Brutigam (Section of Surgery, Charit Universittsmedizin Berlin, Berlin, Germany) for providing primary keratinocytes; N

Brutigam (Section of Surgery, Charit Universittsmedizin Berlin, Berlin, Germany) for providing primary keratinocytes; N. virus-induced c-FLIP downregulation in epithelial cells and many other cell types that do not undergo apoptosis after HSV-1 contamination. Microarray analyses revealed that HSV-1-encoded latency-associated transcript (LAT) sequences, which can substitute for c-FLIP as an inhibitor of caspase-8-mediated apoptosis, are much less abundant in iDCs as compared to epithelial cells. Finally, iDCs infected with an HSV-1 LAT knockout mutant showed increased apoptosis when compared to iDCs infected with the corresponding wild-type HSV-1. Taken together, our results demonstrate that apoptosis of HSV-1-infected iDCs requires both c-FLIP downregulation and diminished expression of viral LAT. Herpes simplex virus type 1 (HSV-1) is an important human pathogen and causes mucocutaneous lesions in the orolabial region (48). Less often, HSV contamination can result in life-threatening encephalitis in otherwise healthy individuals or disseminated contamination in immunocompromised persons. Neonates infected during delivery can also develop severe disseminated contamination. After primary contamination of epithelial cells, HSV-1 establishes a state of latency in ganglia and persists for the lifetime of the host (59). In response to certain stimuli, the Isosteviol (NSC 231875) computer virus reactivates from latency without neuronal death. Thereafter, newly assembled daughter virions migrate Isosteviol (NSC 231875) down the axon IL17RC antibody toward epithelial tissue, causing recurrent infections of mucocutaneous regions and computer virus shedding. Around the molecular level, HSV-1 consists of more than 80 genes that are expressed sequentially in a strongly regulated cascade (23,24). Apoptosis of host cells represents an important defense mechanism against viral invasion by preventing viral replication and dissemination. The extrinsic pathway of apoptosis induction is usually brought on by ligation of death receptors (36) or by injection of granzymes (63). Intrinsic triggers of apoptosis such as DNA damage, oxidative stress, deprivation of growth factors, and viral contamination disrupt the integrity of the mitochondrial membrane, resulting in release of cytochromecinto the cytoplasm (46,68). Transduction of the apoptosis-inducing signal within the cell occurs via initiator caspases: caspase-8 in the extrinsic pathway and caspase-9 in the intrinsic pathway. Both activate downstream effector caspases that cleave cellular substrates, leading Isosteviol (NSC 231875) to the characteristic Isosteviol (NSC 231875) morphological and biochemical features of apoptosis. The apoptosis signaling network is usually negatively regulated by caspase inhibitor molecules such as the cytokine response modifier A (CrmA) and the cellular FLICE-inhibitory protein (c-FLIP) (3,6). Both splice variants of the cellular FLICE-inhibitory protein, the long form (c-FLIPL) and the short form (c-FLIPS), represent important inhibitors of death receptor-induced apoptosis (38). They act as catalytically inactive caspase-8 homologues, which compete with caspase-8 at the level of the death-inducing signaling complex (DISC) to prevent its activation. Viruses, including HSV-1, have evolved numerous strategies to counteract apoptosis (3,6,45). Apoptosis is usually induced in response to HSV-1 at several signaling checkpoints, and the computer virus in turn has evolved multiple mechanisms that block apoptosis to prevent premature cell death (20). It has been shown that HSV-1 renders infected cells resistant to apoptosis induced by cytotoxic T lymphocytes (31). Moreover, studies in rabbits (52) and mice (1,10) have shown that latently infected neurons are resistant to apoptosis, although only noncoding latency-associated transcript (LAT) and no viral proteins could be detected. A major immune evasion strategy of HSV-1 is usually to attack immune cells, including T cells (21,27,28,57), B cells (21), and macrophages (18). Most importantly, like many other herpesviruses (8), HSV-1 also targets human immature dendritic cells (iDCs) (9,39,41,44,55,60) and induces apoptosis (9,44,55). As these cells play a central role in the induction of efficient immune responses (54,64), HSV-1-induced apoptosis of iDCs represents a mechanism of immune evasion giving the computer virus the opportunity to replicate efficiently in epithelial cells and establish latency in neurons. We have previously shown that HSV-1-induced apoptosis of iDCs is usually associated with decreased expression of the antiapoptotic cellular c-FLIP protein (44). In this study, we further analyze the mechanism of c-FLIP downregulation in iDCs and investigate whether c-FLIP downregulation is sufficient to drive uninfected iDCs into apoptosis. Furthermore, we show that many different cell types other than iDCs, including epithelial cells, also decrease expression of c-FLIP protein in response to the computer virus, although they do not undergo apoptosis after contamination. Finally, we address the question why iDCs but not epithelial cells, the main factory for production of HSV daughter virions, are prone to apoptosis after HSV contamination. == MATERIALS AND METHODS == == Cells. == Monocytes were obtained from buffy coats of healthy donors (DRK, Berlin, Germany) by density gradient centrifugation and subsequent isolation with Monocyte isolation kit II Isosteviol (NSC 231875) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Monocytes were then differentiated into iDCs in RPMI.

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