Group B Streptococcus induces macrophage apoptosis by calpain activation.

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.

Effect of trichostatin a and 5'-azacytidine on transgene reactivation in U937 transduced cells.

In mammals, methylation of DNA within regulatory sites and histone deacetylase recruitment in transcriptional repressing domains are involved in the loss of the expression of retroviral DNA or repeat arrays transferred in cells for therapeutic purposes. Various investigation results suggest that methylation/deacetylation events are modulated by extracellular and cytoplasmic signal transduction pathways closely involved in regulating cell differentiation. To analyse gene silencing mechanisms and assess if potential pharmacological treatment affects gene silencing kinetics we transduced U937 myelomonocytic cells with a bicistronic retroviral construct carrying the herpes simplex virus thymidine kinase (HSV-TK) and beta-galactosidase (Lac-Z) genes. This vector can be employed in vivo and in vitro to render transduced cell populations susceptible to ganciclovir (GCV). We verified the effect of the histone deacetylase inhibitor Trichostatin A (TSA) alone or combined with 5'-azacytidine (5'aza-C) on transcription downmodulation. Our results indicate that in our in vitro model TSA is able to reactivate transgene expression, more efficiently and with quicker kinetics (12-24h) than 5'aza-C (36-48 h). The effect is dose dependent (between 1 and 50 nM), with no relevant toxicity. Treatment with both drugs is synergistic in gene reactivation in terms of extension and persistence, with low toxicity and no relevant differentiating effects. The cells in which transgene expression has been reactivated undergo progressive silencing, but once weekly drug treatment can maintain high transgene expression levels for more than 90 days with no evidence of selection. The results obtained by treating U937 transduced clones with TSA and/or 5'aza-C together with IL-3, G-CSF or GM-CSF cytokines suggest that transduced U937 differentiation levels do not affect basal expression, but render these cells more responsive to reactivation by TSA or TSA plus 5'aza-C, but not to 5'aza-C alone. In conclusion, the results suggest that in vitro inhibition of histone deacetylase by TSA can interfere with gene silencing mechanisms affecting 5' Moloney murine leukaemia virus long terminal repeat (MoMuLV-LTR) driven transgene expression thus providing the rationale for TSA and/or 5'aza-C administration in animal models for the translation on gene therapy applications.

Involvement of mitogen-activated protein kinases in Group B Streptococcus-induced macrophage apoptosis.

We previously demonstrated that Group B Streptococcus (GBS), a pathogen that causes serious neonatal infections, induces macrophage apoptosis by beta-hemolysin to avoid the host immune response. GBS-induced macrophage apoptosis is characterized by a calcium increase and is caspase-independent. This study reports the involvement of c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), three members of mitogen-activated protein kinases (MAPKs) family, in GBS-induced macrophage apoptosis. Our data indicate that during induction of apoptosis live GBS stimulates a strong persistent activation of JNK and p38 with concomitant inhibition of ERK. The time courses of MAPKs activation strongly correlate with GBS-induced macrophage apoptosis and are macrophage:GBS ratio-dependent. In fact, when GBS does not cause macrophage apoptosis, e.g. low macrophage:GBS ratio or non hemolytic GBS (gGBS), it induces a transient activation of JNK, p38, and ERK MAPKs. These latter results indicate that sustained and persistent activation of JNK and p38 and inhibition of ERK are involved in the GBS-induced macrophage apoptotic process and suggest that the time course and balance of MAPKs activation are critical for different macrophage responses to GBS (apoptosis versus antimicrobicidal activity). This study indicates a correlation between MAPKs activation and GBS-induced macrophage apoptosis. However, since neither ERK nor p38 inhibitors had an effect on GBS-induced apoptosis, their role in the complex signal network leading to GBS-induced macrophage apoptosis remains to be defined.

Group B streptococcus (GBS) modifies macrophage phosphatidylserine metabolism during induction of apoptosis.

Group B streptococcus (GBS) induced macrophage apoptosis by which it could avoid host defence mechanisms. Macrophages, which constitutively express phosphatidylserine (PtdSer) on the outer leaflet of plasma membrane, increased PtdSer exposure during GBS-induced apoptosis. Induction of apoptosis decreased PtdSer radioactivity of macrophages incubated with [(3)H]serine. The effect appeared not due to increasing conversion of PtdSer to phosphatidylethanolamine or phosphatidylcholine nor to the release of radioactive membrane vesicles. The radioactivity in lysoPtdSer was also reduced. These results confirm that induction of apoptosis involves a modification of PtdSer metabolism and point out the typical features of the GBS-induced apoptosis with respect to other models of apoptosis.