Aureus-induced Apoptosis Research Articles

Mangiferin Inhibits Apoptosis and Autophagy Induced by Staphylococcus aureus in RAW264.7 Cells.

PurposeStaphylococcus aureus (S. aureus) is an important bacterial pathogen, which creates infective inflammation to human being and animals. Mangiferin (MG) is one of the natural flavonoids with anti-inflammatory, anti-bacterial, and anti-oxidative properties. However, the anti-apoptosis and anti-autophagy of MG are unknown. Hence, this study was aimed to research the inhibition of MG on S. aureus-induced apoptosis and autophagy in RAW264.7 cells.MethodsThe RAW264.7 cells were pretreated with MG, or pretreated with SP600125 or anisomycin synchronously, and then infected with S. aureus (MOI=100:1). The viability and proliferation status of RAW264.7 cells were detected by MTT and EdU assay. The relative expression of TNF-α, IL-6 and IL-10 protein was tested with ELISA. The levels of Bax, Bcl-2, caspase-3, c-Jun N-terminal kinase (JNK), extracellular-regulated protein kinase (ERK), p38, LC3, Beclin-1, p62, phosphorylated JNK, phosphorylated p38 and phosphorylated ERK in cells were detected by Western blotting. The apoptosis rate of RAW264.7 cells was analyzed by flow cytometric assay.ResultsThe study showed that MG significantly attenuated RAW264.7 cells apoptosis and autophagy caused by S. aureus. MG alleviated S. aureus-induced apoptosis by down-regulating the protein level of active caspase-3 and Bax and up-regulating the level of Bcl-2. MG also inhibited S. aureus-induced autophagy via decreasing the protein level of LC3-II/LC3-I and Beclin-1 or increasing the protein expression of p62. This protective role was dependent on the up-regulation of JNK signal pathway, which was confirmed by using JNK agonist and inhibitor.ConclusionOur results demonstrated that MG might protect RAW264.7 cells from S. aureus-induced apoptosis and autophagy via inhibiting JNK/Bax-dependent signal pathway. Therefore, MG may be a potential agent against pathological cell damage induced by S. aureus infection.

Sulforaphane reduces intracellular survival of Staphylococcus aureus in macrophages through inhibition of JNK and p38 MAPK‑induced inflammation.

Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP-1-derived macrophages, primary human peripheral blood mononuclear cell-derived macrophages, and primary mouse bone marrow derived-macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus-induced caspases-3/7-dependent cell apoptosis, potentially through downregulation of microRNA (miR)-142-5p and miR-146a-5p. As SFN is a well-known activator of nuclear factor erythroid 2-related factor 2 (Nrf2), Nrf2−/− BMDMs were used to demonstrate that the SFN-mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2-deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus-induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), as well as for the M1 markers C-C motif chemokine receptor 7, IL-23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen-activated protein kinase (MAPK) (p38) and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus-induced proinflammatory gene expression levels compared with those observed in the SFN-pretreated macrophages. Furthermore, THP-1-derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK-inflammatory response and the S. aureus-induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.

Inhibitory Effect of Lactobacillus plantarum Extracts on HT-29 Colon Cancer Cell Apoptosis Induced by Staphylococcus aureus and Its Alpha-Toxin.

Staphylococcus aureus plays an important role in sepsis, septic shock, pneumonia, and wound infections. Here, we demonstrate that Lactobacillus plantarum extracts inhibited S. aureusinduced cell death of a human epithelial cell line, HT-29. In particular, we have shown that S. aureus-induced cell death was abolished by neutralization of α-toxin, indicating that α-toxin is the major mediator of S. aureus-induced cell death. DNA fragmentation experiment and caspase assay revealed that the S. aureus-induced cell death was apoptosis. L. plantarum extracts inhibited the generation of effector caspase-3 and the initiator caspase-9 in S. aureusor α-toxin-induced cell death. Moreover, expression of Bcl-2, an anti-apoptotic protein, was activated in L. plantarum extract-treated cells as compared with the S. aureus- or α-toxintreated only cells. Furthermore, S. aureus-induced apoptosis was efficiently inhibited by lipoteichoic acid and peptidoglycan of L. plantarum. Together, our results suggest that L. plantarum extracts can inhibit the S. aureus-mediated apoptosis, which is associated with S. aureus spreading, in intestinal epithelial cells, and may provide a new therapeutic reagent to treat bacterial infections.

Staphylococcus aureusInduces Apoptosis in Primary Bovine Mammary Epithelial Cells Through Fas-FADD Death Receptor-Linked Caspase-8 Signaling

Infections with Staphylococcus aureus, a common inducer of mastitis, often result in mammary gland damage and death of various cell types. Although S. aureus was suggested to induce apoptosis in a bovine mammary epithelial cell (BMEC) line, MAC-T, it is unknown whether primary BMECs (pBMECs) apoptosis is triggered by S. aureus and the associated underlying molecular mechanisms have not been determined. Here, we demonstrated that S. aureus induced apoptosis in pBMECs in a time- and dose-dependent manner. Further, S. aureus-induced apoptosis in pBMECs was associated with activation of caspase-3 and caspase-8, but caspase-9 was not. In addition, pBMECs apoptosis was mitigated by caspase-3 and caspase-8 inhibitors, suggesting that apoptosis is initiated via caspase-8 activation. Moreover, S. aureus infection significantly increased expressions of Fas and Fas-associated death domain (FADD) of pBMECs. Taken together, our results demonstrated that S. aureus induced apoptosis in pBMECs via the Fas-FADD death receptor and subsequently triggered the caspase-8-dependent signaling.

Baicalin inhibits Staphylococcus aureus-induced apoptosis by regulating TLR2 and TLR2-related apoptotic factors in the mouse mammary glands

Baicalin, the major active constituent of the isolated root of Scutellaria baicalensis, is widely used in China and Southeast Asian countries. Evidence has indicated that baicalin has multiple biological activities, including anti-apoptotic properties. Mastitis is a severe problem in humans and other animals and is characterized by mammary gland cell apoptosis. Staphylococcus aureus (S. aureus) is the major pathogen that causes mastitis. This study was designed to evaluate the protective effects of baicalin on the mammary glands during S. aureus-induced mastitis. In the present study, a mouse model was infected with S. aureus to induce mammary gland injury. Baicalin treatment was administered between 6 and 24h after infection. Toll-like receptor 2, p53, BAX, BCL-2 and caspase-3 expression were analyzed using qPCR and Western blotting. The results indicated that baicalin significantly attenuated pathological damage and cell death in the mammary glands. Further studies revealed that baicalin down-regulated the expression of Toll-like receptor 2 (TLR2) and the phosphorylation of p53 in the mammary glands after S. aureus-induced mastitis. Baicalin also promoted the expression of BCL-2 at the mRNA and protein levels but inhibited BAX and caspase-3 (CASP-3) cleavage. Baicalin inhibited apoptosis and had protective effects on mammary gland tissues during S. aureus-induced mastitis. These effects were displayed by reductions in TLR2 expression and p53 phosphorylation and the regulation of apoptosis-related factors (BCL-2, BAX and CASP-3) in mammary gland tissues.

Staphylococcus aureus induces apoptosis of human monocytic U937 cells via NF- κB signaling pathways

Staphylococcus aureus ( S. aureus) is an opportunistic pathogen and the major causative agent of numerous infections. S. aureus have been shown to induce apoptosis in many cell types. However, the mechanisms regulating primary monocytes and human monocytic U937 cell death following S. aureus challenge are unknown. In this study, we found that infection of primary monocytes and human monocytic U937 cells with S. aureus induced rapid cell death in a dose- and time-dependent manner displaying the characteristic features of apoptosis. Studying the underlying mechanisms we found that the S. aureus-induced apoptosis was associated with a more prominent reduction in expression of the anti-apoptotic protein nuclear transcription factor- κB (NF- κB). Because expressions of anti-apoptotic Bcl-2, survivin, inhibition of apoptosis protein (cIAP), and X-chromosome-linked inhibitor of apoptosis protein (XIAP) are regulated by NF- κB, S. aureus decreased the levels of these proteins in U937 cells through inhibition of NF- κB. Moreover, S. aureus induced apoptotic genes (Bax and caspase-3) expression, and exposure of primary monocytes and U937 cells to S. aureus led to caspase-3 activity. Collectively, these data define a pathway that infection of primary monocytes and U937 cells with S. aureus induces a caspase-dependent inhibition of NF- κB.

Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

To explore the mechanisms involved in Staphylococcus aureus (S. aureus) invading human monocytic U937 cells. S. aureus were added to U937 cells at multiplicity of infections (MOI) of 20:1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry analysis. Akt and nuclear factor-kappaB (NF-kappaB) activities were detected by Western blotting. Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated (phosphorylated) Akt and NF-kappaB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-kappaB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. S. aureus can stimulate the apoptosis of U937 cells. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-kappaB.

C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Involvement of ?5?1-integrin and TNF-? in Staphylococcus aureus ?-toxin-induced death of epithelial cells

Staphylococcus aureus causes suppurative infections which are often associated with tissue destruction and cell death. In the present study, we investigated the molecular and cellular basis of S. aureus-induced apoptosis and death in a human lung epithelial cell line (A549). We found that staphylococcal alpha-toxin is an important mediator of cytotoxicity in these epithelial cells. Specifically, we found that downregulating alpha-toxin production eliminated the cytotoxicity of S. aureus, whereas the addition of alpha-toxin to the cell culture medium significantly increased cell death in a dose-dependent manner. Importantly, we found that alpha-toxin-mediated cell death may partially function through alpha5beta1-integrin, because both the beta1-integrin antibody and the ligand fibronectin inhibited the cytotoxicity of alpha-toxin. Furthermore, we found that the overexpression of the inflammatory cytokine interferon (TNF)-alpha is associated with alpha-toxin-induced cell death, because both the TNF-alpha release inhibitor and antibody effectively inhibited the cytotoxicity of alpha-toxin. In contrast, the cytotoxicity of alpha-toxin was enhanced by the inhibition of the MAPK p38 and NF-kappaB pathways. Taken together, our results suggest that the activation of the MAPK p38 and NF-kappaB pathways are stress responses for survival, rather than direct contributes to alpha-toxin-induced cell death, and that the interaction of alpha-toxin with alpha5beta1-integrin and overproduction of TNF-alpha may contribute to destruction of epithelial cells during S. aureus infection.

Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells

Staphylococcus aureus infections can result in sepsis and septic shock associated with vascular damage and multiple organ failure. Apoptosis appears to play a key role during sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. In contrast to leukocytes, in human umbilical vein endothelial cells and two endothelial cell lines neither purified alpha-toxin nor staphylococcal supernatants were sufficient to induce apoptosis. Apoptosis induction instead required staphylococcal invasion as well as signals from metabolically active intracellular staphylococci. Only strongly haemolytic and invasive staphylococci, but not non-invasive strains induced apoptosis that was caspase-dependent but Fas-independent. However, only a subgroup of clinical isolates with an invasive and haemolytic phenotype induced apoptosis. Expression of alpha-toxin in a non-haemolytic strain partially restored apoptosis induction, suggesting a role of alpha-toxin as a trigger of apoptosis. Furthermore, infection of endothelial cells with isogenic mutants of various regulator genes revealed that apoptosis induction was dependent on the global regulator agr and the alternative sigma factor sigB, but not influenced by sarA. Together, our results indicate that the ability of S. aureus to induce apoptosis in endothelial cells is determined by multiple virulence factors.

Fas (CD95)-Fas ligand interactions are responsible for monocyte apoptosis occurring as a result of phagocytosis and killing of Staphylococcus aureus.

Human peripheral blood monocytes become apoptotic following phagocytosis of Staphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis of S. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 prevents S. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis of S. aureus involves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

Heat-shocked monocytes are resistant to Staphylococcus aureus-induced apoptotic DNA fragmentation due to expression of HSP72.

Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5 degrees C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.