Acceleration of Bone Repair in NOD/SCID Mice by Human Monoosteophils, Novel LL-37-Activated Monocytes

<div><p>Background</p><p>An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function.</p><p>Methods and Findings</p><p>Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not <i>MAPK13</i> KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries.</p><p>Conclusion</p><p>Human derived monoosteophils, characterized as CD45⁺α3⁺α3β⁺CD34⁻CD14⁻BAP (bone alkaline phosphatase)⁻ cells, can function in an animal model of bone injury.</p></div

Monoosteophils release high level of osteopontin, osteoactivin, cartilage glycoprotein-39, chitinase 1, MMP-7 and CCL22.

<p>Monocytes were incubated in the absence or presence of LL-37 (5 µM), GM-CSF (20 ng/mL), GM-CSF+IL-4 (both 20 ng/mL), M-CSF (50 ng/mL) or M-CSF +RANKL (both at 25 ng/mL) for 1, 3 or 6 days. Supernatants were harvested and proteins were detected by using ELISA Kits (n = 3). CHI3L1: cartilage glycoprotein-39.</p

Monoosteophils accelerate cortical bone repair in the drilled-hole bone defect model.

<p>NOD/SCID mice were anesthetized with isoflurane, and holes (0.9 mm) were created in the mid-diaphysis of femur. <b>A–B</b>. Holes were filled with Matrigel (Ctrl), Matrigel +6 d Monoosteophils (6 d MOP, 3×10⁶ cells), Matrigel+1 d Monoosteophils (1 d MOP, 3×10⁶ cells), or Matrigel +1 d Monocytes (1 d Mo, 3×10⁶ cells). After 14 days, femurs were harvested and observed by visual analysis (<b>A</b>) and low resolution µCT (<b>B</b>). <b>C–D</b>. Holes were filled with Matrigel+1 d Monoosteophils (1 d MOP, 3×10⁶ cells) or Matrigel +1 d Monocytes (1 d Mo, 3×10⁶ cells). Bone repair was monitored by serial µCT using coronal (<b>C</b>) and transverse plane imaging (<b>D</b>) and quantitated as remaining defect area (mm²) (<b>E</b>) at days shown in the figure.</p

Differentiation markers and proliferative capacity of monoosteophils.

<p><b>A.</b> Monocytes were incubated in the absence or presence of LL-37 (5 µM), GM-CSF (20 ng/mL), M-CSF (50 ng/mL), or M-CSF and RANKL (both at 25 ng/mL) for 6 days. Surface staining of Integrin α3 and α3β1 were analyzed using flow cytometry<b>. B–C.</b> Monocytes were incubated in the presence of 5 µM LL-37 for different days and proliferative capacity was detected using either (<b>B</b>) FITC BrdU/7AAD flow kit or (<b>C</b>) Cell proliferation dye eFluor 670. Data shown were from three independent experiments.</p

pSmad1/5/8 and MAPK13 signaling in the differentiation of monoosteophils.

<p>Human monocytes were incubated in the absence or presence of LL-37 (5 µM). Cells were harvested on days 0–6 and pSmad1/5/8 (<b>A</b>) and MAPK13 (<b>B</b>) were analyzed by western blot. (<b>C</b>) Mouse monocytes were isolated from bone marrow of <i>MAPK13</i> KO or wild type mice, cultured at the concentration of 1×10⁶ cells/mL in absence or presence of 5 µM CRAMP (murine LL-37) for 6 days, and observed using phase contrast microscopy (magnification, 200×). Data shown are representative of three independent experiments.</p

<i>CEACAM1</i> mRNA isoform expression in BAC2-12 transgenic mouse liver, kidney and small intestine.

<p><b>A.</b> RT-PCR analysis for <i>CEACAM1</i> isoforms in transgenic (1–3) or wild type mice (4–6) in liver (1, 4), kidney (2, 5), and small intestine (3, 6). <b>B.</b> RT-PCR analysis for <i>Ceacam1</i> isoforms in transgenic (7–9) or wild type mice (10–12) in liver (7,10), kidney (8,11), and small intestine (9,12).</p

Immunohistochemistry staining of blood neutrophils from BAC2-12 transgenic and wild type mice.

<p><b>A</b>. Transgenic mouse blood neutrophils stained with anti-human CEACAM1 antibody. Mag. 200X (inset photo enlarged 4X). <b>B</b>. Wild type mouse blood neutrophils stained with anti-human CEACAM1. Mag. 200X. <b>C</b>. Transgenic mice blood neutrophils stained with anti-mouse CEACAM1. Mag. 200X (inset photo enlarged 4X). <b>D</b>. Human neutrophils stained with anti-human CEACAM1. Mag. 200X (inset photo enlarged 4X).</p

Transgenic mouse screening.

<p><b>A</b>. PCR screening for <i>CEACAM1</i> expression. Pups 1 to 12 were born after pronuclear microinjection of BAC2. Founders 1, 6 and 12 were positive using <i>CEACAM1</i> primers. <b>B</b>. Western blots of feces using anti-human CEACAM1 mAb demonstrated that BAC2 founders 1, 6 and 12 expressed human CEACAM1 in their gastrointestinal tracts. <b>C</b>. PCR analysis of the F1 generation from founder 12. About 50% of the pups were positive (left side) for human <i>CEACAM1</i> and 100% positive for murine CEACAM1 (right side). <b>D</b>. Western blot analysis of feces using anti-human CEACAM1 mAb. Pups 7, 8, 12, 13, 14, and 18 from founder BAC2-12 were positive. Most fecal samples were collected in the morning (no label) or in the evening (7P, 8P) to determine if time of collection affected the analysis.</p

Immunohistochemistry staining for human and murine CEACAM1 in kidney and liver of BAC2-12 transgenic and wild type mice.

<p><b>A</b>. Wild type mouse kidney. <b>B</b>. Transgenic mouse kidney. <b>C</b>. Transgenic mouse kidney with isotype control antibody. <b>D</b>. Wild type mouse liver. <b>E</b>. Transgenic mouse liver. <b>F</b>. Transgenic mouse liver with isotype control antibody. Note that human CEACAM1 expression is higher than murine CEACAM1 in transgenic mouse kidney. However, in the liver, murine and human CEACAM1 expression in transgenic mice was similar. Bowman's capsule in the kidney is indicated by an arrow (<b>B</b>). Mag. 200X.</p

Confocal analysis of transgenic mouse neutrophils undergoing binding of <i>E. coli</i> expressing recombinant Opa₅₂ protein.

<p><b>Panel 1</b>. <i>E. coli</i> vector control or Opa₅₂ phagocytosed by human neutrophils. <b>A</b>. Vector control <i>E. coli</i> (FITC labeled, green). <b>B</b>. Same as A, stained with anti-human CEACAM1 mAb Alexa 555 labeled (red). <b>C</b>. Same as A, stained with DAPI (blue). <b>D</b>. Overlay of A–C on phase contrast image. Mag 300X. <b>E</b>. Opa₅₂<i>E. coli</i> (FITC labeled, green). <b>F</b>. Same as E, stained with anti-human CEACAM1 Mab Alexa 555 labeled (red). <b>G</b>. Same as E, stained with DAPI (blue). <b>H</b>. Overlay of E–G on phase contrast image. Mag 300X. <b>Panel 2</b>. <i>E. coli</i> vector control or Opa₅₂ phagocytosed by neutrophils from transgenic (A–D) or wild type (E–H) mice. <b>A, E.</b> Vector control <i>E. coli</i> (overlay with FITC, Alexa 555, and DAPI on phase contrast image). <b>B, F.</b> vector control <i>E. coli</i> (FITC labeled, green). <b>C, G.</b> Opa₅₂<i>E. coli</i>. (overlay with FITC, Alexa 555, and DAPI on phase contrast image) <b>D, H.</b> Opa₅₂ expressing <i>E. coli</i> (FITC staining).</p