Brendan Marshall

Cutting Edge: Induced Indoleamine 2,3 Dioxygenase Expression in Dendritic Cell Subsets Suppresses T Cell Clonal Expansion

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8α, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.

TRYPTOPHAN CATABOLISM AND T CELL RESPONSES

Cells expressing indoleamine 2,3 dioxygenase (IDO) play key roles in regulating adaptive immune responses orchestrated by T cells. In this report we discuss our working model, the tryptophan depletion hypothesis, to explain links between IDO expression and inhibition of T cell responses. We posit that IDO+ cells, particularly professional antigen presenting cells (APCs) promote T cell entry but block cell cycle progression due to tryptophan catabolism. We discuss experimental evidence supporting predictions from the tryptophan depletion hypothesis and the implications that this model has for understanding the origin of tolerant states that explain immunological paradoxes, such as fetal survival, tumor persistence and failure to eradicate pathogens like HIV that cause persistent infections.

Regulation of prostaglandin synthesis and cell adhesion by a tryptophan catabolizing enzyme

BackgroundThe tryptophan catabolizing enzyme, indoleamine 2,3, dioxygenase (IDO) is one of two mammalian enzymes, which can catabolize the rarest essential amino acid, tryptophan. IDO is inducible by cytokines such as interferon-γ and plays a role in inflammation and maternal tolerance of fetal allografts, although its exact mode of action is unclear. Therefore, we investigated the circumstances under which IDO is expressed in vitro together with the effects of overexpression of IDO on the growth and morphology of cells.ResultsOverexpression of IDO in the murine macrophage cell line RAW 264.7 and the murine fibrosarcoma cell line MC57, resulted in the growth of macroscopic cell foci, with altered cell adhesion properties. The expression of IDO was also detected during adhesion of wild type, nontransfected cells in tissue culture to standard cell growth substrates. Inhibition of this expression, likewise resulted in alterations in cell adhesion. Overexpression of IDO or inhibition of endogenous IDO expression was accompanied by changes in metalloproteinase expression and also in the expression and activity of the cyclooxygenase enzymes. In the case of RAW cells, IDO effects on cell growth could be reversed by adding back prostaglandins.ConclusionsThese results suggest that catabolism of the rarest essential amino acid may regulate processes such as cell adhesion and prostaglandin synthesis.

Ceramide activates lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that are hallmarks of human cancer. MDSCs inhibit cytotoxic T lymphocytes (CTLs) and NK cell functions to promote tumor immune escape and progression, and therefore are considered key targets in cancer immunotherapy. Recent studies determined a key role of the apoptosis pathways in tumor-induced MDSC homeostasis and it is known that ceramide plays a key role in regulation of mammalian cell apoptosis. In this study, we aimed to determine the efficacy and underlying molecular mechanism of ceramide in suppression of MDSCs. Treatment of tumor-bearing mice with LCL521, a lysosomotropic inhibitor of acid ceramidase, significantly decreased MDSC accumulation in vivo. Using a MDSC-like myeloid cell model, we determined that LCL521 targets lysosomes and increases total cellular C16 ceramide level. Although MDSC-like cells have functional apoptosis pathways, LCL521-induced MDSC death occurs in an apoptosis- and necroptosis-independent mechanism. LCL521 treatment resulted in an increase in the number of autophagic vesicles, heterolysosomes and swollen ERs. Finally, concomitant inhibition of cathepsin B and cathepsin D was required to significantly decrease LCL521-induced cell death. Our observations indicate that LCL521 targets lysosomes to activate cathepsin B and cathepsin D, resulting in interrupted autophagy and ER stress that culminates in MDSC death. Therefore, a ceramidase inhibitor is potentially an effective adjunct therapeutic agent for suppression of MDSCs to enhance the efficacy of CTL-based cancer immunotherapy.

An Outer Mitochondrial Translocase, Tom22, Is Crucial for Inner Mitochondrial Steroidogenic Regulation in Adrenal and Gonadal Tissues

ABSTRACT After cholesterol is transported into the mitochondria of steroidogenic tissues, the first steroid, pregnenolone, is synthesized in adrenal and gonadal tissues to initiate steroid synthesis by catalyzing the conversion of pregnenolone to progesterone, which is mediated by the inner mitochondrial enzyme 3β-hydroxysteroid dehydrogenase 2 (3βHSD2). We report that the mitochondrial translocase Tom22 is essential for metabolic conversion, as its knockdown by small interfering RNA (siRNA) completely ablated progesterone conversion in both steroidogenic mouse Leydig MA-10 and human adrenal NCI cells. Tom22 forms a 500-kDa complex with mitochondrial proteins associated with 3βHSD2. Although the absence of Tom22 did not inhibit mitochondrial import of cytochrome P450scc (cytochrome P450 side chain cleavage enzyme) and aldosterone synthase, it did inhibit 3βHSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3βHSD2 is localized at the inner mitochondrial space (IMS), where it interacts through a specific region with Tom22 with its C-terminal amino acids and a small amino acid segment of Tom22 exposed to the IMS. Therefore, Tom22 is a critical regulator of steroidogenesis, and thus, it is essential for mammalian survival.

Murine cytomegalovirus infection and apoptosis in organotypic retinal cultures.

PURPOSE An organotypic retinal culture model was used to determine the pattern of murine cytomegalovirus (MCMV) infection and whether apoptosis is induced in MCMV-infected cultured retinas. METHODS Retinas harvested from C57BL/6 mice were individually cultured at 37 degrees C on 3-microm filter inserts placed in 24-well plates. Some retinas were infected with MCMV (5 x 10(5) PFU/well). At days 4, 7, and 11 after infection (pi), the culture medium and cultured retinas were collected for examination. RESULTS Replicating virus was recovered and viral early antigen (EA)- and late antigen (LA)-positive cells were observed in the MCMV-infected retinal cultures. Most MCMV-infected cells were glia and horizontal cells. Infection resulted in atrophy of the photoreceptor cells and cytomegaly. Apoptosis of uninfected bystander cells, including photoreceptor cells and horizontal cells, was observed. TNF-alpha was produced by activated microglia during MCMV infection of the retina. Mouse apoptosis microarray studies, caspase activity studies, and RT-PCR studies showed that the genes involved in both the death receptor-mediated apoptotic pathway and the mitochondrial pathway were upregulated. CONCLUSIONS Many aspects of MCMV infection of retinal cultures parallel those observed during MCMV retinitis in mice. Thus, this in vitro system may be used to explore the role of apoptosis of uninfected retinal cells and the contribution of cytokines and other modulators to the pathogenesis of CMV retinitis.

Decreased protein nitration in macrophages that overexpress indoleamine 2, 3-dioxygenase.

The activity of indoleamine 2, 3-dioxygenase (IDO; E.C. 1.13.11.42) catalyzes the oxidative cleavage of tryptophan to form kynurenine. IDO activity consumes superoxide anions; therefore, we postulated that over-expression of IDO might mitigate superoxide-anion dependent, oxidative modification of cellular proteins in vitro. We prepared and characterized RAW 264.7 macrophages that were stably transfected with either an IDO expression vector or the control (empty) vector. We detected IDO mRNA, protein, and enzyme activity in the IDO-transfected macrophages, but not in the macrophages transfected with the empty vector. To generate superoxide anions in situ, we treated the IDO-and control-transfected cultures with xanthine or hypoxanthine, and then used ELISA methods to quantitate the relative levels of oxidatively modified proteins in total cell lysates. The levels of protein carbonyls were similar in IDO-transfected and vector-transfected macrophages; however, protein nitration was significantly less in IDO-transfected cells compared to control transfectants. In addition, steady-state levels of superoxide anions were significantly lower in the IDO-transfected cultures compared with control transfectants. Our results are consistent with the concept that, besides degrading tryptophan, IDO activity may protect cells from oxidative damage.

Mitochondrial metabolic regulation by GRP78

ER chaperone GRP78 is the central regulator of adrenals and gonadal steroid synthesis for mammalian survival. Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity.

Virus spread and immune response following anterior chamber inoculation of HSV-1 Lacking the Beclin-binding domain (BBD)

The autophagy response induced by HSV-1 infection is antagonized by the Beclin-binding domain (BBD). The purpose of this study was to determine if lack of the BBD affects viral spread and immune response in the eyes and brain. Our results showed that lack of the BBD increases autophagy response and activation of NLRP3 inflammasome, which in turn induces a more rapid innate immune response mediated by macrophage/microglia and NK cells in the injected eye, limiting virus replication and retinal damage. We conclude that autophagy plays a role in controlling HSV-1 infection by more rapid induction of the innate immune response.

Lack of TNF-α Promotes Caspase-3–Independent Apoptosis during Murine Cytomegalovirus Retinitis

PURPOSE Both caspase-dependent and caspase-independent apoptosis contribute to retinal damage during murine cytomegalovirus (MCMV) retinitis, and TNF-α is among the inducers of apoptosis. The aim of this study was to determine the contribution of TNF-α by studying virus replication and apoptosis in immunosuppressed (IS) TNF-α(-/-) mice. METHODS IS TNF-α(-/-) mice or wild-type mice were inoculated with MCMV by the supraciliary route. Injected eyes were examined by plaque assay, electron microscopy, Western blot analysis (caspase-3, caspase-8, caspase-12, Bid, NF-κB, cFlip, XIAP), staining for MCMV early antigen, and TUNEL assay. RESULTS Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retinas of IS TNF-α(-/-) mice than in those of wild-type mice. The level of active caspase-3 was similar in both groups; however, more activated proteins for genes involved in the mitochondrial pathway (cleaved caspase-8, tBid) and endoplasmic reticulum (ER) stress (cleaved caspase-12) and, though less active, NF-κB subunits and antiapoptotic proteins (XIAP and cFlip) were detected in the TNF-α(-/-) eyes compared with wild-type mice. CONCLUSIONS Although TNF-α is an inducer of apoptosis, the results of this study suggest that TNF-α is also antiapoptotic by the following mechanism: TNF-α activation of NF-κB promotes the production of the antiapoptosis genes, c-flip or XIAP, which, in turn, inhibit the activation of caspase-8 and the mitochondrial pathway or the activation of caspase-12 and ER stress.