labeled osteoblasts were incubated with macrophages identified that macrophages rapidly efferocytose apoptotic osteoblasts (Fig. 3, Supplemental Videos 1–5).

Studies to determine whether increased bone marrow SPMs facilitate macrophage efferocytosis revealed that RvD1 and RvD2 increased uptake of apoptotic osteoblasts (Fig. 4). Of note, RvD1 (100 pM–10 nM) and RvD2 (100 pM–100 nM) increased efferocytosis of osteoblasts (30–60%) to a similar extent as efferocytosis of apoptotic polymorphonuclear neutrophils (∼30%) (12). These experiments demonstrate the ability of macrophages at physiologically relevant concentrations, based on our LC-MS-MS quantification, to engulf apoptotic osteoblasts, a process facilitated by Rvs produced in the local environment. Other studies identified that RvD1 reduced macrophage-derived TNF-α, promoting the resolution of inflammation, and that the process of efferocytosis involves subsets of macrophages reprogrammed from CD11b^high to CD11b^low (13, 14).

The process of bone remodeling has been likened to inflammation, where the inflammatory stage of “injury” correlates with pressure and microfracture. The “reaction” stage correlates with osteoclastic bone resorption, and the “repair” stage correlates with osteoblastic bone formation (15). PTH is a potent inducer of bone remodeling and is well known to increase bone formation through mechanisms that are still not entirely clear. PTH drives robust increases in bone accrual during wound healing (16, 17); hence, our interest was in determining whether PTH was associated with LMs responsible for the resolution of inflammation. PTH stimulates IL-6 and CCL2 production from osteoblasts, which recruits and activates myeloid cells (18, 19). During wound healing, macrophages phagocytose debris and apoptotic cells. PTH has robust anabolic actions during traumatic injuries associated with increased apoptosis, such as bone marrow ablation (20). Hence, PTH could act in the marrow to recruit macrophages and promote the release of SPMs that facilitate efferocytosis. Such a proresolving circuit would be enhanced when PTH is administered during osseous wound healing where it expedites skeletal homeostasis in favor of anabolism. In summary, the bone marrow has a unique profile of specialized LMs, and PTH increases select SPMs. Bone marrow macrophages efferocytose apoptotic osteoblasts, which is facilitated by RvD1 and RvD2. Taken together with reports that macrophages produce TGFβ during efferocytosis and that TGFβ recruits mesenchymal stem cells to renew osteoblasts (21), these results suggest a new feedback system for the resolution of inflammation and restoration of skeletal homeostasis.

We thank M. Shinohara, M. Kibi, and H. Arnardottir for cell procurement.

C.N.S. is an inventor on patents (Rvs) assigned to Brigham and Women’s Hospital and licensed to Resolvyx Pharmaceuticals. C.N.S. is a scientific founder of Resolvyx Pharmaceuticals and owns equity in the company. C.N.S.'s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. The other authors have no financial conflicts of interest.