Murine Bone Marrow Macrophages Research Articles

Intact O-antigen is critical structure for the exceptional tubular shape of outer membrane vesicles in Francisella tularensis

Francisella tularensis is a highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20–250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host–pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs.

Host P2X7R-p38MAPK axis mediated intra-macrophage leishmanicidal activity of Spergulin-A

Current drugs are inefficient for the treatment of visceral leishmaniasis an immunosuppressive ailment caused by Leishmania donovani. Regrettably, there is no plant-origin antileishmanial drug present. P2X7R is constitutively present on macrophage surfaces and can be a putative therapeutic target in intra-macrophage pathogens with function attributes towards inflammation, host cell apoptosis, altered redox, and phagolysosomal maturation by activating p38MAPK. Here we demonstrated that the initial interaction of Spergulin-A (Sp A), a triterpenoid saponin with RAW 264.7 macrophages was mediated through P2X7R involving the signaling cascade intermediates Ca++, p38MAPK, and NF-κβ. Phospho (P)-p38MAPK involvement is shown to have specific and firm importance in leishmanial killing with increased NF-κβp65. Phago-lysosomal maturation by Sp A also campaigns for another contribution of P2X7R. In vivo evaluation of the anti-leishmanial activity of Sp A was monitored through expression analyses of P2X7R, P-p38MAPK, and NF-κβp65 in murine spleen and bone-marrow macrophages and supported Sp A being a natural compound of leishmanicidal functions which acted through the P2X7R-p38MAPK axis.

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function.

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.

HIF-1α activator DMOG inhibits alveolar bone resorption in murine periodontitis by regulating macrophage polarization

Periodontitis is initiated by serious and sustained bacterial infection and ultimately results in chronic immune-mediated inflammation, tissue destruction, and bone loss. The pathogenesis of periodontitis remains unclear. Host immunological responses to periodontal bacteria ultimately determine the severity and mechanisms governing periodontitis progression. This study aimed to clarify the effect of the hypoxia-inducible factor-1α (HIF-1α) activator dimethyloxalylglycine (DMOG) on a mouse periodontitis model and its underlying role in macrophage polarization. qRT-PCR analysis showed that DMOG inhibited the M1-like polarization of both RAW264.7 macrophages and murine bone marrow macrophages (BMMs) and downregulated TNF-α, IL-6, CD86, and MCP-1 expression in vitro. Immunofluorescence staining and flow cytometry also confirmed the less percentage of F4/80 + CD86 + cells after DMOG treatment. The phosphorylation of NF-κB pathway was also inhibited by DMOG with higher level of HIF-1α expression. Furthermore, mice treated with DMOG showed decreased alveolar bone resorption in the experimental periodontitis model, with significant increases in alveolar bone volume/tissue volume (BV/TV) and bone mineral density (BMD). DMOG treatment of mice decreased the ratio of M1/M2 (CD86+/CD206+) macrophages in periodontal tissues, resulting in the downregulation of proinflammatory cytokines such as TNF-α and IL-6 and increased levels of anti-inflammatory factors such as IL-4 and IL-10. DMOG treatment promoted the number of HIF-1α-positive cells in periodontal tissues. This study demonstrated the cell-specific roles of DMOG in macrophage polarization in vitro and provided insight into the mechanism underlying the protective effect of DMOG in a model of periodontitis.

Biological Evaluation and Transcriptomic Analysis of Corylin as an Inhibitor of Osteoclast Differentiation.

Corylin, a flavonoid isolated from the fruit of Psoralea corylifolia, has an osteogenic effect on osteoblasts in vitro and bone micromass ex vivo. However, the effect and mechanism of corylin in regulating osteoclastogenesis remain unknown. By using murine bone marrow macrophages as the osteoclast precursor, corylin was found to inhibit the receptor activator of nuclear factor (NF) κB ligand (RANKL)-induced osteoclast differentiation via down-regulating osteoclastic marker genes. In parallel, F-actin formation and osteoclast migration were diminished in corylin-treated cultured osteoclasts, and subsequently the expressions of osteoclastic proteins were suppressed: the suppression of protein expression was further illustrated by transcriptomic analysis. Furthermore, corylin inhibited the nuclear translocation of p65, giving rise to a restraint in osteoclastic differentiation through the attenuation of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells c1 (NFATc1). There was no obvious change in apoptosis when the RANKL-induce osteoclasts were cultured in the presence of corylin. The finding supports the potential development of corylin as an osteoclast inhibitor against osteoporosis.

The downregulation of IGFBP3 by TGF-β signaling in oral cancer contributes to the osteoclast differentiation

Oral squamous cell carcinoma (OSCC) frequently invades nearby bone and bone involvement determines the prognosis of patients. Growth factors, stored in the bone matrix and released during bone destruction, are known as key components in the bone-tumor interaction. However, the coordination of growth factor signals and the precise mechanism of bone destruction in oral cancer are still unclear. In the study, we investigated the differential cytokine expression profile of oral cancer cells by TGF-β treatment and the function of altered expression of cytokines on the osteoclast differentiation. We established TGFBR2-knockdown cells using small hairpin RNA. TGF-β was treated to both TGFBR2 expressing and knockdown cells and the culture supernatants were analyzed using a cytokine array kit. We found that the TGF-β inhibited IGFBP3 level and enhanced MMP9 level. We confirmed this regulation of IGFBP3 and MMP9 by TGF-β using ELISA and zymography, respectively. IGFBP3 is known as to modulate the bioavailability of IGF1, which is abundant in the bone microenvironment and regulates osteoclast differentiation. Therefore, we further analyzed the function of IGFBP3 on osteoclastogenesis. Although IGFBP3 increased the viability of murine bone marrow macrophages, the osteoclast differentiation of these cells was blocked by IGFBP3 in a dose-dependent manner. These results revealed a novel pathway for the regulation of osteoclastogenesis by oral cancer cells, which may be a new therapeutic target for osteolysis induced by oral cancer infiltrating into the bone.

TGF-β in the Secretome of Irradiated Peripheral Blood Mononuclear Cells Supports In Vitro Osteoclastogenesis.

Osteoclastogenesis required for bone remodeling is also a key pathologic mechanism of inflammatory osteolysis being controlled by paracrine factors released from dying cells. The secretome of irradiated, dying peripheral blood mononuclear cells (PBMCs) has a major impact on the differentiation of myeloid cells into dendritic cells, and macrophage polarization. The impact on osteoclastogenesis, however, has not been reported. For this aim, we used murine bone marrow macrophages exposed to RANKL and M-CSF to initiate osteoclastogenesis, with and without the secretome obtained from γ-irradiated PBMCs. We reported that the secretome significantly enhanced in vitro osteoclastogenesis as determined by means of histochemical staining of the tartrate-resistant acid phosphatase (TRAP), as well as the expression of the respective target genes, including TRAP and cathepsin K. Considering that TGF-β enhanced osteoclastogenesis, we confirmed the TGF-β activity in the secretome with a bioassay that was based on the increased expression of IL11 in fibroblasts. Neutralizing TGF-β by an antibody decreased the ability of the secretome to support osteoclastogenesis. These findings suggested that TGF-β released by irradiated PBMCs could enhance the process of osteoclastogenesis in vitro.

Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro.

Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1β and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.

Metformin directly suppresses atherosclerosis in normoglycaemic mice via haematopoietic adenosine monophosphate-activated protein kinase.

AimsAtherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis.Methods and resultsNormoglycaemic Ldlr−/− hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10−11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 μM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects.ConclusionMetformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK.

Hypoallergenic infant formula lacks transforming growth factor beta activity and has a lower anti-inflammatory activity than regular infant formula

Hypoallergenic formulas are recommended for infants who are not breastfed and cannot tolerate cow milk formulas due to allergy. These formulas are hydrolyzed to break down larger protein chains into shorter, easy-to-digest, and potentially less allergenic proteins. Hydrolysis, however, possibly occurs at the expense of the transforming growth factor beta (TGF-β) and anti-inflammatory activity that is inherent in regular formula. Our objective was to determine the TGF-β and the anti-inflammatory activity of commercially available hypoallergenic and regular formulas. Human gingival fibroblasts were incubated with reconstituted formulas followed by detection of TGF-β target genes and activation of Smad2/3 signaling. Gingival fibroblasts and the oral squamous cell carcinoma cell line HSC-2 were also exposed to formulas before adding interleukin (IL)1β and tumor necrosis factor (TNF)α to provoke expression of pro-inflammatory cytokines. For murine bone marrow-derived macrophages, pro-inflammatory cytokine expression was stimulated with saliva. Changes in p65 nuclear translocation and phosphorylation of smad3 and p38 were analyzed by immunostaining. Our study demonstrated that regular formula, but not hypoallergenic formula, enhanced the expression of TGF-β target genes IL11, PRG4, and NOX4 in gingival fibroblasts. Hypoallergenic formulas also failed to initiate nuclear translocation of Smad2/3 and phosphorylation of Smad3. Moreover, regular formulas were more potent than hypoallergenic formulas in reducing the expression of pro-inflammatory cytokines in gingival fibroblasts, HSC-2 epithelial cells, and murine bone marrow macrophages. Hypoallergenic and regular formulas had a similar capacity to reduce p65 nuclear translocation and phosphorylation of p38 in fibroblasts. These findings suggest that hypoallergenic formulas lack in vitro TGF-β activity and have a lower anti-inflammatory activity compared with regular formulas.

Thrombin fine tunes innate immune cell function

Abstract Thrombin is the main effector protease in the coagulation cascade. It can also signal via protease activating receptors (PAR). The presence of these receptors on the surface of innate immune cells has been well reported but the functional consequence of activation has yet to be fully defined. This study aims to investigate the role thrombin has on innate immune cell function using murine bone marrow macrophages (BMM). In Vitro, stimulating mature MCSF-cultured BMM with thrombin did not affect gross markers of macrophage polarisation (iNOS or CD206). The stimulated cell supernatants contained increased amounts of Interferon gamma (IFNγ) but reduced IL10. Thrombin increased IFNγ receptor expression at the cell surface. Thrombin-treated cells had increased lipid rich microdomains by Cholera Toxin B staining and increased co-localisation of the LPS receptor within the lipid rafts. Compared to untreated cells, thrombin stimulated cells were highly sensitive to low dose M1 polarising stimuli, as evidenced by iNOS expression. The thrombin treated cells down regulated surface ABCA1 expression, but this was prevented by transfecting cells with siRNA against Cullin 3. This preserved ABCA1 expression and prevented the increase in lipid rich microdomains after thrombin stimulation and was associated with the loss of heightened sensitivity to low dose M1 stimuli. Taken together this shows a clear pro inflammatory signal on the thrombin treated cells and to date the first description of ABCA1’s key role in thrombin mediated inflammatory signalling.

Bacterial RNA Contributes to the Down-Modulation of MHC-II Expression on Monocytes/Macrophages Diminishing CD4+ T Cell Responses.

Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.

Breast cancer cell-derived exosomal miR-20a-5p promotes the proliferation and differentiation of osteoclasts by targeting SRCIN1.

Bone metastasis of breast cancer makes patients suffer from pain, fractures, spinal cord compression, and hypercalcemia, and is almost incurable. Although the mechanisms of bone metastasis in breast cancers have been studied intensively, novel specific target will be helpful to the development of new therapeutic strategy of breast cancer. Herein, we focused on the microRNA of tumor cell‐derived exosomes to investigate the communication between the bone microenvironment and tumor cells. The expression of miR‐20a‐5p in the primary murine bone marrow macrophages (BMMs), MCF‐10A, MCF‐7, and MDA‐MB‐231 cell lines, as well as the cell‐derived exosomes were assessed by qRT‐PCR. Transwell assays were used to evaluate the effects of miR‐20a‐5p on tumor cell migration and invasion. The expression of exosomes marker including CD63and TSG101 was detected by Western Blot. Cell cycle distribution of BMMs was analyzed by flow cytometry. 3‐UTR luciferase reporter assays were used to validate the putative binding between miR‐20a‐5p and SRCIN1. MiR‐20a‐5p was highly expressed in breast tumor tissues and the exosomes of MDA‐MB‐231 cells. MiR‐20a‐5p promoted migration and invasion in MDA‐MB‐231 cells, and the proliferation and differentiation of osteoclasts. MDA‐MB‐231 cell‐derived exosomes transferred miR‐20a‐5p to BMMs and facilitated the osteoclastogenesis via targeting SRCIN1. The present work provides evidence that miR‐20a‐5p transferred from breast cancer cell‐derived exosomes promotes the proliferation and differentiation of osteoclasts by targeting SRCIN1, providing scientific foundations for the development of exosome or miR‐20a‐5p targeted therapeutic intervention in breast cancer progression.

Abstract 3005: High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid cells

Abstract Introduction: Exosomes are 30-150 nm membrane-bound vesicles that mediate intercellular communication and are secreted in abundance by neoplastic cells. Tumor-derived exosomes are capable of transmitting pro-malignant signals to target cells at local or distant sites to enhance tumor invasiveness and promote metastasis. A sub-set of this pro-malignant intercellular communication is dependent upon target cell cholesterol homeostasis. Here, we show that prostate cancer exosome communication with myeloid cells can be inhibited by treatment with high density lipoprotein mimetic nanoparticles (HDL NPs), which reduce cholesterol in myeloid cells in a targeted fashion. Methods: Exosomes were isolated by ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells and used for all experiments. In vitro assays were conducted using murine bone marrow macrophages obtained from culture of total murine bone marrow in M-CSF for 7 days. Confocal microscopy and flow cytometry were used for in vitro and in vivo uptake assays. Osteoclastogenesis assays were performed using a commercially available TRAP staining kit (Sigma-Aldrich). NF-kB signaling experiments were performed using a reporter human monocyte cell line (THP1-Dual). HDL NPs were synthesized using 5 nm gold nanoparticle templates, apolipoprotein A-1, and phospholipids. Results: HDL NPs were found to inhibit the cellular uptake of prostate cancer exosomes in mouse bone marrow macrophages in vitro. Furthermore, HDL NPs inhibited exosome-induced osteoclastogenesis and exosome-induced monocyte NF-kB signaling. Finally, HDL NP-mediated inhibition of exosome communication was found to be dependent upon scavenger receptor type B-1 (SR-B1). SR-B1 was shown to be expressed ubiquitously in mouse bone marrow macrophages; and HDL NPs were unable to inhibit exosome communication in bone marrow macrophages derived from SR-B1-/- mouse bone marrow. Conclusion: These data demonstrate that HDL NPs inhibit prostate cancer exosome communication with murine myeloid cells, as evidenced by cellular uptake, osteoclastogenesis, and NF-kB signaling. Moreover, SR-B1 is required for HDL NP inhibition of PCa exosome communication. In sum, these results indicate that target cell cholesterol homeostasis may be important for exosome-mediated signaling in prostate cancer, particularly in immune cells and in the bone microenvironment, and that HDL NPs are potent inhibitors of PCa exosome-mediated signaling. Citation Format: Stephen E. Henrich, Kaylin M. McMahon, Michael P. Plebanek, C. Shad Thaxton. High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3005.

Milk modulates macrophage polarization in vitro

ObjectiveMilk holds an anti-inflammatory response that is particularly important to protecting infants against necrotizing enterocolitis. Milk might also exert anti-inflammatory effects in adulthood, including the oral cavity where macrophages of the oral mucosal control innate immunity defense. It remains unknown, however, whether milk can modulate the local inflammatory response by affecting the polarization of macrophages. Material and MethodsTo determine whether pasteurized human milk and pasteurized cow milk can provoke macrophage polarization, murine bone marrow macrophages and RAW264.7 cells were exposed to human saliva or the inflammatory cytokines IL1β and TNFα. Activation of pro-(M1) inflammatory response is indicated by the expression of IL1 and IL8. To determine polarization towards a M2 phenotype, the expression of arginase 1 (ARG1) and chitinase-like 3 (Chil3) was determined by reverse transcriptase PCR and immunoassay. Western blot was done on phosphorylated p38 and JNK. ResultsAqueous fractions of human milk and cow milk from different donors, respectively, significantly decreased the inflammatory response of primary macrophages and RAW264.7 cells when exposed to saliva or IL1 and TNFα. Similar to IL4, human milk and cow milk caused a robust expression of ARG1 and Chil3 in primary macrophages. The polarization of macrophages by pasteurized milk occurred independent of the phosphorylation of p38 and JNK. ConclusionThese data suggest that pasteurized milk, independent of the origin, can cause the polarization of macrophages from a pro-inflammatory M1 towards a pro-resolving M2 phenotype. Thus, milk might have a protective role for the oral cavity by modulation of the macrophage-based innate immune system.

SAT-558 Tsh Modulation Of Bone Biology - Further Evidence From A Recombinant Tsh-β Variant.

We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919-26, 2013 and Endocrinology 157:3658-67, 2016). Furthermore, we have also shown by molecular modelling that both TSH-β and TSH-βv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH-β variant we have inserted the gene sequence together with a FLAG tag into the P-secTag2 vector which was then cloned into mammalian HEK-293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH-β and the TSH-βv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH-β polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH-β and TSH-βv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8-fold increase in luciferase activity with TSH-β and a 4-fold increase with TSH-βv. These data demonstrate the inherent bioactivity of single TSH-β chains which was mirrored by TSH-βv. Local paracrine action by macrophage derived TSH-βv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.

(-)-Epigallocatechin-3-Gallate Decreases Osteoclastogenesis via Modulation of RANKL and Osteoprotegrin.

Osteoporosis is the second most common epidemiologic disease in the aging population worldwide. Previous studies have found that frequent tea drinkers have higher bone mineral density and less hip fracture. We previously found that (−)-epigallocatechin gallate (EGCG) (20–100 µmol/L) significantly suppressed receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis and pit formation via inhibiting NF-κB transcriptional activity and nuclear transport of NF-κB in RAW 264.7 cells and murine primary bone marrow macrophage cells. The most important regulation in osteoclastogenesis is the receptor activator of nuclear factor-kB/RANKL/osteoprotegrin (RANK/RANKL/OPG) pathway. In this study, we used the coculture of RAW 264.7 cells and the feeder cells, ST2, to evaluate how EGCG regulated the RANK/RANKL/OPG pathway in RAW 264.7 cells and ST2 cells. We found EGCG decreased the RANKL/OPG ratio in both mRNA expression and secretory protein levels and eventually decreased osteoclastogenesis by TRAP (+) stain osteoclasts and TRAP activity at low concentrations—1 and 10 µmol/L—via the RANK/RANKL/OPG pathway. The effective concentration can be easily achieved in daily tea consumption. Taken together, our results implicate that EGCG could be an important nutrient in modulating bone resorption.

Effects of retinoids on physiologic and inflammatory osteoclastogenesis in vitro.

Increased intake of vitamin A (retinoids) is associated with decreased bone mass and increased fracture risk in humans. Mechanistic studies in rodents have shown that hypervitaminosis A results in decreased bone mass caused by an increase in periosteal osteoclasts while simultaneously decreasing endocortic osteoclasts. In vivo and ex vivo bone organ cultures have demonstrated that excess retinoids increase osteoclast formation due to increased receptor activator of nuclear factor kappa B-ligand (RANKL) expression. In vitro, studies using murine bone marrow macrophages (BMM) have shown that retinoids inhibit osteoclast formation induced by recombinant RANKL. These opposing in vivo/ex vivo versus in vitro effects may elucidate why excess retinoids affect periosteal and endocortic osteoclast formation differently. In addition, it has been reported that retinoids can inhibit osteoclast formation under inflammatory conditions such as experimentally induced arthritis in mice. In the present study, we have compared the effect of all-trans-retinoic acid (ATRA) on physiologically and inflammatory induced osteoclastogenesis. ATRA inhibited physiologically induced (RANKL) osteoclast formation of human peripheral blood monocytes and mouse BMM as well as human monocytes stimulated with the pro-inflammatory compounds, TNF-α and LPS. The inhibition was due to impeded differentiation, rather than fusion, of mononucleated progenitor cells. ATRA disrupted differentiation by interfering with osteoclastogenic intracellular signaling. In line with this view, overexpression of Tnfrsf11a (encodes for RANK) in BMM could not overcome the inhibition of osteoclastogenesis by ATRA. The data suggest that ATRA inhibits both physiologic and inflammatory osteoclast differentiation of progenitors from the bone marrow and peripheral blood.

Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics.

ABSTRACTOsteoclasts are bone‐resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M‐CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts in the presence of RANKL, but M‐CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high‐throughput proteomics strategy, we quantified the global protein changes in osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL‐dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M‐CSF were significantly different, including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M‐CSF‐dependent signaling and functions. © 2018 American Society for Bone and Mineral Research. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Development of a Janus Kinase Inhibitor Prodrug for the Treatment of Rheumatoid Arthritis.

While highly efficacious in treating rheumatoid arthritis (RA), the approved Janus kinase (JAK) inhibitor, Tofacitinib (Tofa, CP-690 550), has dose-dependent toxicities that limit its clinical application. In this study, we have examined whether a prodrug design that targets arthritic joints would enhance Tofa's therapeutic efficacy, which may provide an opportunity for future development of safer Tofa dosing regimens. A prodrug of Tofa (P-Tofa) was synthesized by conjugating the drug to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an acid cleavable carbamate linker. The therapeutic efficacy of a single dose of P-Tofa was compared to the dose-equivalent daily oral administration of Tofa in an adjuvant-induced arthritis (AA) rat model. Saline treated AA rats and age-matched healthy rats were used as controls. Observational analyses support the superior and sustained efficacy of a single dose P-Tofa treatment compared to the dose-equivalent daily Tofa administration in ameliorating joint inflammation. Micro-CT and histological analyses demonstrated that the P-Tofa treatment provided a structural preservation of the joints better than that of the dose-equivalent Tofa. Optical imaging, immunohistochemistry, and fluorescence-activated cell sorting analyses attribute P-Tofa's superior therapeutic efficacy to its passive targeting to arthritic joints and inflammatory cell-mediated sequestration. In vitro cell culture studies reveal that the P-Tofa treatment produced sustained the inhibition of JAK/STAT6 signaling in IL-4-treated murine bone marrow macrophages, consistent with a gradual subcellular release of Tofa. Collectively, a HPMA-based nanoscale prodrug of P-Tofa has the potential to enhance the therapeutic efficacy and widen the therapeutic window of Tofa therapy in RA.