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

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

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

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

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

RNAi reduction of SHP-1 leads to elevated Syk activation and augmented inflammasome activation.

<p>(<b>A</b>) Immunoblot analysis of p-Syk, Syk, SHP-1 and GAPDH of LPS treated neutrophils from WT mice reconstituted with shRNA control retroviral vector transduced WT BM cells (WT/(shcontrol)), WT mice reconstituted with shSHP-1 retroviral vector transduced WT BM cells (WT/(shSHP-1)) compared with WT mice. (<b>B</b>) ROS production by LPS treated neutrophils from WT mice, WT/(shcontrol) and WT/(shSHP-1) chimera measured by MFI of fluorescent probe H2DCFDA using FACS. (<b>C</b>) Quantification of lysosome destabilization of LPS treated neutrophils from WT mice, WT/(shcontrol) and WT/(shSHP-1) chimera measured by MFI of LysoSensor Green using FACS. (<b>D</b>) Immunoblot analysis showing capase-1 activation of LPS treated neutrophils from WT mice, WT/(shcontrol) and WT/(shSHP-1). (<b>E</b>) IL-1β production of LPS treated neutrophils from WT mice, WT/(shcontrol) and WT/(shSHP-1) chimera. Data are representative of 3 different experiments (<b>A</b> and <b>D</b>) and p values (<b>B, C</b> and <b>E</b>) were calculated by a 2-tailed T-test *0.01</p

Loss of CEACAM1 leads to elevated Syk activation and enhanced inflammasome activation.

<p>(<b>A</b>) Immunoblot analysis of WT and Ceacam1^−/− (KO) neutrophils with p-Syk (Y525/526), Syk and GAPDH antibodies. (<b>B</b>) Immunoblot analysis of TLR4, Syk, p-Syk and CEACAM1 in WT and Ceacam1^−/− (KO) neutrophils with or without LPS treatment (100 ng/ml) after immunoprecipitation with TLR4 antibody. (<b>C</b>) Confocal microscopy showing LPS induced lysosomal destabilization in WT and Ceacam1^−/− neutrophils (DQ-Ovalbumin, 10 mg/ml; green) with or without LPS (100 ng/ml), cell membranes were stained with fluorescent cholera toxin B-subunit (red). (<b>D</b>) Quantification of lysosomal destabilization of WT and Ceacam1^−/− neutrophils as measured by MFI of LysoSensor Green using FACS. (<b>E</b>) IL-1β production in the supernatants of LPS treated WT and Ceacam1^−/− neutrophils under different treatment conditions. (<b>F</b>) Immunoblot analysis showing caspase-1 activation of LPS treated WT and Ceacam1^−/− neutrophils under different treatment conditions. (<b>G</b>) IL-1β production in the supernatants of LPS treated WT neutrophils with or without glibenclamide treatment (250 µM). Data are representative of 3 different experiments and p values (<b>C</b>) were calculated by a 2-tailed T-test *0.01</p

Model for the inhibition of the inflammasome in neutrophils by CEACAM1.

<p>(<b>A</b>) LPS binds to TLR (the usual downstream effects such as activation of NFκB are not shown for clarity). (<b>B</b>) The complex recruits and activates Syk (pSyk) which in the absence of CEACAM1 fully activates the inflammasome that includes ROS production from the mitochondrion and cathepsin B from the lysosome. The activated inflammasome converts pro-caspase-1 to active caspase-1, which in turn converts pro-IL-1β to active IL-1β. (<b>C</b>) In the presence of CEACAM1, both Syk and CEACAM1 are phosphorylated when LPS binds to TLR4. CEACAM1 recruits SHP1 via its phosphorylated ITIM. SHP1 dephosphorylates pSyk, reducing the production of ROS and lysosome disruption, which in turn, reduces the activity of the inflammasome.</p

LPS induced neutrophil inflammasome activation depends on ROS production and lysosome destabilization.

<p>(<b>A</b>) IL-1β production in the supernatants of wild type (WT), P2X7^−/− and Cybb^−/− neutrophils under different treatment conditions: LPS (100 ng/ml), KN62 (2 µM, neutrophils were pre-treated for 30 minutes before LPS treatment), APDC (100 µM), bafilomycin A (125 nM), z-YVAD-fmk (1 mM). (<b>B</b>) Immunoblot analysis showing caspase-1 activation of WT, P2X7^−/− and Cybb^−/− neutrophils under different treatment conditions. (<b>C</b>) ROS production by LPS treated WT and CEACAM1^−/− neutrophils with or without APDC (100 µM) as measured by MFI of fluorescent probe H2DCFDA using FACS. Data are representative of 3 different experiments (<b>B</b>) and p values (<b>A</b> and <b>C</b>) were calculated by a 2-tailed T-test, **0.001</p

CEACAM1 down-regulates Syk activation through ITIM recruitment of SHP-1.

<p>(<b>A</b>) Immunoblot analysis of p-Tyr, Syk, SHP-1 and CEACAM1 in WT and Ceacam1^−/− (KO) neutrophils with or without LPS treatment (100 ng/ml) after IP with anti-CEACAM1 antibody. (<b>B</b>) Immunoblot analysis of Syk, SHP-1 in WT and Ceacam1^−/− neutrophils with or without LPS after IP with anti-SHP-1 antibody. (<b>C</b>) Immunoblot analysis of p-Syk, Syk and GAPDH in neutrophils from Ceacam1^−/− mice reintroduced with empty vector (KO/(emp)), CEACAM1-2L (KO/(CC1-2L)), CEACAM1-4L (KO/(CC1-4L)), CEACAM1-2S (KO/(CC1-2S)), CEACAM1-4S (KO/(CC1-4S)), ITIMs mutated CEACAM1-2L (KO/(CC1-2 Lm)) and ITIMs mutated CEACAM1-4L (KO/(CC1-4 Lm)) with or without LPS treatment. (<b>D</b>) Immunoblot analysis of the p-Tyr, Syk, SHP-1 and CEACAM1 in neutrophils from KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras after IP with CEACAM1 antibody. (<b>E</b>) IL-1β production in the supernatants of LPS treated neutrophils from KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras. (<b>F</b>) Immunoblot analysis showing caspase-1 activation of LPS treated neutrophils KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras. Data are representative of 3 different experiments and p values (<b>E</b>) were calculated by a 2-tailed T-test ***p≤0.001.</p