Signaling Pathways In Monocytes Research Articles

Human amniotic mesenchymal stem cell-conditioned medium inhibited LPS-induced cell injury and inflammation by modulating CD14/TLR4-mediated signaling pathway in monocytes

Human amniotic mesenchymal stem cells (hAMSCs) have attracted considerable attention as a promising regenerative therapy. Many studies reported that the conditioned medium of hAMSCs (AM-CM) exerted anti-inflammatory and immunomodulatory functions, while its underlying mechanism is poorly understood. In this study, we first confirmed that AM-CM (25%, 50%, 100%) was optimal for anti-inflammation at 24 h. Lipopolysaccharide (LPS)-induced alteration of cell morphology, the decrease of cell proliferation, and the upregulation of cell apoptosis were significantly reversed in AM-CM-treated THP-1 cells. 25% and 50% AM-CM significantly decreased LPS-induced intracellular reactive oxygen species (ROS) production and proinflammatory cytokines secretion. Mechanistically, we found that AM-CM treatment suppressed LPS-induced activation of MAPK and NF-κB pathways by inhibiting CD14/TLR4 in THP-1 cells. Meanwhile, activation of NLRP3 inflammasome was also dose-dependently attenuated by AM-CM treatment. Thus, AM-CM may exert positive influences on the inflammation microenvironment and provide a novel strategy for improving tissue regeneration.

Exosomal NAMPT from chronic lymphocytic leukemia cells orchestrate monocyte survival and phenotype under endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress has been reported to be transmitted from tumor cells to immune cells via exosome and implicated in immune escape. However, the influence of ER stress on monocytes in chronic lymphocytic leukemia (CLL) cells is largely unknown. Here, we observed the expression of ER stress markers (GRP78, ATF6, PERK, IRE1a, and XBP1s) in CLL cells. The increasing mRNA expression of these ER stress response components was positively correlated with more aggressive disease. Exosome from ER stress inducer tunicamycin (TM)-primed CLL cells (ERS-exo) up-regulated the expression of ER stress marker on monocytes, indicating ER stress is transmissible in vitro via exosome. Treatment with ERS-exo promoted the survival of monocytes and induced phenotypic changes with a significantly larger percentage of CD14+ CD16+ monocytes. Finally, we identified exosome-mediated transfer of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) from ER stressed CLL cells into monocytes as a novel mechanism through which ERS-exo regulated monocytes. Exosomal eNAMPT up-regulated nicotinamide adenine dinucleotide (NAD+ ) production which subsequently activated SIRT1-C/EBPβ signaling pathway in monocytes. Our results suggest the role of ER stress in mediating immunological dysfunction in CLL.

Executable models of immune signaling pathways in HIV-associated atherosclerosis

Atherosclerosis (AS)-associated cardiovascular disease is an important cause of mortality in an aging population of people living with HIV (PLWH). This elevated risk has been attributed to viral infection, anti-retroviral therapy, chronic inflammation, and lifestyle factors. However, the rates at which PLWH develop AS vary even after controlling for length of infection, treatment duration, and for lifestyle factors. To investigate the molecular signaling underlying this variation, we sequenced 9368 peripheral blood mononuclear cells (PBMCs) from eight PLWH, four of whom have atherosclerosis (AS+). Additionally, a publicly available dataset of PBMCs from persons before and after HIV infection was used to investigate the effect of acute HIV infection. To characterize dysregulation of pathways rather than just measuring enrichment, we developed the single-cell Boolean Omics Network Invariant Time Analysis (scBONITA) algorithm. scBONITA infers executable dynamic pathway models and performs a perturbation analysis to identify high impact genes. These dynamic models are used for pathway analysis and to map sequenced cells to characteristic signaling states (attractor analysis). scBONITA revealed that lipid signaling regulates cell migration into the vascular endothelium in AS+ PLWH. Pathways implicated included AGE-RAGE and PI3K-AKT signaling in CD8+ T cells, and glucagon and cAMP signaling pathways in monocytes. Attractor analysis with scBONITA facilitated the pathway-based characterization of cellular states in CD8+ T cells and monocytes. In this manner, we identify critical cell-type specific molecular mechanisms underlying HIV-associated atherosclerosis using a novel computational method.

Regulating monocyte infiltration and differentiation: Providing new therapies for colorectal cancer patients with COVID-19.

The outbreak of coronavirus disease 2019 (COVID-19) is a significant challenge for clinicians, especially for immunocompromised cancer patients. By analyzing the impact of COVID-19 on the immune microenvironment of colorectal cancer (CRC) patients at the tissue level and single-cell level, we found that CRC patients are more easily infected by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), but promotion of infiltration and differentiation of monocytes makes them more likely to develop severe COVID-19. Because of the continuing activation of nuclear factor (NF)-κB and C-C chemokine receptor type 5 (CCR5) signaling pathways in monocytes, imbalance of macrophage polarization can aggravate the cytokine release syndrome. Therefore, regulating the infiltration and differentiation of monocytes is helpful for the treatment of COVID-19 in CRC patients.

Association of Circulating Sex Hormones With Inflammation and Disease Severity in Patients With COVID-19

Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (β = -0.43; 95% CI, -0.52 to -0.17; P < .001), C-reactive protein (β = -0.38; 95% CI, -0.78 to -0.16; P = .004), interleukin 1 receptor antagonist (β = -0.29; 95% CI, -0.64 to -0.06; P = .02), hepatocyte growth factor (β = -0.46; 95% CI, -0.69 to -0.25; P < .001), and interferon γ-inducible protein 10 (β = -0.32; 95% CI, -0.62 to -0.10; P = .007). Estradiol and IGF-1 concentrations were not associated with COVID-19 severity in men. Testosterone, estradiol, and IGF-1 concentrations were similar in women with and without severe COVID-19. Gene set enrichment analysis revealed upregulated hormone signaling pathways in CD14+CD16- (ie, classical) monocytes and CD14-CD16+ (ie, nonclassical) monocytes in male patients with COVID-19 who needed intensive care unit treatment vs those who did not. In this single-center cohort study of patients with COVID-19, lower testosterone concentrations during hospitalization were associated with increased disease severity and inflammation in men. Hormone signaling pathways in monocytes did not parallel serum hormone concentrations, and further investigation is required to understand their pathophysiologic association with COVID-19.

Monocytes From Patients With Macrophage Activation Syndrome and Secondary Hemophagocytic Lymphohistiocytosis Are Hyperresponsive to Interferon Gamma.

ObjectiveTo investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.MethodsFresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.ResultsMonocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001]. Statistically significant correlations between IFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.ConclusionOur data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.

Induction of NF-κB inflammatory pathway in monocytes by microparticles from patients with systemic lupus erythematosus

BackgroundElevated levels of circulating microparticles (MPs) and molecules of the complement system have been reported in patients with systemic lupus erythematosus (SLE). Moreover, microparticles isolated from patients with SLE (SLE-MPs) contain higher levels of damage-associated molecular patterns (DAMPs) than MPs from healthy controls (CMPs). We hypothesize that the uptake of MPs by monocytes could contribute to the chronic inflammatory processes observed in patients with SLE. Therefore, the aim of this study was to evaluate the expression of activation markers, production of proinflammatory mediators, and activation of the NF-κB signaling pathway in monocytes treated with CMPs and SLE-MPs. MethodologyMonocytes isolated from healthy individuals were pretreated or not with pyrrolidine dithiocarbamate (PDTC) and cultured with CMPs and SLE-MPs. The cell surface expression of CD69 and HLA-DR were evaluated by flow cytometry; cytokine and eicosanoid levels were quantified in culture supernatants by Cytokine Bead Array and ELISA, respectively; and the NF-κB activation was evaluated by Western blot and epifluorescence microscopy. ResultsThe cell surface expression of HLA-DR and CD69, and the supernatant levels of IL-6, IL-1β, PGE2, and LTB4 were higher in cultures of monocytes treated with SLE-MPs than CMPs. These responses were blocked in the presence of PDTC, a pharmacological inhibitor of the NF-κB pathway, with concomitant reduction of IκBα and cytoplasmic p65, and increased nuclear translocation of p65. ConclusionsThe present findings indicate that significant uptake of SLE-MPs by monocytes results in activation, production of inflammatory mediators, and triggering of the NF-κB signaling pathway.

Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-γ-Dependent Functional Reprogramming of Myeloid Cells

Regulatory T (Treg) cell infiltration of solid tumors often correlates with poor prognosis, but their tumor-suppressive function lacks mechanistic understanding. Through a combination of transgenic mice, cell fate mapping, adoptive transfer, and co-injection strategies, we demonstrate that Treg cell ablation-dependent anti-tumor effects in murine breast cancer require intratumoral recruitment of CCR2+ inflammatory monocytes, which primarily differentiate into tumor-associated macrophages (TAMs), and lead to reprogramming of their function in an IFN-γ-dependent manner. Furthermore, transcriptomic signatures from murine TAMs in Treg cell-ablated conditions correlate with increased overall survival in human breast cancer. Our studies highlight the strong myeloid dependency of breast cancer and provide the basis for the development of therapeutic strategies based on manipulation of the IFN-γ signaling pathway in monocytes.

Zinc Silicate/Nano-Hydroxyapatite/Collagen Scaffolds Promote Angiogenesis and Bone Regeneration via the p38 MAPK Pathway in Activated Monocytes.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-β1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

Induction of COX-2 gene transcription and prostaglandin E2 (PGE2) production in primary human monocytes by MDP adjuvant: role of MAPK and p300 HAT

Abstract Muramyl dipeptide (MDP) is the minimal structure responsible for adjuvant activity of peptidoglycans. Early clinical trials of vaccines formulated with MDP revealed adjuvant-induced pyrogenic response. Here we studied signaling pathway leading to production of mediator of fever, PGE2, in human monocytes activated with MDP. MDP alone induced nuclear translocation of NF-kB but only minimal levels of COX-2 mRNA expression and PGE2 production in monocytes. T cell conditioned medium (TCM) prepared from CD3 bead-purified T cells dramatically upregulated COX-2 mRNA and PGE2 in MDP-activated monocytes but did not increase nuclear NF-kB. COX-2 mRNA expression and PGE2 production in monocytes activated with MDP and TCM were sensitive to the inhibitors of ERK1/2, p38, and JNK MAPK. Western blotting confirmed that MDP alone and TCM alone induced phosphorylation of p38 and SAPK/JNK and of ERK1/2, respectively. In addition to MAPK, the role of p300 histone acetyltransferase (HAT) was studied by ChIP assay. TCM induced binding of p300 to a COX-2 promoter. Inhibition of p300 blocked binding of p300 to COX-2 promoter, acetylation of H3 and H4 histones, upregulation of COX-2 mRNA and of PGE2 in monocytes activated with MDP/TCM. Importantly, inhibitors of either p38 or ERK1/2 prevented recruitment of p300 to COX-2 promoter. Taken together these results suggest cooperation between two signaling pathways in monocytes: nuclear translocation of NF-kB and activation of SAPK/JNK and p38 MAPK are induced by MDP, while activation of ERK1/2 and recruitment of p300 to COX-2 promoter are induced by TCM. These two pathways are equally important for p300-facilitated acetylation of H3 and H4 histones at the COX-2 promoter and for up-regulated COX-2/PGE2 production.

Circulatory heavy metals (cadmium, lead, mercury, and chromium) inversely correlate with plasma GST activity and GSH level in COPD patients and impair NOX4/Nrf2/GCLC/GST signaling pathway in cultured monocytes

This study aims to examine the hypothesis that circulatory heavy metals may be associated with lung function decline and lower plasma GST activity and GSH level in COPD patients via activating monocytes mediated by impairing the NOX4/Nrf2/GCLC/GST signaling pathway. Results showed that the blood levels of heavy metals (cadmium, lead, mercury, and chromium) were significantly higher in COPD patients of coal mine site compared to the healthy controls. The levels of heavy metals in COPD patients were significantly and negatively correlated with lung function, GST activity, and GSH level. Using flowcytometry, fluorescence spectroscopy, and immunoblotting studies we have further demonstrated that treatment with individual heavy metals dose-dependently increased the NOX4 protein expression, intracellular ROS production, and decreased the Nrf2, GCLC, and GST protein expression, GST activity, and GSH level in THP-1 monocytes. None of the treatment caused any change in cell viability compared to control. In conclusion, this study suggests that circulatory heavy metals in COPD patients of coal mine site weakened the lung function, decreased the plasma GST activity and GSH level via impairing the NOX4/Nrf2/GCLC/GST signaling pathway in monocytes, which may cause monocyte activation and initiate the COPD pathophysiology.

MiR-199b-5p Regulates Immune-Mediated Allograft Rejection after Lung Transplantation Through the GSK3β and NF-κB Pathways.

Emerging evidence indicates that acute rejection mainly associated with the inflammatory response is an independent risk factor for chronic rejection after lung transplantation. Monocytes are the main pro-inflammatory leukocytes infiltrating around the lesions and play vital roles in triggering the acute rejection. In the rat lung transplantation model, lipopolysaccharide (LPS)-induced severe acute rejection was strongly associated with advanced chronic rejection. The exact regulatory mechanism of pro-inflammation in monocytes is not yet clear. In this study, we identified a novel anti-inflammatory effect of miR-199b-5p (miR-199b) through the GSK3β and NF-κB pathways. THP-1 monocytes treated with LPS showed a significant decrease in miR-199b that is inversely correlated to GSK3β expression and NF-κB activation. Furthermore, the NF-κB-associated inflammatory response was reduced due to the overexpression of miR-199b targeting GSK3β, which was rescued by the inhibition of miR-199b. These results indicated that miR-199b attenuated the inflammatory response at least partly through the GSK3β/NF-κB signaling pathways in monocytes. Our data point toward a potentially important role for miR-199b in the inhibition of rejection after lung transplantation.

Differences in TLR7/8 activation between monocytes and macrophages

The recognition of single-stranded RNA by TLR7/8 leads to the production of NF-κB-mediated cytokines and type I IFNs. However, the role of TLR7/8 activation in monocytes and macrophages is still unclear. The aim of this study was to investigate the differences in the activation of TLR7/8 between these two cell types. Microarray analysis, qRT-PCR and flow cytometry were used to analyse TLR7/8 signalling pathways in monocytes and macrophages after stimulation with agonists. Our data indicated that TLR8 agonists activated the NF-κB- and IRF-mediated pathways in THP-1 cells, whereas TLR7 agonists did not. However, silent TLR8 and enhanced TLR7 expression could increase TLR7-induced NF-κB activation in monocytes. TLR7 and TLR8 agonists induced NF-κB activation but no ISG response in PMA-differentiated THP-1 cells. The mRNA levels of pro-inflammatory cytokine were elevated upon CL075 stimulation in macrophages compared to monocytes. Thus, TLR7 and TLR8 might modulate different immune responses in monocytes and macrophages.

Inhibition of BAFF binding to its receptor, BR3, by low molecular weight compounds results in suppression of activation of monocytes and B cells in vitro and in vivo model of autoimmune diseases.

Abstract Background and Purpose We have demonstrated that the elevated expression of BAFF receptor (BR3) on monocytes is involved in the pathogenesis of primary Sjogren’s syndrome and BR3 is a therapeutic target to treat pSS. In our previous study, we have discovered two pyrrolopryimidine derivatives, BIK12 and BIK13, which inhibit binding of BAFF to BR3, using our original high-throughput screening system. In this study, we investigated inhibitory effects of these compounds on BAFF signaling pathways in monocytes and B cells in vitro. In addition, we analyzed the effects of the compounds on serum level of an anti-dsDNA antibody in autoimmune disease model mice. Methods Peripheral monocytes were stimulated with soluble BAFF (sBAFF) and cultured in vitro with or without peripheral B cells in the presence of BIK-12 or BIK-13. IL-6 production by monocytes and IgG production by B cells were measured by ELISA. MRL/lpr and NZBWF1 mice were treated with these compounds three times a week at a dose of 1.0 or 0.2 mg/kg for 6 months. The titer of an anti-ds DNA antibody was measured by ELISA. Results and Discussion sBAFF-induced IL-6 production by peripheral monocytes and IgG production by B cells co-cultured with sBAFF-stimulated peripheral monocytes were significantly suppressed by BIK12 and BIK13 in a dose dependent manner. Interestingly, the titer of an anti-dsDNA antibody in both MRL/lpr and NZBWF1 mice received BIK13 was significantly lower than control mice after 16 weeks’ treatment, indicating that the compound was also efficacious in vivo.

Activation of the JAK-STAT Signaling Pathway after In Vitro Stimulation with IFNß in Multiple Sclerosis Patients According to the Therapeutic Response to IFNß.

Interferon beta (IFNß) is a common treatment used for multiple sclerosis (MS) which acts through the activation of the JAK-STAT pathway. However, this therapy is not always effective and currently there are no reliable biomarkers to predict therapeutic response. We postulate that the heterogeneity in the response to IFNß therapy could be related to differential activation patterns of the JAK-STAT signaling pathway. Our aim was to evaluate the basal levels and the short term activation of this pathway after IFNß stimulation in untreated and IFNß treated patients, as well as according to therapeutic response. Therefore, cell surface levels of IFNAR subunits (IFNAR1 and IFNAR2) and the activated forms of STAT1 and STAT2 were assessed in peripheral blood mononuclear cells from MS patients by flow cytometry. Basal levels of each of the markers strongly correlated with the expression of the others in untreated patients, but many of these correlations lost significance in treated patients and after short term activation with IFNß. Patients who had undergone IFNß treatment showed higher basal levels of IFNAR1 and pSTAT1, but a reduced response to in vitro exposure to IFNß. Conversely, untreated patients, with lower basal levels, showed a greater ability of short term activation of this pathway. Monocytes from responder patients had lower IFNAR1 levels (p = 0.039) and higher IFNAR2 levels (p = 0.035) than non-responders just after IFNß stimulation. A cluster analysis showed that levels of IFNAR1, IFNAR2 and pSTAT1-2 in monocytes grouped 13 out of 19 responder patients with a similar expression pattern, showing an association of this pattern with the phenotype of good response to IFNß (p = 0.013). Our findings suggest that an activation pattern of the IFNß signaling pathway in monocytes could be associated with a clinical phenotype of good response to IFNß treatment and that a differential modulation of the IFNAR subunits in monocytes could be related with treatment effectiveness.

Manipulation of necroptosis by Porphyromonas gingivalis in periodontitis development.

To eliminate invading pathogens and keep homeostasis, host employs multiple approaches such as the non-inflammation associated-apoptosis, inflammation associated-necroptosis and pyroptosis, etc. Necroptosis is known as a highly pro-inflammatory form of cell death due to the release of massive damage-associated molecular patterns (DAMPs). For the first time, we reported that Porphyromonas gingivalis induced cellular necroptosis through receptor-interacting protein 1 (RIP1)/RIP3/mixed lineage kinase domain-like (MLKL) signaling pathway in monocytes. Necroptosis in THP-1 cells was induced by MLKL phosphorylation in vitro. P. gingivalis treated-THP-1 cells exhibited lower cell death rate with pretreatment of inhibitors RIP1 and MLKL, accompanied with attenuated TNF-α and IL-6 expressions. Moreover, the necroptosis risk was also reduced via gene silencing by RIP3 or MLKL in the P. gingivalis treated-THP-1 cell lines. We further explored P. gingivalis-induced necroptosis in animal models in vivo. Firstly, C57BL/6 mice were injected with P. gingivalis in the subcutaneous chamber model. Animals pretreated with MLKL inhibitor exhibited significantly enhanced P. gingivalis clearance; in addition, levels of TNF-α and IL-6 were notably decreased by 60% via MLKL inhibition. Secondly, P. gingivalis-induced periodontitis was utilized to investigate necroptosis related-periodontopathogensis. Positive staining of phosphorylated MLKL in mice periodontitis biopsies was detected to a higher degree, while larger amount of alveolar bone loss was observed in MLKL (-) group comparing to those in the MLKL (+) group. These findings may suggest that P. gingivalis play essential roles in necroptosis process during periodontitis, and our research may shed light on the further work on the related periodontopathogenesis investigation.

Crosslinking of Fc Gamma-Rllb and Fc Epsilon-RI Binding Peptides Inhibits Osteoclast Formation in Multiple Myeloma through Inactivation of the ITAM Signaling Pathway

Introduction: Overactivity of osteoclasts resulting in bone destruction is a hallmark of multiple myeloma (MM). Receptor for activation of NF-kB ligand (RANKL) and monocyte colony stimulating factor (MCSF) signaling pathways both promote proliferation and survival of the precursors of the osteoclast lineage, and have been widely investigated in MM. The third pathway involved in osteoclast differentiation is the immunoreceptor tyrosine-based activation motif (ITAM) with c-Fms signaling. ITAM and its inhibitor ITIM provide the basis for two opposed signaling modules that duel for control of osteoclast formation. Human monocyte/macrophage expresses the low-affinity FcγRIIb and high-affinity Fcε receptor 1 (FcεRI). Both receptors mediate Syk phosphorylation to activate or inactivate downstream ITAM or ITIM signaling molecules. In this study, we determined the effects of an IgG(CH2-CH3) and IgE(CH2-CH3-CH4) fusion protein that activates the ITIM inhibitory pathway on downstream signaling of Syk and osteoclast formation in monocytes from MM patients.Methods: We constructed IgG(CH2-CH3) with an IgE(CH2-CH3-CH4) fusion protein using standard cloning techniques. We evaluated the fusion protein on osteoclast formation using cells from either human monocytes isolated from MM patients' peripheral blood mononuclear cells (PBMCs) or bone marrow (BM) MCs with an anti-CD14 micro-bead affinity column and magnetic bead selection (Miltenyi Biotec, Auburn, CA). The monocytes were cultured on slide-culture dishes (2 X 105 cells/well). The cells were treated with the fusion protein or with IgE or IgG and subsequently treated with 50ng/ml RANKL (receptor for activation of nuclear factor kB and 10ng/ml MCSF (monocyte colony stimulating factor) in order to stimulate osteoclast formation at the beginning of the culture and during a medium change after 3 days with the same amount of growth factors added. The cells were fixed for tartrate resistant acid phosphatase (TRAP)-staining assay on day 21. To investigate ITIM signaling pathway we determined Syk phosphorylation of monocytes treated or without treated with fusion protein by Western blot analysis.Results: We found that in a concentration-dependent fashion, the fusion protein inhibited osteoclast cell formation from CD14+ MCs from PB or BM exposed to RANKL and MCSF. We further analyzed the effects on the FcγRIIb-SHIP signaling pathway in monocytes induced with 50ng/ml RANKL and 10ng/ml MCSF following exposure to fusion protein or control IgG or IgE. The results showed that the monocytes showed markedly lower Syk phosphorylation following exposure to the fusion protein (100-200ng/ml). There was no change of Syk phosphorylationl in monocytes treated with IgG or IgE or IgG with IgE.Conclusions: The results of our study show that intact human IgG or IgE does not affect the ITAM or ITIM signaling pathways. However, a fusion protein consisting of IgG(CH2-CH3) with IgE(CH2-CH3-CH4) showed the ability to activate the ITIM inhibition pathway through FcγRIIb to reduce osteoclast formation. Thus, blockage of ITAM may be treating novel treatment for preventing bone loss for MM patients. DisclosuresNo relevant conflicts of interest to declare.

Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery.

Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient's immune state before surgery and predict recovery is highly desirable in perioperative medicine. To evaluate a patient's presurgical immune state, cell-type-specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue. Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14 monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples. Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients' preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.

(E)-3-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one ameliorates the collagen-arthritis via blocking ERK/JNK and NF-κB signaling pathway

Our previous report has shown a natural pyranochalcones-derived compound, (E)-3-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (5b), that exerted protection against carrageenan-induced hind paw edema and adjuvant-induced arthritis. In this study, collagen-induced arthritis (CIA) model was used to further examine the anti-arthritic effects of 5b in vivo; the underlying molecular mechanisms of action were also investigated using a murine monocytic cell line, RAW264.7 cells. Here we showed that oral administration of 5b (20mg/kg) significantly suppressed the progression of arthritis. Improvement in disease severity was accompanied by inhibition of CD68-positive cells in knee joint and reduced pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in serum. In vitro, 5b suppressed expressions of iNOS, cyclooxygenase-2 (COX-2), TNF-α, IL-6 and IL-1β as well as productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated macrophages. This compound also significantly suppressed LPS-induced NF-κB activation, including phosphorylation of I-κB, degradation of I-κB, and nuclear translocation of p65 and p50. Treatment with 5b also blocked LPS-induced expression of TLR4 remarkably, suppressed degradation of IRAKs and phosphorylations of JNK and ERK, but had little effect to p38 kinase activation. These findings indicated that 5b might be a therapeutic agent for rheumatoid arthritis, and exerted an anti-inflammatory effect mainly through mediating TLR4, NF-κB and ERK/JNK signaling pathways in monocytes.

Expression and activation of intracellular receptors TLR7, TLR8 and TLR9 in peripheral blood monocytes from HIV-infected patients.

TLR´s play a role in host defense in HIV infection recognizing the viral DNA or RNA. Their activation induces a signaling pathway that includes the proteins MyD88, IRAK4, TRAF6 and the transcription factor NF-kBp65. To determine the expression of TLR7, TLR8 and TLR9, and activation of its signaling pathway in monocytes from patients infected with HIV. Methods. Expression of TLR7, TLR8 and TLR9 was determined in monocytes from HIV-infected patients (n= 13) and control subjects (n= 13), which were activated with specific ligands. The expression of MyD88 and NF-kBp65 were determined by flow cytometry; IRAK4 and TRAF6 were studied by immunoblotting. No statistical difference was found in the expression of TLR7, 8 and 9 in monocytes from patients compared to controls, but we observed the non-significant increased expression of TLR9 in patients. The activation showed no significant difference in the expression of MyD88 and NF-kBp65 in patients when compared to controls, but were decreased in stimulated cells over non-stimulated cells. IRAK4 and TRAF6 were not detected. No statistical difference was observed in the expression of intracellular TLRs, MyD88 and NFkBp65 in monocytes from patients compared to controls. This was probably due to effective antiretroviral therapy being received at the time of study entry. Additional studies are needed under controlled conditions that include infected patients with and without ARVT, responders and non-responders, and work with different cell populations.