Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid

We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-alpha mRNA expression and tumor necrosis factor-alpha production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-alpha compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-alpha and IL-1alpha mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products significantly suppressed tumor necrosis factor-alpha gene expression and tumor necrosis factor-alpha production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-alpha inhibitory products to evade the host's immune system, and that tumor necrosis factor-alpha mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.     

Endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the IL-2/IL-4 receptor gamma chain

In vitro monocyte-derived macrophages (MDMac) and synovial fluid macrophages from inflamed joints differ from monocytes in their responses to interleukin 4 (IL-4). While IL-4 can suppress LPS-induced interleukin beta (IL-beta) and tumour necrosis factor alpha (TNF-alpha) production by monocytes, IL-4 can suppress LPS-induced IL-1 beta, but not TNFalpha production by the more differentiated cells. Recently we reported a correlation between the ability of IL-4 to regulate TNFalpha production by monocytes and the expression of the IL-4 receptor gamma chain or gamma common (gamma c chain). Like MDMac, interferon alpha (IFNalpha)-treated monocytes expressed less IL-4 receptor gamma c chain, reduced levels of IL-4-activated STAT6 and IL-4 could not suppress LPS-induced TNFalpha production. In addition, like monocytes and MDMac, IFNalpha-treated monocytes expressed normal levels of the IL-4 receptor alpha chain and IL-4 significantly suppressed LPS-induced IL-1 beta production. With addition of IFNalpha-neutralizing antibodies, the ability of IL-4 to suppress LPS-induced TNFalpha production with prolonged monocyte culture was restored. Detection of IFNalpha in synovial fluids from inflamed joints further implicates IFNalpha in the inability of IL-4 to suppress TNFalpha production by synovial fluid macrophages. This study identifies a mechanism for the differential expression of gamma c and varied responses to IL-4 by human monocytes compared with MDMac.     

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1β acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.     

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.     

Suberoylanilide hydroxamic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing nuclear factor-kappaB activation

Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials. How SAHA mediates its effects is poorly understood. We found that in several human cancer cell lines, SAHA potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents and inhibited TNF-induced invasion and receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of anti-apoptotic (IAP1, IAP2, X chromosome-linked IAP, Bcl-2, Bcl-x(L), TRAF1, FLIP, and survivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc), and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascular endothelial growth factor) gene products. Because several of these genes are regulated by NF-kappaB, we postulated that SAHA mediates its effects by modulating NF-kappaB and found that SAHA suppressed NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, lipopolysaccharide, H(2)O(2), phorbol myristate acetate, and cigarette smoke; the suppression was not cell type-specific because both inducible and constitutive NF-kappaB activation was inhibited. We also found that SAHA had no effect on direct binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. Furthermore, SAHA inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and the p65 subunit of NF-kappaB. Overall, our results indicated that NF-kappaB and NF-kappaB-regulated gene expression inhibited by SAHA can enhance apoptosis and inhibit invasion and osteoclastogenesis.     

Stat3-dependent induction of interleukin-3 receptor expression in leukemia inhibitory factor-stimulated M1 mouse leukemia cells

M1 mouse leukemia cells differentiate to macrophages/monocytes by the stimulation of interleukin-6 (IL-6)/leukemia inhibitory factor (LIF). To identify new LIF-induced genes, we have performed representational difference analysis using M1 cells and cloned mouse interleukin-3 (IL-3) receptor beta subunit gene. The mRNA expression of both IL-3 receptor (IL-3R) alpha and beta subunits is upregulated after 1 h stimulation of LIF and remains to be elevated along the differentiation of M1 cells. This induction is almost completely suppressed in M1 cells expressing a dominant negative form of Stat3. Furthermore, we show that IL-3-induced Stat5 phosphorylation increases in LIF-stimulated M1 cells. These results suggest that Stat3 may play a role in the differentiation of myeloid cells by regulating IL-3R expression.     

MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.     

Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKL‐induced osteoclastogenesis

Diabetes results in increased fracture risk, and advance glycation endproducts (AGEs) have been implicated in this pathophysiology. S100 proteins are ligands for the receptor of AGEs (RAGE). An intracellular role of the S100 family member S100A4 (Mts1) to suppress mineralization has been described in pre‐osteoblastic MC3T3‐E1 cells. However, S100 proteins could have additional effects on bone. The goal of the current study was to determine effects of increased extracellular S100 on osteoclastogenesis. We first determined the direct effects of S100 on pre‐osteoclast proliferation and osteoclastic differentiation. RANKL‐treated RAW 264.7 cell proliferation and TRAP activity were significantly inhibited by S100, and the number and size of TRAP‐positive multinucleated cells were decreased. We then determined whether S100 could affect osteoclastogenesis by an indirect process by examining effects of conditioned media from S100‐treated MC3T3‐E1 cells on osteoclastogenesis. In contrast to the direct inhibitory effect of S100, the conditioned media promoted RAW 264.7 cell proliferation and TRAP activity, with a trend toward increased TRAP‐positive multinucleated cells. S100 treatment of the MC3T3‐E1 cells for 14 days did not significantly affect alkaline phosphatase, M‐CSF, or OPG gene expression. RANKL was undetectable in both untreated and treated cells. The treatment slightly decreased MC3T3‐E1 cell proliferation. Interestingly, S100 treatment increased expression of RAGE by the MC3T3‐E1 cells. This suggested the possibility that S100 could increase soluble RAGE, which acts as a decoy receptor for S100. This decrease in availability of S100, an inhibitor of pre‐osteoclast proliferation, could contribute to osteoclastogenesis, ultimately resulting in increased bone resorption. J. Cell. Biochem. 107: 917–925, 2009. © 2009 Wiley‐Liss, Inc.