Monocyte Apoptosis Research Articles

Acute Exercise-Induced Oxidative Stress Does Not Affect Immediate or Delayed Precursor Cell Mobilization in Healthy Young Males.

Exercise is known to acutely and transiently mobilize precursor cells to the peripheral blood. To date, the underlying mechanisms have not yet been fully elucidated and we hypothesized that exercise-induced oxidative stress could be a mobilizing agent, either directly or via circulating apoptotic cells as mediators. The aim of the study was to assess the effect of acute exercise-induced oxidative stress on numbers of circulating angiogenic precursor cells (CACs), circulating non-angiogenic precursor cells (nCACs), mesenchymal precursor cells (MPCs), mature endothelial cells (ECs), and mononuclear cells (MNCs), as well as their apoptotic subsets. Healthy, young males (n = 18, age: 24.2 ± 3.5 years) completed two identical, standardized incremental cycling tests. The first, un-supplemented control test was followed by a 7-day-long supplementation of vitamin C (1,000 mg/day) and E (400 I.U./day), immediately preceding the second test. Blood samples were collected before, directly after, 30, 90, 180, and 270 min after exercise, and aforementioned circulating cell numbers were determined by flow cytometry and a hematology analyzer. Additionally, total oxidative capacity (TOC) and total antioxidative capacity (TAC) were measured in serum at all timepoints. Antioxidative supplementation abolished the exercise-induced increase in the oxidative stress index (TOC/TAC), and reduced baseline concentrations of TOC and TOC/TAC. However, it did not have any effect on CACs, nCACs, and MPC numbers or the increase in apoptotic MNCs following exercise. Our results indicate that exercise-induced oxidative stress is neither a main driver of lymphocyte and monocyte apoptosis, nor one of the mechanisms involved in the immediate or delayed mobilization of precursor cells.

In vitro effects of conjugated linoleic acid (CLA) on inflammatory functions of bovine monocytes

The conjugated linoleic acid (CLA) isomers, a group of naturally occurring isomers of the essential fatty acid (FA) linoleic acid, have received special attention in animal and human nutrition. Although they have long been used as dietary integrators in dairy cows, the effects of CLA isomers on bovine immune cells remain mostly undisclosed. The present study aimed to cover this gap and investigate the in vitro effects of CLA on inflammatory functions, including chemotaxis, phagocytosis, killing capability, and extracellular respiratory burst of purified bovine monocytes (CD14+). The apoptosis rate of monocytes was addressed as well. Once assessed, the effects of different concentrations (10, 50, 100, and 500 μM) of the 2 main CLA isomers, namely cis-9,trans-11 and trans-10,cis-12, the experiments were carried out using a concentration of 50 μM of the CLA isomers, both individually and in a mixture (50:50). The immunomodulatory activities of linoleic acid, an essential FA, and stearic acid, a saturated FA, were also investigated. Only the 50:50 CLA mixture was able to reduce monocyte apoptosis and to increase the extracellular respiratory burst during experimental proinflammatory conditions, as assessed by measuring production of reactive oxygen species. Linoleic acid and CLA had no effects on chemotaxis, phagocytosis, or killing capability. Remarkably, treatment of monocytes with stearic acid significantly reduced their chemotactic capability. The present results demonstrated that CLA isomers do have immunomodulatory effects on some functions of bovine monocytes, and that the mixture of the 2 CLA isomers is more effective than the CLA isomers individually.

Apoptosis and secondary necrosis of neutrophils and monocytes in the immunopathogenesis of rheumatoid arthritis: a cohort study.

Rheumatoid arthritis (RA) is a progressive chronic inflammatory and autoimmune joint disease. Neutrophils and monocytes are the main target cells of innate immune defense that modulate the course of inflammatory rheumatic diseases. Dysfunctional phagocytosis is a common feature in RA. The aim of this study was to evaluate the diagnostic value of apoptotic changes in neutrophils and monocytes and their relationship with rheumatoid activity measured by the DAS28 score. We used the APOLECT flow cytometric assay for evaluating primary necrotic, apoptotic, and secondary necrotic neutrophils and monocytes determination in RA patients compared with healthy controls. The apoptotic granulocytes were greater in RA patients compared to healthy controls (0.76 ± 0.15% vs. 0.58 ± 0.17%, P < 0.05). The percentage of primary necrotic granulocytes was significantly elevated in RA patients compared to healthy controls (3.84 ± 0.5% vs. 1.96 ± 0.33%). No significant difference was noted for primary necrotic monocytes. The number of secondary necrotic granulocytes and monocytes was high in RA patients (0.94 ± 0.15% vs. 0.4 ± 0.06% and 4.83 ± 1.06% vs. 1.8 ± 0.33%, respectively). The obtained results suggest that neutrophils and monocytes undergo apoptotic modifications which are accompanied by secondary necrotic cells formation in RA. These shifts may lead to autoantigen accumulation that results in the progressive course RA.

The effects of IL-4 and RANKL on viability of giant cell granuloma patients' monocytes

Giant cell granuloma is a non-malignant lesion characterized by proliferation of granulation tissues with multinucleated giant cells. Given the contribution of IL-4 and RANKL to the disease pathogenesis, we investigated effects caused by synergism between these factors on the viability and apoptosis of monocytes from GCG patients. In this study, the isolated monocytes were treated with IL-4 and RANKL. MTT and Annexin V-FITC/PI staining were used to investigate the viability and apoptosis of monocytes, respectively. In monocytes isolated from both GCG patients and healthy controls, treatment with IL-4 and RANKL led to increased viability and decreased apoptosis, even though these changes were significantly more obvious in patient-derived monocytes. Our results demonstrated that treatment with IL-4 and RANKL yields enhanced viability and reduced apoptosis in monocytes isolated from GCG patients compared with that of healthy individuals. These findings might provide evidence for the potential role of IL-4 and RANKL in the pathogenesis of GCG.

Chronic mild hypoxia accelerates recovery from preexisting EAE by enhancing vascular integrity and apoptosis of infiltrated monocytes

While several studies have shown that hypoxic preconditioning suppresses development of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), no one has yet examined the important clinically relevant question of whether mild hypoxia can impact the progression of preexisting disease. Using a relapsing-remitting model of EAE, here we demonstrate that when applied to preexisting disease, chronic mild hypoxia (CMH, 10% O2) markedly accelerates clinical recovery, leading to long-term stable reductions in clinical score. At the histological level, CMH led to significant reductions in vascular disruption, leukocyte accumulation, and demyelination. Spinal cord blood vessels of CMH-treated mice showed reduced expression of the endothelial activation molecule VCAM-1 but increased expression of the endothelial tight junction proteins ZO-1 and occludin, key mechanisms underlying vascular integrity. Interestingly, while equal numbers of inflammatory leukocytes were present in the spinal cord at peak disease (day 14 postimmunization; i.e., 3 d after CMH started), apoptotic removal of infiltrated leukocytes during the remission phase was markedly accelerated in CMH-treated mice, as determined by increased numbers of monocytes positive for TUNEL and cleaved caspase-3. The enhanced monocyte apoptosis in CMH-treated mice was paralleled by increased numbers of HIF-1α+ monocytes, suggesting that CMH enhances monocyte removal by amplifying the hypoxic stress manifest within monocytes in acute inflammatory lesions. These data demonstrate that mild hypoxia promotes recovery from preexisting inflammatory demyelinating disease and suggest that this protection is primarily the result of enhanced vascular integrity and accelerated apoptosis of infiltrated monocytes.

Membrane particles derived from mesenchymal stromal cells as a novel cell free therapy for immunomodulation and regeneration

Background & Aim Mesenchymal stromal cells (MSC) are widely studied for their immunomodulatory and regenerative properties. However, MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Recent work demonstrated that inactivated MSC which lost their capacity to secrete factors maintain their regulatory capacity, suggesting that cell membrane dependent interactions with immune cells are responsible for the regulatory effects. We propose a new cell-free therapy based on the generation of small plasma Membrane Particles (MP) from adipose tissue MSC. The aim of this work is to study the therapeutic potential of MP for immune and degenerative disorders Methods, Results & Conclusion MP from adipose tissue MSC were generated by hypotonic shock and extrusion. Parameters such as size (by nanosight), morphology (by cryo-electron microscopy), and enzyme activity were analysed. The interaction of MP with cells was analysed by confocal microscopy. The therapeutic potential of MP was tested in three in vitro models:1.Immune cells were treated with MP and monocyte apoptosis were performed by flow cytometry.2.Inflammed endothelial cells (EC) model (EC+TNFα). Monocyte transmigration through an EC monolayer, and EC angiogenesis capacity were analyzed by microscopy. Endothelial layer integrity was evaluated by colorimetric assay.3.Human lung fibroblasts from biopsies of Idiopathic pulmonary fibrosis patients were stimulated with TGFb to increase the expression of profibrotic molecules (collagen I, and III, fibronectin, PDL-1, tenascin-c and MMP1). The protein gene expression was analysed by PCR. The results showed that the average size of MP was 120 nm, and they showed a spherical shape. MP exhibited ATPase and nucleotidase activity, indicating they are enzymatically active. MP bound to the cell membranes of monocytes and were internalized by EC and fibroblasts. With respect to the in vitro functional immune studies, MP induced apoptosis of proinflammatory monocytes. On EC, MP decreased the permeability of the EC monolayer reducing the number of transmigrating monocytes. They also stimulated the formation of angiogenic structures in vitro.In the fibrosis model, MP decreased the expression of profibrotic proteins in fibroblasts such as collagen II, tenascin-c, and PDL-1. In conclusion, MP have immunomodulatory and regenerative properties and have potential as a novel cell-free therapy for treatment of immunological and degenerative disorders.

CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

BackgroundRecent studies have revealed the involvement of circular RNAs (circRNAs) in the control and elimination of invading Mycobacterium tuberculosis (Mtb) by macrophages. However, the regulatory mechanism of circAGFG1 in macrophages infected by Mtb has not been fully explored. In this study, we sought to investigate the role of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages and reveal its the molecular mechanism.MethodsThe expression of circAGFG1 in macrophages from patients with active tuberculosis and in Mtb-treated macrophages in vitro was explored. Then, the effect of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages was evaluated by flow cytometry, electron microscope, immunofluorescence, and Western blotting. Bioinformatics analysis was used to identify and validate the downstream regulatory pathway of circAGFG1, miRNA-1257/Notch. For further analysis, the role of miRNA-1257 on autophagy and apoptosis was assessed.ResultsIn vitro, ectopic expression of circAGFG1 upregulated autophagy and reduced apoptosis significantly in Mtb-infected cells. Notch levels were discovered to be increased by the silencing effect of circAGFG1 on miRNA-1257 expression. miRNA-1257 was found to noticeably reduce autophagy and promote macrophage apoptosis. Increased circAGFG1 expression, decreased monocyte apoptosis, and enhanced autophagy were found in macrophages from patients with active tuberculosis.ConclusionsIn active tuberculosis, circAGFG1 enhances autophagy and reduces apoptosis via the miRNA-1257/Notch axis; this provides new therapeutic targets for tuberculosis patients.

HIPK3 Mediates Inflammatory Cytokines and Oxidative Stress Markers in Monocytes in a Rat Model of Sepsis Through the JNK/c-Jun Signaling Pathway.

Sepsis is a fetal immunological disorder and its complication worsens in the patients with hemodialysis which may increase the risk of death. In the present study, we aimed to investigate the effect of homeodomain-interacting protein kinase 3 (HIPK3) on inflammatory factors and oxidative stress markers in monocytes of rats with sepsis by regulating the c-Jun amino-terminal kinase (JNK)/c-Jun signaling pathway. A rat model of sepsis was initially established using cecal ligation and puncture (CLP) and was further identified by enlarged spleen tissues, inflammation, and oxidative stress. Monocytes were isolated from rats with CLP-induced sepsis. HIPK3 was observed to be downregulated while JUN was upregulated in monocytes from rats with CLP-induced sepsis. Furthermore, isolated monocytes were transduced with lentiviral vectors expressing HIPK3 or shRNA against HIPK3 to explore the effect of HIPK3 on viability and apoptosis of monocytes as well as inflammatory factors and oxidative stress markers. The obtained data exhibited that overexpression of HIPK3 or inhibition of the JNK signaling pathway enhanced proliferation, reduced apoptosis of monocytes, alleviated inflammation, and oxidative stress injury. Consistently, our results may provide evidence that HIPK3 could inhibit the JNK/c-Jun signaling pathway, thereby potentially retarding the progression of sepsis.

Resinous adhesive systems differentially affect the expression of cytokines by human monocytes stimulated or not with Streptococcus mutans in vitro

ObjectivesThe polymerization of adhesive systems is incomplete and the residual monomers that have been released have a cytotoxic capacity. Some teeth develop into pulp necrosis after composite resin restorations. Considering frequent pulpal inflammation in response to cariogenic bacteria, substances released from the patches could affect the cells of the inflammatory infiltrate and interfere with the mechanisms of defense against microorganisms and protection of pulpal tissue. The aim of this study was to evaluate the effect of substances released by different resinous adhesive systems on cell viability and cytokine expression by human monocytes stimulated in vitro with Streptococcus mutans. DesignPeripheral blood mononuclear cells from 10 healthy subjects were stimulated with S. mutans and then incubated with supernatants obtained from the Single Bond Universal (SBU) or Clearfil SE Bond (CSEB) adhesive systems for eight hours. Staining with Annexin V and 7AAD for analysis of apoptosis were performed and detection of monocytes expressing cytokines IL-1α, IL-6, IL-8, IL-10, IL-12 and TNF-α were performed by flow cytometry. ResultsNo treatment significantly affected apoptosis in monocytes. SBU supernatant increased the frequency of monocytes expressing IL-8 and decreased the monocytes expressing IL-10. Considering S. mutans-stimulated cells, while SBU increased the frequency of IL-8+ monocytes, CSEB reduced the frequency of IL-6 and TNF-α positive monocytes. ConclusionsProducts released from SBU seem to induce proinflammatory effects on monocytes while those from CSEB show an anti-inflammatory outcome. These effects may interfere in the control of cytokine-mediated immunoinflammatory pulp reactions, both in the presence and absence of stimulation by cariogenic bacteria.

Down-regulation of lincRNA-EPS regulates apoptosis and autophagy in BCG-infected RAW264.7 macrophages via JNK/MAPK signaling pathway

Macrophages play a major role in the control and elimination of invading Mycobacterium tuberculosis (Mtb). Long intergenic noncoding RNA erythroid prosurvival (lincRNA-EPS) plays an important role in regulating various biologic processes in macrophages, including inflammatory responses, cell apoptosis, and autophagy. Whereas the effect of lincRNA-EPS in regulating the immune response of macrophages to Mtb is little studied. This study aimed to explore lincRNA-EPS expression in monocytes from patients with active pulmonary tuberculosis (PTB) and from healthy individuals. We also sought to investigate the effect of lincRNA-EPS on Bacillus Calmette-Guérin (BCG)-infected macrophages apoptosis and autophagy. Our study found that lincRNA-EPS expression was down-regulated in the monocytes from patients with active PTB compared with healthy individuals, accompanied by significant attenuated monocyte apoptosis and enhanced autophagy. In vitro, knockdown of lincRNA-EPS inhibited apoptosis and promoted autophagy in BCG-infected RAW264.7 macrophages. Moreover, we revealed that lincRNA-EPS regulated apoptosis and autophagy of BCG-infected RAW264.7 macrophages via JNK/MAPK signaling pathway. In conclusion, our findings demonstrated that knockdown of lincRNA-EPS inhibits apoptosis and enhances autophagy by activating the JNK/MAPK signaling pathway in BCG-infected RAW264.7 macrophages. Suggesting that lincRNA-EPS could serve as a new potential therapeutic target for PTB.

Circulating adult stem and progenitor cell numbers\u2014can results be trusted?

BackgroundWithin the last years, the interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown. Cell estimates are often derived by determining the subgroup of CPC as percent lymphocytes (LYM) or mononuclear cells (MNC) via flow cytometry and back calculation over whole blood (WB) cell counts. However, results might depend on the used cell isolation technique and/or gating strategy. We aimed to investigate MNC loss and apoptosis during the flow cytometry sample preparation process preceded by either density gradient centrifugation (DGC) or red blood cell lysis (RBCL) and the potential difference between results derived from back calculation at different stages of cell isolation and from WB.MethodsHuman blood was subjected to DGC and RBCL. Samples were stained for flow cytometry analysis of CPC (CD34+/CD45dim) and apoptosis analysis (Annexin V) of MNC and CPC subsets. MNC and LYM gating strategies were compared.ResultsBoth DGC as well as RBCL yielded comparable CPC concentrations independent of the gating strategy when back calculated over WB values. However, cell loss and apoptosis differed between techniques, where after DGC LYM, and monocyte (MONO) concentrations significantly decreased (p < 0.01 and p < 0.05, respectively), while after RBCL LYM concentrations significantly decreased (p < 0.05) and MONO concentrations increased (p < 0.001). LYM apoptosis was comparable between techniques, but MONO apoptosis was higher after DGC than RBCL (p < 0.001).ConclusionsInvestigated MNC counts (LYM/MONO ratio) after cell isolation and staining did not always mimic WB conditions. Thus, final CPC results should be corrected accordingly, especially when reporting live CPC concentrations after DGC; otherwise, the CPC regenerative potential in circulation could be biased. This is of high importance in the context of non-invasively induced CPC mobilization such as by acute physical exercise, since these cell changes are small and conclusions drawn from published results might affect further applications of physical exercise as non-invasive therapy.

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

Familial Mediterranean fever (FMF) is the most frequent hereditary systemic autoinflammatory syndrome. FMF is usually caused by biallelic mutations in the MEFV gene, encoding Pyrin. Conclusive genetic evidence lacks for about 30% of patients diagnosed with clinical FMF. Pyrin is an inflammasome sensor maintained inactive by two kinases (PKN1/2). The consequences of MEFV mutations on inflammasome activation are still poorly understood. Here, we demonstrate that PKC superfamily inhibitors trigger inflammasome activation in monocytes from FMF patients while they trigger a delayed apoptosis in monocytes from healthy donors. The expression of the pathogenic p.M694V MEFV allele is necessary and sufficient for PKC inhibitors (or mutations precluding Pyrin phosphorylation) to trigger caspase‐1‐ and gasdermin D‐mediated pyroptosis. In line with colchicine efficacy in patients, colchicine fully blocks this response in FMF patients’ monocytes. These results indicate that Pyrin inflammasome activation is solely controlled by Pyrin (de)phosphorylation in FMF patients while a second control mechanism restricts its activation in healthy donors/non‐FMF patients. This study paves the way toward a functional characterization of MEFV variants and a functional test to diagnose FMF.

Long non-coding RNA-HOTAIR promotes the progression of sepsis by acting as a sponge of miR-211 to induce IL-6R expression.

Sepsis remains the primary cause of death in intensive care units and multiple long non-coding RNAs (lncRNAs) have been demonstrated to be dysregulated in samples of patients with sepsis. However, whether lncRNA-HOTAIR is involved in the etiology of sepsis remains unclear. The aim of the present study was to investigate the role of HOTAIR in sepsis and to reveal the associated mechanisms. A bioinformatics analysis and dual-luciferase reporter assay was performed to evaluate the interaction between HOTAIR and miR-211, as well as miR-211 and IL-6R. An animal model of sepsis was established in mice via cecal ligation and puncture. Interferon (IFN)-γ, interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, IL-1β, IL-6 receptor (R), microRNA (miR)-211 and HOTAIR expression was measured using reverse transcription-quantitative PCR. Cellular proliferation and apoptosis of monocytes were assessed using cell counting kit-8 assay and flow cytometry, respectively. miR-211 was revealed to be targeted by HOTAIR and IL-6R. The expression of IFN-γ, IL-6, IL-17, TNF-α, IL-1β, IL-6R and HOTAIR was significantly upregulated in the septic mice, whereas miR-211 expression was downregulated. The overexpression of hox transcript antisense RNA (HOTAIR) and knockdown of miR-211 were associated with an increased expression of IFN-γ, IL-6, IL-17, TNF-α, IL-1β and IL-6R in monocytes, while the overexpression of miR-211 exhibited the opposite effect. HOTAIR overexpression and miR-211 knockdown significantly inhibited cellular proliferation and promoted monocyte apoptosis, whereas the overexpression of miR-211 exhibited the opposite effects in monocytes. Therefore, HOTAIR may promote the progression of sepsis by indirectly regulating the expression of IL-6R via miR-211.

Simvastatin Enhances the Immune Response Against Mycobacterium tuberculosis.

Tuberculosis remains a serious threat worldwide. For this reason, it is necessary to identify agents that shorten the duration of treatment, strengthen the host immune system, and/or decrease the damage caused by the infection. Statins are drugs that reduce plasma cholesterol levels and have immunomodulatory, anti-inflammatory and antimicrobial effects. Although there is evidence that statins may contribute to the containment of Mycobacterium tuberculosis infection, their effects on peripheral blood mononuclear cells (PBMCs) involved in the immune response have not been previously described. Using PBMCs from 10 healthy subjects infected with M. tuberculosis H37Rv, we analyzed the effects of simvastatin on the treatment of the infections in an in vitro experimental model. Direct quantification of M. tuberculosis growth (in CFU/mL) was performed. Phenotypes and cell activation were assessed via multi-color flow cytometry. Culture supernatant cytokine levels were determined via cytokine bead arrays. The induction of apoptosis and autophagy was evaluated via flow cytometry and confocal microscopy. Simvastatin decreased the growth of M. tuberculosis in PBMCs, increased the proportion of NKT cells in culture, increased the expression of co-stimulatory molecules in monocytes, promoted the secretion of the cytokines IL-1β and IL-12p70, and activated apoptosis and autophagy in monocytes, resulting in a significant reduction in bacterial load. We also observed an increase in IL-10 production. We did not observe any direct antimycobacterial activity. This study provides new insight into the mechanism through which simvastatin reduces the mycobacterial load in infected PBMCs. These results demonstrate that simvastatin activates several immune mechanisms that favor the containment of M. tuberculosis infection, providing relevant evidence to consider statins as candidates for host-directed therapy. They also suggest that future studies are needed to define the roles of statin-induced anti-inflammatory mechanisms in tuberculosis treatment.

TNF-α-elicited miR-29b potentiates resistance to apoptosis in peripheral blood monocytes from patients with rheumatoid arthritis.

CD14-positive monocytes from patients with rheumatoid arthritis (RA) are more resistant to apoptosis, which promotes their persistence at the inflammatory site and thereby contributes crucially to immunopathology. We sought to elucidate one mechanism underlying this unique pathogenesis: resistance to apoptosis and the potential involvement of miR-29b in this process. CD14-positive peripheral blood monocytes (PBMs) from RA patients were observed to be resistant to spontaneous apoptosis compared to PBMs from healthy volunteers. Intriguingly, expression of miR-29b was significantly upregulated in PBMs from RA patients than those from healthy volunteers, and this upregulation was correlated with RA disease activity. Functionally, forced expression of the exogenous miR-29b in CD14-positive Ctrl PBMs conferred resistance to spontaneous apoptosis and Fas-induced death, thereafter enhancing the production of major proinflammatory cytokines in there cells. Following identification of the potential miR-29b target transcripts using bioinformatic algorithms, we showed that miR-29b could directly bind to the 3'-UTR of the high-mobility group box-containing protein 1 (HBP1) and inhibited its transcription in PBMs. Importantly, stable expression of the exogenous HBP1 in differentiated THP-1 monocytes effectively abolished miR-29b-elicited resistance to Fas-induced apoptosis. Finally, among patients with RA and good clinical responses to immunotherapy, expression levels of miR-29b were significantly compromised in those treated with infliximab (a TNF-α inhibitor) but not in those treated with tocilizumab (a humanized mAb against the IL-6 receptor), pointing to a potential association between miR-29b activation and TNF-α induction. The available data collectively suggest that TNF-α-elicited miR-29b potentiates resistance to apoptosis in PBMs from RA patients via inhibition of HBP1 signaling, and testing patients for miR-29b/HBP1 expression ratios may provide more accurate prognostic information and could influence the recommended course of immunotherapy.

Polyubiquitination of p62/SQSTM1 is a prerequisite for Fas/CD95 aggregation to promote caspase-dependent apoptosis in cadmium-exposed mouse monocyte RAW264.7 cells

Cadmium(Cd) induces cytotoxicity via autophagy-induced apoptosis in non-activated mouse monocytes; however, the molecular mechanism remains unclear. Here, we show that autophagy induces Fas (CD95/APO-1)-mediated apoptosis by promoting accumulation of p62/SQSTM1 in response to Cd. Cd produced tumor necrosis factor (TNF)-α, peaking at 6 h, and exhibiting a concentration-dependent increase. Immunoblot analysis revealed polyubiquitinated (polyUb) full-length Fas (antibody clone G-9) and reduced cytosolic Fas (antibody clone M-20) in Cd-exposed RAW264.7 cells. The accumulation of polyUb-Fas was transient and positively correlated with polyUb-p62 and polyUb-proteins. Autophagy inhibition via chemical and genetic modulation suppressed Cd-induced polyUb-p62, polyUb-Fas, and polyUb-protein levels, whereas the level of cytosolic Fas recovered to that of the control. Immunofluorescence (IF) staining for full-length Fas, p62, and ubiquitin revealed an aggregated pattern in Cd-induced apoptotic cells, which was inhibited by blocking autophagy. Fas colocalized with microtubule-associated protein 1 light chain (LC)-3B. IF staining and immunoprecipitation assays revealed colocalization and interaction among p62, Ub, and Fas. Knockdown of p62 reduced the binding of Ub and Fas. Together, these data suggest that polyUb-p62 targets Fas and recruits it to autophagosomes, where Fas transiently aggregates to promote apoptosis and is degraded with polyUb-p62. In conclusion, autophagy regulates C-terminal cytosolic Fas aggregation via p62 polyubiquitination, which is required for apoptosis and may play a critical role in the production of select cytokines.

The Role of Distinctive Sphingolipids in the Inflammatory and Apoptotic Effects of Electronegative LDL on Monocytes.

Electronegative low-density lipoprotein (LDL(−)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(−)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(−) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP), and N,N-dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(−) had an increased content in Cer and Sph compared to LDL(+). LDL(−)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(−) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(−). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(−) on monocytes, whereas Sph plays a more important role in LDL(−)-induced inflammation, and S1P counteracts apoptosis.

Vimentin modulates apoptosis and inflammatory cytokine release by a human monocytic cell line (THP-1) in response to lipopolysaccharides in vitro.

Background:It has recently been recognized that serum vimentin is elevated in infectious diseases, and that vimentin plays a role in regulating neutrophils and macrophages associated inflammation. However, the mechanisms are unclear. This study was designed to explore the role of vimentin in regulating monocyte survival or apoptosis as well as inflammatory cytokine secretion in response to lipopolysaccharides (LPSs).Methods:A human monocytic leukemia cell line (THP-1) was transfected with vimentin-specific small interfering RNA (siRNA) or vimentin over-expressing plasmid. Apoptosis was assessed by TdT-mediated dUTP Nick-End Labeling (TUNEL) and DNA content assay. Immunoblotting was performed to detect apoptosis-associated proteins. Cytokines (interleukin [IL]-6, IL-10, and tumor necrosis factor α [TNF-α]) were measured by enzyme-linked immuno sorbent assay. Two-way analysis of variance followed by Student's t test was used to compare means between different groups.Results:Suppression of vimentin in THP-1 cells resulted in increased apoptotic response in the presence of LPS, while over-expression of vimentin could prevent the cells from apoptosis in response to LPS. LPS alone or suppression of vimentin resulted in significant up-regulation of caspase-3 (1.42 ± 0.20 of LPS alone and 1.68 ± 0.10 of vimentin suppression vs. control, t = 5.21 and 10.28, respectively, P < 0.05). In addition, pro-inflammatory cytokines (IL-6 and TNF-α) was significantly increased (IL-6: 577.90 ± 159.90 pg/day/105 cells vs. 283.80 ± 124.60 pg/day/105 cells of control, t = 14.76, P < 0.05; TNF-α: 54.10 ± 5.80 vs. 17.10 ± 0.10 pg/day/105 cells of control, t = 6.71, P < 0.05), while anti-inflammatory cytokine (IL-10) was significantly up-regulated in the THP-1 cells that over-expressed vimentin (140.9 ± 17.2 pg/day/105 cells vs. undetectable in control cells).Conclusions:In summary, the vimentin may regulate innate immunity through modulating monocytes viability as well as inflammatory response in sepsis through shifting the balance of pro-inflammatory and anti-inflammatory cytokines.

H7N9 influenza A virus activation of necroptosis in human monocytes links innate and adaptive immune responses

We previously demonstrated that avian influenza A H7N9 virus preferentially infected CD14+ monocyte in human peripheral blood mononuclear cells (PBMCs), which led to apoptosis. To better understand H7N9 pathogenesis in relation to monocyte cell death, we showed here that extensive phosphorylation of mixed lineage kinase domain-like (MLKL) protein occurred concurrently with the activation of caspases-8, -9 and -3 in H7N9-infected monocytes at 6 h post infection (hpi), indicating that apoptosis and necroptosis pathways were simultaneously activated. The apoptotic morphology was readily observed in H7N9-infected monocytes with transmission electron microscopy (TEM), while the pan-caspase inhibitor, IDN6556 (IDN), accelerated cell death through necroptosis as evidenced by the increased level of pMLKL accompanied with cell swelling and plasma membrane rupture. Most importantly, H7N9-induced cell death could only be stopped by the combined treatment of IDN and necrosulfonamide (NSA), a pMLKL membrane translocation inhibitor, but not by individual inhibition of caspase or RIPK3. Our data further showed that activation of apoptosis and necroptosis pathways in monocytes differentially contributed to the immune response of monocytes upon H7N9 infection. Specifically, caspase inhibition significantly enhanced, while RIPK3 inhibition reduced the early expression of type I interferons and cytokine/chemokines in H7N9-infected monocytes. Moreover, culture supernatants from IDN-treated H7N9-infected monocyte promoted the expression of co-stimulatory molecule CD80, CD83 and CD86 on freshly isolated monocytes and monocyte-derived dendritic cells (MDCs) and enhanced the capacity of MDCs to induce CD3+ T-cell proliferation in vitro. In contrast, these immune stimulatory effects were abrogated by using culture supernatants from H7N9-infected monocyte with RIPK3 inhibition. In conclusion, our findings indicated that H7N9 infection activated both apoptosis and necroptosis in monocytes. An intact RIPK3 activity is required for upregulation of innate immune responses, while caspase activation suppresses the immune response.

Impact of different emulsifiers on biocompatibility and inflammatory potential of Perfluorohexyloctane (F6H8) emulsions for new intravenous drug delivery systems.

BackgroundEmulsions on the basis of Perfluorohexyloctane (F6H8), a semifluorinated alkane (SFA), have shown to dissolve and transport highly lipophilic compounds. It is unknown how F6H8-containing emulsions (F6H8-cEM) interact with compartment blood, the reticuloendothelial system (RES), or influence injured organs in vivo. The current study was conducted to investigate the in vitro biocompatibility of F6H8-cEM and their drug delivery properties. Afterward, an in vivo study was performed as a proof-of-concept study in a rat model of acute kidney injury (AKI), which focused on the potential influence of F6H8-cEM on inflammation in an injured organ.MethodsTwo different F6H8-cEM were stabilized by the emulsifying agents Poloxamer 188 (Pluronic® F68) or lecithin (S75). The two resulting emulsions F6H8-Pluronic or F6H8-lecithin were tested in vitro for the potential modulation of acute inflammation via whole blood assay, FACS, and ELISA. Antioxidant capacity and drug delivery properties were measured with an oxidation assay. Secondly, AKI was induced in the rats, which were treated with the F6H8-lecithin emulsion. Renal function and inflammation were assessed.ResultsBoth F6H8-cEM were phagocytized by monocytes and both dose-dependently affected apoptosis (Annexin V binding) in monocytes. TNF-α expression increased dose-dependency for F6H8-Pluronic emulsion but not for F6H8-lecithin in a whole blood assay. Both F6H8-cEM were able to carry α-tocopherol as a model drug. Animals with AKI treated with the F6H8-lecithin emulsion showed a significantly better renal function and less infiltration of inflammatory cells in renal tissue compared to the control, while inflammatory markers in renal tissue, except HO-1, were not affected by F6H8-lecithin.ConclusionsPluronic® F68 does not seem suitable as a biocompatible surfactant for F6H8-cEM. The injured kidney was not negatively influenced by the F6H8-lecithin emulsion. Lecithin-stabilized F6H8-cEM could be tested for preclinical studies as a carrier system for lipophilic agents.