Chikungunya-induced cell death is limited by ER and oxidative stress-induced autophagy.

It has been recognized that macroautophagy constitutes an important survival mechanism that allows both the maintenance of cellular homeostasis and the regulation of programmed cell death pathways (e.g., apoptosis). Although several pathogens have been described to induce autophagy, the prosurvival function of this process in infectious models remains poorly characterized. Our recent studies on chikungunya virus (CHIKV), the causative agent of major epidemics in India, Southeast Asia and southern Europe, reveal a novel mechanism by which autophagy limits the cytopathic effects of CHIKV by impinging upon virus-induced cell death pathways.

Chikungunya virus-induced autophagy delays caspase-dependent cell death.

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.

A whodunit: an appointment with death.

This is the tale of murder, suicide, evolution, and resurrection, taking place in four parts, and all in the name of antigen cross-priming. We invite you to explore the dark mysteries lurking within each of us as you are guided through circuitous cellular pathways in a merciless fight for survival... with viral immunity being the grand finale?!

Receptor-mediated phagocytosis elicits cross-presentation in nonprofessional antigen-presenting cells.

In cross-presentation by dendritic cells (DCs), internalized proteins are retrotranslocated into the cytosol, degraded by the proteasome, and the generated antigenic peptides bind to MHC class I molecules for presentation on the cell surface. Endoplasmic reticulum (ER) contribution to phagosomal membranes is thought to provide antigen access to the ER-associated degradation (ERAD) machinery, allowing cytosolic dislocation. Because the ERAD pathway is present in all cell types and exogenous antigens encounter an ER-containing compartment during phagocytosis, we postulated that forcing phagocytosis in cell types other than DCs would render them competent for cross-presentation. Indeed, FcRgammaIIA expression endowed 293T cells with the capacity for both phagocytosis and ERAD-mediated cross-presentation of an antigen provided as an immune complex. The acquisition of this ability by nonprofessional antigen-presenting cells suggests that a function potentially available in all cell types has been adapted by DCs for presentation of exogenous antigens by MHC class I molecules.

Hsp90-mediated cytosolic refolding of exogenous proteins internalized by dendritic cells.

Dendritic cells efficiently internalize exogenous protein antigens by fluid-phase uptake and receptor-mediated endocytosis. Such antigens contribute to cross-presentation by being translocated into the cytosol for proteasomal degradation, which liberates immunogenic peptides that can bind to major histocompatibility complex (MHC) class I molecules after being transported into the endoplasmic reticulum (ER). MHC class I-peptide complexes are then expressed on the cell surface and presented to CD8+ T cells. Here we show that internalized proteins can have an alternative fate. After internalization, proteins are first unfolded to allow translocation into the cytosol using a pathway related to ER-associated degradation (ERAD). Subsequently the unfolded proteins can undergo cytosolic refolding assisted by the chaperone Hsp90. These observations not only clarify the cellular processes regulating cytosolic access following endocytosis, but also demonstrate that functional proteins can potentially regain their activity in the cytosol of dendritic cells.

A role for the endoplasmic reticulum protein retrotranslocation machinery during crosspresentation by dendritic cells.

Crosspresentation of exogenous antigens (Ags) to CD8(+) T cells by dendritic cells generally requires their entry into the cytosol. Here we show that both soluble and phagocytosed extracellular Ags accessed the cytosol via molecular components required for endoplasmic reticulum (ER)-associated degradation (ERAD). Exogenous Pseudomonas aeruginosa Exotoxin A, which inhibits protein translocation from the ER to the cytosol, abrogated crosspresentation. Exotoxin A also prevented the transporter associated with antigen processing (TAP) inhibitor, ICP47, from entering the cytosol and blocking TAP-mediated peptide transport. In an in vitro model of retrotranslocation, the AAA ATPase p97, an enzyme critical for ERAD, was the only cytosolic cofactor required for protein export from isolated phagosomes. Functional p97 was also required for crosspresentation but not conventional presentation. Thus, crosspresentation appears to result from an adaptation of the retrotranslocation mechanisms involved in the degradation of misfolded ER proteins.

Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles.

CD8(+) T-cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8(+) T cells against these cell-associated antigens, exogenous antigens must be cross-presented by professional antigen-presenting cells (APCs). While cross-presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein-based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow-derived dendritic cells (BMDCs), the MHC class I presentation of PLGA-encapsulated ovalbumin (OVA) stimulated T cell interleukin-2 secretion at 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead-associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle-mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Mechanisms of MHC class I-restricted antigen processing and cross-presentation.

In this review, we discuss recent data from our laboratory that address two aspects of major histocompatibility complex (MHC) class I-restricted antigen processing. First, we consider the nature of the peptide-loading complex, which is the assembly of proteins in the endoplasmic reticulum (ER) into which newly synthesized MHC class I-beta(2) microglobulin (beta(2)m) heterodimers are incorporated, and the mechanisms involved in MHC class I assembly and peptide loading that are facilitated by the peptide-loading complex. Second, we discuss mechanisms of cross-presentation, the phenomenon whereby extracellular and luminal protein antigens can be processed by antigen-presenting cells, particularly dendritic cells, and presented by MHC class I molecules to CD8(+) T cells. The focus of the discussion is mainly on the human MHC class I system.

Regulation of microtubule stability and mitotic progression by survivin.

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which has been implicated in both preservation of cell viability and regulation of mitosis in cancer cells. Here, we show that HeLa cells microinjected with a polyclonal antibody to survivin exhibited delayed progression in prometaphase (31.5 +/- 6.9 min) and metaphase (126.8 +/- 73.8 min), as compared with control injected cells (prometaphase, 21.5 +/- 3.3 min; metaphase, 18.9 +/- 4.5 min; P < 0.01). Cells injected with the antibody to survivin displayed short mitotic spindles severely depleted of microtubules and occasionally underwent apoptosis without exiting the mitotic block or thereafter. Forced expression of survivin in HeLa cells profoundly influenced microtubule dynamics with reduction of pole-to-pole distance at metaphase (8.57 +/- 0.21 microm versus 10.58 +/- 0.19 microm; P < 0.0001) and stabilization of microtubules against nocodazole-induced depolymerization in vivo. These data demonstrate that survivin functions at cell division to control microtubule stability and assembly of a normal mitotic spindle. This pathway may facilitate checkpoint evasion and promote resistance to chemotherapy in cancer.

Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function.

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala(3)-Ile(19) exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence, nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys(57)-Trp(67) identified a cytosolic pool of survivin, which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala(3)-Ile(19), Met(38)-Thr(48), Pro(47)-Phe(58) and Cys(57)-Trp(67) identified both survivin pools within the same mitotic cell. A ratio of approximately 1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34(cdc2), and was phosphorylated by p34(cdc2) on Thr(34), in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34(cdc2) and was not phosphorylated on Thr(34). Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.