Modulating the phenotype and function of bone marrow-derived macrophages via mandible and femur osteoblasts.

Various studies have been investigated the phenotypic and functional distinctions of craniofacial and long bone cells involved in bone regeneration. However, the process of bone tissue regeneration after bone grafting involves complicated interactions between different cell types at the donor-recipient site. Additionally, differences in alterations of the immune microenvironment at the recipient site remained to be explored. Osteoblasts (OBs) and macrophages (MØ) play essential roles in the bone restoration and regeneration processes in the bone and immune systems, respectively. The modulation of MØ on OBs has been extensively explored in the literature, whereas limited research has been conducted on the influence of OBs on the MØ phenotype and function. In the present study, OBs from the mandible and femur (MOBs and FOBs, respectively) promoted cranial defect regeneration in rats, with better outcomes noted in the MOBs-treated group. After MOBs transplantation, a significant inflammatory response was induced, accompanied by an early increase in IL-10 secretion. And then, there was an upregulation in M2-MØ-related cell markers and inflammatory factor expression. Condition media (CM) of OBs mildly inhibited apoptosis in MØ, enhanced their migration and phagocytic functions, and concurrently increased iNOS and Arg1 expression, with MOB-CM demonstrating more pronounced effects compared to FOB-CM. In conclusion, our investigation showed that MOBs and FOBs have the ability to modulate MØ phenotype and function, with MOBs exhibiting a stronger regulatory potential. These findings provide a new direction for improving therapeutic strategies for bone regeneration in autologous bone grafts from the perspective of the immune microenvironment.

Engineering immunomodulatory and osteoinductive implant surfaces via mussel adhesion-mediated ion coordination and molecular clicking.

Immune response and new tissue formation are important aspects of tissue repair. However, only a single aspect is generally considered in previous biomedical interventions, and the synergistic effect is unclear. Here, a dual-effect coating with immobilized immunomodulatory metal ions (e.g., Zn2+) and osteoinductive growth factors (e.g., BMP-2 peptide) is designed via mussel adhesion-mediated ion coordination and molecular clicking strategy. Compared to the bare TiO2 group, Zn2+ can increase M2 macrophage recruitment by up to 92.5% in vivo and upregulate the expression of M2 cytokine IL-10 by 84.5%; while the dual-effect of Zn2+ and BMP-2 peptide can increase M2 macrophages recruitment by up to 124.7% in vivo and upregulate the expression of M2 cytokine IL-10 by 171%. These benefits eventually significantly enhance bone-implant mechanical fixation (203.3 N) and new bone ingrowth (82.1%) compared to the bare TiO2 (98.6 N and 45.1%, respectively). Taken together, the dual-effect coating can be utilized to synergistically modulate the osteoimmune microenvironment at the bone-implant interface, enhancing bone regeneration for successful implantation.

NLRP3 Triggers Attenuate Lipocalin-2 Expression Independent with Inflammasome Activation.

Lipocalin-2 (LCN2), a small secretory glycoprotein, is upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 inhibits bacterial growth by iron sequestration and regulates the innate immune system. Inflammasome activates the inflammatory caspases leading to pyroptosis and cytokine maturation. This study examined the effects of inflammasome activation on LCN2 secretion in response to TLR signaling. The triggers of NLRP3 inflammasome activation attenuated LCN2 secretion while it induced interleukin-1ß in mouse macrophages. In mice, NLRP3 inflammasome activation inhibited TLR-mediated LCN2 secretion. The inhibition of NLRP3 triggers on LCN2 secretion was caused by the inhibited transcription and translation of LCN2. At the same time, no changes in the other cytokines and IκBζ, a well-known transcriptional factor of Lcn2 transcription, were observed. Overall, NLRP3 triggers are a regulator of LCN2 expression suggesting a new linkage of inflammasome activation and LCN2 secretion in the innate immunity.

Exosomal miR-335 derived from mature dendritic cells enhanced mesenchymal stem cell-mediated bone regeneration of bone defects in athymic rats.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect. METHODS: The BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss®) and then implanted into the femoral bone defect of athymic rats. RESULTS: mDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo. CONCLUSION: mDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335.

Epothilone B prevents lipopolysaccharide-induced inflammatory osteolysis through suppressing osteoclastogenesis via STAT3 signaling pathway.

Inflammatory osteolysis is a common osteolytic specificity that occurs during infectious orthopaedic surgery and is characterized by an imbalance in bone homeostasis due to excessive osteoclast bone resorption activity. Epothilone B (Epo B) induced α-tubulin polymerization and enhanced microtubule stability, which also played an essential role in anti-inflammatory effect on the regulation of many diseases. However, its effects on skeletal system have rarely been investigated. Our study demonstrated that Epo B inhibited osteoclastogenesis in vitro and prevented inflammatory osteolysis in vivo. Further analysis showed that Epo B also markedly induced mature osteoclasts apoptosis during osteoclastogenesis. Mechanistically, Epo B directly suppressed osteoclastogenesis by the inhibitory regulation of the phosphorylation and activation of PI3K/Akt/STAT3 signaling directly, and the suppressive regulation of the CD9/gp130/STAT3 signaling pathway indirectly. The negative regulatory effect on STAT3 signaling further restrained the translocation of NF-κB p65 and NFATc1 from the cytosol to the nuclei during RANKL stimulation. Additionally, the expression of osteoclast specific genes was also significantly attenuated during osteoclast fusion and differentiation. Taken together, these findings illustrated that Epo B protected against LPS-induced bone destruction through inhibiting osteoclastogenesis via regulating the STAT3 dependent signaling pathway.

Comparison of post-traumatic changes in circulating and bone marrow leukocytes between BALB/c and CD-1 mouse strains.

This manuscript emerged from a larger third-party funded project investigating a new poly-trauma model and its influence upon secondary sepsis. The present sub-study compared selected leukocyte subpopulations in the circulation and bone marrow after polytrauma in BALB/c versus CD-1 mice. Animals underwent unilateral femur fracture, splenectomy and hemorrhagic shock. We collected blood and bone marrow for flow cytometry analysis at 24h and 48h post-trauma. Circulating granulocytes (Ly6G+CD11+) increased in both strains after trauma. Only in BALB/c mice circulating CD8+ T-lymphocytes decreased within 48h by 30%. Regulatory T-cells (Tregs, CD4+CD25+CD127low) increased in both strains by approx. 32%. Circulating Tregs and lymphocytes (CD11b-Ly6G-MHC-2+) were always at least 1.5-fold higher in BALB/c, while the bone marrow MHC-2 expression decreased in CD-1 mice (p<0.05). Overall, immune responses to polytrauma were similar in both strains. Additionally, BALB/c expressed higher level of circulating regulatory T-cells and MHC-2-positive lymphocytes compared to CD-1 mice.

Trauma Severity and Its Impact on Local Inflammation in Extremity Injury-Insights From a Combined Trauma Model in Pigs.

Background: Extremity fracture is frequently seen in multiple traumatized patients. Local post-traumatic inflammatory reactions as well as local and systemic interactions have been described in previous studies. However, trauma severity and its impact on the local immunologic reaction remains unclear. Therefore, fracture-associated local inflammation was investigated in a porcine model of isolated and combined trauma to gain information about the early inflammatory stages. Material and Methods: Polytrauma (PT) consisted of lung contusion, liver laceration, femur fracture, and controlled hemorrhage. Monotrauma (MT) consisted of femur fracture only. The fracture was operatively stabilized and animals were monitored under ICU-standard for 72 h. Blood, fracture hematoma (FH) as well as muscle samples were collected throughout the experimental period. Levels of local and systemic pro- and anti-inflammatory as well as angiogenetic cytokines were measured by ELISA. Results: Both groups showed a significant decrease in pro-inflammatory IL-6 in FH over time. However, concentrations in MT were significantly higher than in PT. The IL-8 concentrations initially decreased in FH, but recovered by the end of the observation period. These dynamics were only statistically significant in MT. Furthermore, concentrations measured in muscle tissue showed inverse kinetics compared to those in FH. The IL-10 did not present statistical resilient dynamics over time, although a slight increase in FH was seen by the end of the observation time in the MT group. Conclusions: Time-dependent dynamics of the local inflammatory response were observed. Trauma severity showed a significant impact, with lower values in pro- as well as angiogenetic mediators. Fracture repair could be altered by these trauma-related changes of the local immunologic milieu, which might serve as a possible explanation for the higher rates of delayed or non-union bone repair in polytraumatised patients.

Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronauts' health and safety. However, consequences on B-cell development and responses are still under-investigated. To fill this gap, we used advanced proteomics analysis of femur bone and marrow to compare mice flown for 1 mo on board the BION-M1 biosatellite, followed or not by 1 wk of recovery on Earth, to control mice kept on Earth. Our data revealed an adverse effect on B lymphopoiesis 1 wk after landing. This phenomenon was associated with a 41% reduction of B cells in the spleen. These reductions may contribute to explain increased susceptibility to infection even if our data suggest that flown animals can mount a humoral immune response. Future studies should investigate the quality/efficiency of produced antibodies and whether longer missions worsen these immune alterations.-Tascher, G., Gerbaix, M., Maes, P., Chazarin, B., Ghislin, S., Antropova, E., Vassilieva, G., Ouzren-Zarhloul, N., Gauquelin-Koch, G., Vico, L., Frippiat, J.-P., Bertile, F. Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

Induction of CD4+CD25+FOXP3+ regulatory T cells by mesenchymal stem cells is associated with modulation of ubiquitination factors and TSDR demethylation.

BACKGROUND: Mesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown. METHODS: We first investigated the role of cell-cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4+ T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4+ T cells collected from the spleens of protected mice. RESULTS: We found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4+ T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro. CONCLUSIONS: Our data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.

Efficacy of intraoperatively prepared cell-based constructs for bone regeneration.

BACKGROUND: Conventional cell-based bone regeneration suffers from the major disadvantage of limited cell supply, time-consuming in vitro expansion cultures, and limited patient-friendliness related to cell isolation and multiple visits to the clinic. Here, we utilized an alternative concept using "easy access cells" that can be obtained in an intraoperative manner to prepare cell-based constructs. METHODS: We used stromal vascular fraction (SVF) from human adipose tissue and human monocytes for intraoperative preparation of bone constructs. Conventional constructs grafted with expanded human adipose tissue mesenchymal stem cells (ADMSCs) derived from the same donor were set as positive controls. Additionally, we combined both cell types either or not with monocytes. The cellular interaction of human SVF and ADMSCs with human monocytes was evaluated in vitro. The feasibility and bone-regenerative capacity of intraoperative constructs were determined histologically and histomorphometrically in a rat femoral condyle bone defect model. RESULTS: SVF displayed equal in vitro osteogenic differentiation compared to donor-matched expanded ADMSCs, which for both was significantly enhanced upon co-culture with monocytes. Moreover, SVF and ADMSCs displayed different immunoregulatory effects on monocytes/macrophages. Upon implantation in rat femoral bone defects, SVF constructs demonstrated superior bone formation compared to ADMSC constructs and cell-free controls; no effects of monocyte addition were observed. CONCLUSION: In conclusion, we here demonstrate the feasibility of intraoperative SVF construct preparation and superior bone-regenerative capacity thereof compared to donor-matched ADMSC constructs. The superiority of SVF constructs was found to be linked to the distinct differences between immunoregulatory effects of SVF and ADMSCs.

Improved Osteogenesis by HVEM-Expressing Allogenic Bone Marrow-Derived Mesenchymal Stem Cells in an Immune Activation Condition and Mouse Femoral Defect Model.

Use of allogeneic mesenchymal stem cells (allo-MSCs) in bone tissue engineering strategies can overcome the limitations associated with autologous MSCs, but unfortunately, the immunogenicity of allo-MSCs leads to a high rate of rejection, unless immunosuppressive agents are used. B and T lymphocyte attenuator (BTLA) is a newly discovered immunoglobulin superfamily inhibitory receptor, and Herpesvirus-entry mediator (HVEM), a member of the tumor necrosis factor receptor family, is the only ligand of BTLA. Both BTLA and HVEM are widely expressed in B and T lymphocytes and other immune cells and play significant roles in the negative regulation of an immunoreaction. Therefore, we hypothesized that MSCs could be modified to maintain their bone differentiation ability through negative regulation of the immune response, and to test this hypothesis, we generated HVEM-expressing MSCs and tested their potential for osteogenic differentiation and bone repair in a simulated immune activation condition in vitro and in a mice femoral defect model. We found that osteogenic differentiation of allo-MSCs was decreased significantly in the activated immune microenvironment and that HVEM expression by allo-MSCs inhibited the immune response, resulting in improved osteogenic differentiation in vitro and new bone formation by allo-MSCs in a mouse femoral defect model. Our results also preliminarily suggested that the mechanism by which HVEM-expressing allo-MSCs overcome inflammation and enhance osteogenesis may be related to inhibition of interleukin-17. Overall, the data obtained in the present study provide support for the further development of HVEM-modified allo-MSCs as potentially ideal seed cells for bone tissue engineering applications.

Impairment of Bone Remodeling in LIGHT/TNFSF14-Deficient Mice.

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.

Decreased bone mineral density in experimental myasthenia gravis in C57BL/6 mice.

Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.

Orthopaedic wear particle-induced bone loss and exogenous macrophage infiltration is mitigated by local infusion of NF-κB decoy oligodeoxynucleotide.

Excessive production of wear particles from total joint replacements induces chronic inflammation, macrophage infiltration, and consequent bone loss (periprosthetic osteolysis). This inflammation and bone remodeling are critically regulated by the transcription factor NF-κB. We previously demonstrated that inhibition of NF-κB signaling by using the decoy oligodeoxynucleotide (ODN) mitigates polyethylene wear particle-induced bone loss using in vitro and in vivo models. However, the mechanisms of NF-κB decoy ODN action, and in particular its impact on systemic macrophage recruitment, remain unknown. In the current study, this systemic macrophage infiltration was examined in our established murine femoral continuous particle infusion model. RAW264.7 murine macrophages expressing a luciferase reporter gene were injected into the systemic circulation. Quantification of bioluminescence showed that NF-κB decoy ODN reduced the homing of these reporter macrophages into the distal femurs exposed to continuous particle delivery. Particle-induced reduction in bone mineral density at the distal diaphysis of the femur was also mitigated by infusion of decoy ODN. Histological staining showed that the decoy ODN infusion decreased osteoclast and macrophage numbers, but had no significant effects on osteoblasts. Local infusion of NF-κB decoy ODN reduced systemic macrophage infiltration and mitigated particle-induced bone loss, thus providing a potential strategy to treat periprosthetic osteolysis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3169-3175, 2017.

NLR2 and TLR3, TLR4, TLR5 Ligands, Injected In Vivo, Improve after 1 h the Efficiency of Cloning and Proliferative Activity of Bone Marrow Multipotent Stromal Cells and Reduce the Content of Osteogenic Multipotent Stromal Cells in CBA Mice.

Ligands NLR2 (muramyldipeptide) and TLR (bacterial LPS, flagellin, CpG-dinucleotide, and Poly I:C) and S. typhimurium antigenic complex by 1.5-3-fold increase the efficiency of cloning and content of multipotent stromal cells (MSC) in the bone marrow of CBA mice as soon as 1 h postinjection. The counts of large colonies (150-500 cells) increased by 2.5-3.3 times in comparison with intact bone marrow cultures at the expense of a decrease in the number of smaller colonies, which attests to enhanced proliferation of stromal cells in the colonies. The efficiency of cloning and hence, MSC content in the femoral bone decreased by 1.2-1.9 times after 3 h and increased again after 24 h to the level 1.3-1.5 times higher than the level 1 h postinjection (LPS, Poly I:C, and S. typhimurium antigenic complex). The dynamics of bone marrow MSC cloning efficiency after 1-3 h corresponded to the dynamics of serum cytokine concentrations during the same period. However, the levels of serum cytokines after 24 h in general were similar to those in intact mice or were lower. The concentrations of osteogenic multipotent stromal cells in the bone marrow decreased 2-3-fold after 3 h and thus persisted by 24 h postinjection. Twofold (at 24-h interval) and a single injection of S. typhimurium antigenic complex to mice led to a significant increase of cloning efficiency, observed as early as just 1 h postinjection (1.9 and 1.5 times, respectively). The same picture was observed for serum cytokines. On the whole, injections of TLR and NLR ligands and of S. typhimurium antigenic complex led to stromal tissue activation within 1 h postinjection, this activation consisting in a significant increase of the efficiency of cloning and of MSC count in the bone marrow, and also in an increase in their proliferative activity and a decrease (after 3 h) of osteogenic MSC concentration.

Counts of MSC in the Bone Marrow of Young and Old CBA Mice after a Single Exposure to Osteogenic Stimuli (Curettage, BMP-2 Injection) or Antigens (S. typhimurium Antigenic Complex) and in Heterotopic Bone Marrow Transplants.

The efficiency of cloning and the content of multipotent stromal cells (MSC) in the femoral bone marrow of intact CBA mice was 1.5 times less in old mice (24-36 months) than in young ones (2-3 months). The concentration of osteogenic MSC was higher in old vs. young mice (42±3 vs. 22±2%, respectively). Changes in the total counts of MSC and concentrations of osteogenic MSC in response to osteogenic (curettage, BMP-2) and immunogenic stimuli (S. typhimurium antigenic complex) were similar in young and old mice in comparison with intact controls of respective age. The counts of the total pool of bone marrow MSC and pool of osteogenic MSC in response to osteogenic stimuli were 1.5-2 times less in old vs. young mice. This difference seemed to be a result of age-specific decrease of their bone marrow count but not of age-specific decrease of the MSC functional activity, this leading to a decrease in the transplantability of bone marrow stromal tissue of old mice. Comparison of transplantations "old donor - young recipient" vs. "young donor - young recipient" demonstrated a decrease in the count of nuclear cells (1.8 times), size of bone capsule (2-fold), efficiency of MSC cloning (1.6 times), count of MSC per transplant (2.9 times), and count of osteogenic MSC per transplant (3.3 times). The concentrations of osteogenic MSC in transplants from young and old donors leveled in young recipients, that is, seemed to be regulated by the host. Serum concentrations of IL-10 and TNF-α in intact old mice were at least 2.9 and 2 times higher than in young animals, while the concentrations of almost all the rest studied cytokines (IL-2, IL-5, GM-CSF, IFN-γ, IL-4, IL-12) were lower. Presumably, the decrease in the content of bone marrow MSC and in transplantability of bone marrow stromal tissue in old mice were caused by exhaustion of the MSC pool as a result of age-specific chronic inflammation. These data indicated a close relationship between age-specific changes in the stromal tissue and immune system.

Time Series miRNA-mRNA integrated analysis reveals critical miRNAs and targets in macrophage polarization.

Polarization of macrophages is regulated through complex signaling networks. Correlating miRNA and mRNA expression over time after macrophage polarization has not yet been investigated. We used paired RNA-Seq and miRNA-Seq experiments to measure the mRNA and miRNA expression in bone marrow-derived macrophages over a time-series of 8 hours. Bioinformatics analysis identified 31 differentially expressed miRNAs between M1 and M2 polarized macrophages. The top 4 M1 miRNAs (miR-155-3p, miR-155-5p, miR-147-3p and miR-9-5p) and top 4 M2 miRNAs (miR-27a-5p, let-7c-1-3p, miR-23a-5p and miR-23b-5p) were validated by qPCR. Interestingly, M1 specific miRNAs could be categorized to early- and late-response groups, in which three new miRNAs miR-1931, miR-3473e and miR-5128 were validated as early-response miRNAs. M1 polarization led to the enrichment of genes involved in immune responses and signal transduction, whereas M2 polarization enriched genes involved in cell cycle and metabolic processes. C2H2 zinc-finger family members are key targets of DE miRNAs. The integrative analysis between miRNAs and mRNAs demonstrates the regulations of miRNAs on nearly four thousand differentially expressed genes and most of the biological pathways enriched in macrophage polarization. In summary, this study elucidates the expression profiles of miRNAs and their potential targetomes during macrophage polarization.

Continuous single cell imaging reveals sequential steps of plasmacytoid dendritic cell development from common dendritic cell progenitors.

Functionally distinct plasmacytoid and conventional dendritic cells (pDC and cDC) shape innate and adaptive immunity. They are derived from common dendritic cell progenitors (CDPs) in the murine bone marrow, which give rise to CD11c+ MHCII- precursors with early commitment to DC subpopulations. In this study, we dissect pDC development from CDP into an ordered sequence of differentiation events by monitoring the expression of CD11c, MHC class II, Siglec H and CCR9 in CDP cultures by continuous single cell imaging and tracking. Analysis of CDP genealogies revealed a stepwise differentiation of CDPs into pDCs in a part of the CDP colonies. This developmental pathway involved an early CD11c+ SiglecH- pre-DC stage and a Siglec H+ CCR9low precursor stage, which was followed rapidly by upregulation of CCR9 indicating final pDC differentiation. In the majority of the remaining CDP pedigrees however the Siglec H+ CCR9low precursor state was maintained for several generations. Thus, although a fraction of CDPs transits through precursor stages rapidly to give rise to a first wave of pDCs, the majority of CDP progeny differentiate more slowly and give rise to longer lived precursor cells which are poised to differentiate on demand.

Experimental Simulation of Traumatic Osteomyelitis in Rats.

A model of traumatic osteomyelitis in rats is created. The disease development was evaluated by bone marrow myelograms, results of scintigraphy, and histological studies.

Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice.

ApoE-null (ApoE-KO) mice fed a high-fat diet (HFD) develop atherosclerosis, due in part to activation of vascular inflammation by oxidized low-density lipoprotein. Since bone loss also occurs in these mice, we used them to investigate the impact of oxidized lipids on bone homeostasis and to search for underlying pathogenic pathways. Four-month-old female ApoE-KO mice fed a HFD for three months exhibited increased levels of oxidized lipids in bone, as well as decreased femoral and vertebral trabecular and cortical bone mass, compared with ApoE-KO mice on normal diet. Despite HFD-induced increase in expression of Alox15, a lipoxygenase that oxidizes LDL and promotes atherogenesis, global deletion of this gene failed to ameliorate the skeletal impact of HFD. Osteoblast number and function were dramatically reduced in trabecular and cortical bone of HFD-fed mice, whereas osteoclast number was modestly reduced only in trabecular bone, indicating that an imbalance in favor of osteoclasts was responsible for HFD-induced bone loss. These changes were associated with decreased osteoblast progenitors and increased monocyte/macrophages in the bone marrow as well as increased expression of IL-1ß, IL-6, and TNF. HFD also attenuated Wnt signaling as evidenced by reduced expression of Wnt target genes, and it decreased expression of pro-osteoblastogenic Wnt ligands. These results suggest that oxidized lipids decrease bone mass by increasing anti-osteoblastogenic inflammatory cytokines and decreasing pro-osteoblastogenic Wnt ligands.