Publications by authors named "Karen A Fortner"

28 Publications

  • Page 1 of 1

IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca through the noncanonical STAT3 pathway.

Proc Natl Acad Sci U S A 2021 Sep;118(37)

Immunology and Microbiology Department, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.
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http://dx.doi.org/10.1073/pnas.2103444118DOI Listing
September 2021

T cell homeostatic proliferation promotes a redox state that drives metabolic and epigenetic upregulation of inflammatory pathways in lupus.

Antioxid Redox Signal 2021 Jul 30. Epub 2021 Jul 30.

University of Vermont College of Medicine, 12352, Burlington, Vermont, United States;

Significance: Numerous abnormalities in T cells have been described in patients with systemic lupus erythematosus (SLE), including lymphopenia, DNA demethylation, expression of endogenous retroviruses, increased cell death, enlarged mitochondria, production of reactive oxygen species (ROS), and the appearance of CD4-CD8- T cells. We propose a model in which accelerated homeostatic proliferation of T cells promotes an epigenetic and metabolic program leading to this cluster of abnormalities. Recent Advances: Growing knowledge of the innate immune disorders in SLE has included increased mitochondrial size and ROS production that induces oligomerization of mitochondrial antiviral-signaling (MAVS) protein and type I Interferon production, as well as DNA demethylation, upregulation of inflammatory genes, and expression of certain endogenous retroviruses in SLE peripheral blood mononuclear cells (PBMC). All of these events are part of the cellular program that occurs during homeostatic proliferation of T cells.

Critical Issues: Despite extensive knowledge of the myriad autoantibodies in SLE and other immune abnormalities, a cogent model has been lacking to link the numerous and seemingly disparate immune aberrations. This may partly explain the general lack of new drugs specifically for SLE in over 50 years. A more coherent model of SLE would not only unify the variety of immune abnormalities is SLE, it would also suggest new therapies.

Future Directions: The model of augmented homeostatic proliferation leading to increased mitochondrial mass, ROS, DNA demethylation, and upregulation of inflammatory genes suggests strategic new targets for SLE, including antioxidants and certain inhibitors of metabolism.
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http://dx.doi.org/10.1089/ars.2021.0078DOI Listing
July 2021

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Nat Commun 2021 05 14;12(1):2804. Epub 2021 May 14.

Division of Immunobiology, Department of Medicine, Larner College of Medicine, University of Vermont, Burlington, VT, USA.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.
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http://dx.doi.org/10.1038/s41467-021-23071-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121950PMC
May 2021

Selective DNA Demethylation Accompanies T Cell Homeostatic Proliferation and Gene Regulation in Lupus-Prone Mice.

Immunohorizons 2020 10 23;4(10):679-687. Epub 2020 Oct 23.

Vermont Center for Immunology and Infectious Diseases, Department of Medicine, University of Vermont Larner College of Medicine, Burlington, VT 05405; and

Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4CD8TCR-αβ (DN) T cells that arise from CD8 precursors and are also found in human SLE. We have previously observed that T cell accumulation in mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in DN T cells compared with their CD8 precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis.
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http://dx.doi.org/10.4049/immunohorizons.2000078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141279PMC
October 2020

Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD) by increasing mitochondrial fatty acid oxidation.

Nat Commun 2020 07 3;11(1):3360. Epub 2020 Jul 3.

Department of Medicine, Immunobiology Division, University of Vermont, Burlington, VT, 05405, USA.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.
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http://dx.doi.org/10.1038/s41467-020-16991-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334216PMC
July 2020

Targeting mitochondrial oxidative stress with MitoQ reduces NET formation and kidney disease in lupus-prone MRL- mice.

Lupus Sci Med 2020 04 16;7(1). Epub 2020 Apr 16.

Vermont Center for Immunology and Infectious Diseases, Department of Medicine, University of Vermont Larner College of Medicine, Burlington, VT, USA

Objectives: Recent investigations in humans and mouse models with lupus have revealed evidence of mitochondrial dysfunction and production of mitochondrial reactive oxygen species (mROS) in T cells and neutrophils. This can provoke numerous cellular changes including oxidation of nucleic acids, proteins, lipids and even induction of cell death. We have previously observed that in T cells from patients with lupus, the increased mROS is capable of provoking oligomerisation of mitochondrial antiviral stimulator (MAVS) and production of type I interferon (IFN-I). mROS in SLE neutrophils also promotes the formation of neutrophil extracellular traps (NETs), which are increased in lupus and implicated in renal damage. As a result, in addition to traditional immunosuppression, more comprehensive treatments for lupus may also include non-immune therapy, such as antioxidants.

Methods: Lupus-prone MRL- mice were treated from weaning for 11 weeks with the mitochondria-targeted antioxidant, MitoQ (200 µM) in drinking water. Mice were then assessed for ROS production in neutrophils, NET formation, MAVS oligomerisation, serum IFN-I, autoantibody production and renal function.

Results: MitoQ-treated mice manifested reduced neutrophil ROS and NET formation, decreased MAVS oligomerisation and serum IFN-I, and reduced immune complex formation in kidneys, despite no change in serum autoantibody .

Conclusions: These findings reveal the potential utility of targeting mROS in addition to traditional immunosuppressive therapy for lupus.
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http://dx.doi.org/10.1136/lupus-2020-000387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199895PMC
April 2020

Glycolysis Induces MCJ Expression That Links T Cell Proliferation With Caspase-3 Activity and Death.

Front Cell Dev Biol 2019 11;7:28. Epub 2019 Mar 11.

Vermont Center for Immunology and Infectious Diseases, Larner College of Medicine, University of Vermont, Burlington, VT, United States.

An effective adaptive immune response requires rapid T cell proliferation, followed by equally robust cell death. These two processes are coordinately regulated to allow sufficient magnitude of response followed by its rapid resolution, while also providing the maintenance of T cell memory. Both aspects of this T cell response are characterized by profound changes in metabolism; glycolysis drives proliferation whereas oxidative phosphorylation supports the survival of memory T cells. While much is known about the separate aspects of T cell expansion and contraction, considerably less is understood regarding how these processes might be connected. We report a link between the induction of glycolysis in CD8 T cells and upregulation of the inhibitor of complex I and oxidative phosphorylation, methylation-controlled J protein (MCJ). MCJ acts synergistically with glycolysis to promote caspase-3 activity. Effector CD8 T cells from MCJ-deficient mice manifest reduced glycolysis and considerably less active caspase-3 compared to wild-type cells. Consistent with these observations, in non-glycolytic CD8 T cells cultured in the presence of IL-15, MCJ expression is repressed by methylation, which parallels their reduced active caspase-3 and increased survival compared to glycolytic IL-2-cultured T cells. Elevated levels of MCJ are also observed in the highly proliferative and glycolytic subset of CD4CD8 T cells in Fas-deficient mice. This subset also manifests elevated levels of activated caspase-3 and rapid cell death. Collectively, these data demonstrate tight linkage of glycolysis, MCJ expression, and active caspase-3 that serves to prevent the accumulation and promote the timely death of highly proliferative CD8 T cells.
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http://dx.doi.org/10.3389/fcell.2019.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421275PMC
March 2019

Glycolysis promotes caspase-3 activation in lipid rafts in T cells.

Cell Death Dis 2018 01 19;9(2):62. Epub 2018 Jan 19.

Vermont Center for Immunology and Infectious Diseases, Larner College of Medicine, University of Vermont, Burlington, VT, USA.

Resting T cells undergo a rapid metabolic shift to glycolysis upon activation in the presence of interleukin (IL)-2, in contrast to oxidative mitochondrial respiration with IL-15. Paralleling these different metabolic states are striking differences in susceptibility to restimulation-induced cell death (RICD); glycolytic effector T cells are highly sensitive to RICD, whereas non-glycolytic T cells are resistant. It is unclear whether the metabolic state of a T cell is linked to its susceptibility to RICD. Our findings reveal that IL-2-driven glycolysis promotes caspase-3 activity and increases sensitivity to RICD. Neither caspase-7, caspase-8, nor caspase-9 activity is affected by these metabolic differences. Inhibition of glycolysis with 2-deoxyglucose reduces caspase-3 activity as well as sensitivity to RICD. By contrast, IL-15-driven oxidative phosphorylation actively inhibits caspase-3 activity through its glutathionylation. We further observe active caspase-3 in the lipid rafts of glycolytic but not non-glycolytic T cells, suggesting a proximity-induced model of self-activation. Finally, we observe that effector T cells during influenza infection manifest higher levels of active caspase-3 than naive T cells. Collectively, our findings demonstrate that glycolysis drives caspase-3 activity and susceptibility to cell death in effector T cells independently of upstream caspases. Linking metabolism, caspase-3 activity, and cell death provides an intrinsic mechanism for T cells to limit the duration of effector function.
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http://dx.doi.org/10.1038/s41419-017-0099-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833351PMC
January 2018

The molecular signature of murine T cell homeostatic proliferation reveals both inflammatory and immune inhibition patterns.

J Autoimmun 2017 Aug 24;82:47-61. Epub 2017 May 24.

Department of Medicine, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8 T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4CD8TCRαβ T cell subset that results from extensive homeostatic expansion of CD8 T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.
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http://dx.doi.org/10.1016/j.jaut.2017.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902411PMC
August 2017

IL-6 promotes the differentiation of a subset of naive CD8+ T cells into IL-21-producing B helper CD8+ T cells.

J Exp Med 2016 10 26;213(11):2281-2291. Epub 2016 Sep 26.

Department of Medicine/Immunobiology Program, University of Vermont, Burlington, VT 05405

IL-6 is known to contribute to the differentiation of CD4 T cells into different subsets of effector T helper cells. Less is known about the potential of IL-6 in regulating CD8 T cell effector function. Here, we identify IL-6 as a master regulator of IL-21 in effector CD8 T cells. IL-6 promotes the differentiation of a subset of naive CD8 T cells that express IL-6R into a unique population of effector CD8 T cells characterized by the production of high levels of IL-21 and low levels of IFN-γ. Similar to CD4 T follicular helper (Tfh) cells, IL-21-producing CD8 T cells generated in the presence of IL-6 directly provide help to B cells to induce isotype switching. CD8 T cell-derived IL-21 contributes to the production of protective virus-specific IgG antibodies during influenza virus infection. Thus, this study reveals the presence of a new mechanism by which IL-6 regulates antibody production during viral infection, and a novel function of effector CD8 T cells in the protection against viruses.
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http://dx.doi.org/10.1084/jem.20160417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068236PMC
October 2016

Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

J Innate Immun 2016 19;8(5):479-92. Epub 2016 Jul 19.

Vermont Center for Immunology and Infectious Diseases, Department of Medicine, The University of Vermont College of Medicine, Burlington, Vt., USA.

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress.
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http://dx.doi.org/10.1159/000446498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002261PMC
October 2017

Fine-Tuning of CD8(+) T Cell Mitochondrial Metabolism by the Respiratory Chain Repressor MCJ Dictates Protection to Influenza Virus.

Immunity 2016 06 24;44(6):1299-311. Epub 2016 May 24.

Program in Immunobiology, Department of Medicine, University of Vermont, Burlington, Vermont, 05405 USA. Electronic address:

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.
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http://dx.doi.org/10.1016/j.immuni.2016.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917451PMC
June 2016

Protein disulfide isomerase-endoplasmic reticulum resident protein 57 regulates allergen-induced airways inflammation, fibrosis, and hyperresponsiveness.

J Allergy Clin Immunol 2016 Mar 4;137(3):822-32.e7. Epub 2015 Oct 4.

Department of Pathology and Laboratory Medicine, University of Vermont College of Medicine, Burlington, VT. Electronic address:

Background: Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum-localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown.

Objectives: Here we sought to examine the contribution of airway epithelium-specific ERp57 in the pathogenesis of allergic asthma.

Methods: We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57.

Results: Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite-challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite-induced endoplasmic reticulum stress and airways fibrosis.

Conclusions: Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype.
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http://dx.doi.org/10.1016/j.jaci.2015.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597791PMC
March 2016

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest.

J Cell Physiol 2015 Aug;230(8):1929-43

Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.
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http://dx.doi.org/10.1002/jcp.24927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414874PMC
August 2015

The c-FLIPL cleavage product p43FLIP promotes activation of extracellular signal-regulated kinase (ERK), nuclear factor κB (NF-κB), and caspase-8 and T cell survival.

J Biol Chem 2014 Jan 25;289(2):1183-91. Epub 2013 Nov 25.

From the Vermont Center for Immunology and Infectious Diseases and.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.
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http://dx.doi.org/10.1074/jbc.M113.506428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887185PMC
January 2014

Proliferating γδ T cells manifest high and spatially confined caspase-3 activity.

Immunology 2012 Apr;135(4):276-86

Department of Medicine, Immunobiology Program, The University of Vermont College of Medicine, Burlington, VT 05405, USA.

Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T-cell antigen receptor (TCR) signalling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T-cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4(+) and CD8(+) αβ T cells and γδ T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4(+) T cells manifested the lowest levels of active caspases whereas γδ T cells manifested the highest levels. Further analysis revealed that most of the difference in T-cell subsets was the result of high levels of active caspase-3 in non-dying effector γδ T cells. Despite this, γδ T cells manifested little spontaneous or CD3 restimulation-induced cell death as the result of confinement of active caspases to the cell membrane. By contrast, CD4(+) T cells were highly sensitive to CD3-induced cell death, associated with the appearance of active caspases in the cytoplasm and cleavage of the caspase substrates Bid and ICAD. Hence, the location and amount of active caspases distinguishes effector T-cell subsets and profoundly influences the fate of the T-cell response.
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http://dx.doi.org/10.1111/j.1365-2567.2011.03540.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372744PMC
April 2012

Increased caspase activity primes human Lyme arthritis synovial γδ T cells for proliferation and death.

Hum Immunol 2011 Dec 22;72(12):1168-75. Epub 2011 Sep 22.

Vermont Center for Immunology and Infectious Diseases, University of Vermont College of Medicine, Burlington, VT 05405, USA.

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function and manifesting cytolytic activity. Their numbers often increase during infections, such as human immunodeficiency virus, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we observed that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared with αβ T cells because of increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and because signaling is more efficient through T cell receptor (TCR)-γδ than through TCR-αβ, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than in comparable αβ T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model in which human γδ T cells evolved to function quickly and transiently in an innate fashion.
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http://dx.doi.org/10.1016/j.humimm.2011.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224150PMC
December 2011

Reduced immune response to Borrelia burgdorferi in the absence of γδ T cells.

Infect Immun 2011 Oct 18;79(10):3940-6. Epub 2011 Jul 18.

Vermont Center for Immunology and Infectious Diseases, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.

Little is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cells in vitro are activated by Borrelia burgdorferi in a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cells in vitro to produce cytokines and chemokines that are important for the adaptive immune response. This suggested that in vivo γδ T cells may assist in activating the adaptive immune response. We examined this possibility in vivo and observed that γδ T cells are activated and expand in number during Borrelia infection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borrelia antibodies, cytokines, and chemokines. This paralleled a greater Borrelia burden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.
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http://dx.doi.org/10.1128/IAI.00148-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187251PMC
October 2011

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts.

Int Immunol 2011 Feb 25;23(2):75-88. Epub 2011 Jan 25.

Immunobiology Program, Department of Medicine, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.
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http://dx.doi.org/10.1093/intimm/dxq466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030730PMC
February 2011

Apoptosis regulators Fas and Bim synergistically control T-lymphocyte homeostatic proliferation.

Eur J Immunol 2010 Nov 27;40(11):3043-53. Epub 2010 Oct 27.

Vermont Center for Immunology and Infectious Disease, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.

The size of the peripheral T-lymphocyte compartment is governed by complex homeostatic mechanisms that balance T-cell proliferation and death. Proliferation and survival signals are mediated in part by recurrent self-peptide/MHC-TCR interactions and signaling by the common γ chain-containing cytokine receptors, including those for IL-7 and IL-15. We have previously shown that the death receptor Fas (CD95/APO-1) regulates apoptosis in response to repeated TCR stimulation, whereas the Bcl-2 homology domain 3-only protein Bim mediates cytokine withdrawal-induced apoptosis. We therefore reasoned that these two molecules might cooperate in the regulation of homeostatic proliferation. In this study, we observe that the combined loss of Fas and Bim synergistically enhances the accumulation of T cells in lymphopenic host mice, and this is particularly pronounced for the unusual CD4(-) CD8(-) TCRαβ(+) T cells that are characteristic of Fas-deficient (Fas(lpr/lpr) ) mice. Our findings demonstrate that these CD4(-) CD8(-) TCRαβ(+) T cells arise from homeostatic proliferation of CD8(+) T cells. These studies also underscore the profound rate of baseline T-cell proliferation that likely occurs in wild-type mice even in the absence of foreign antigen, and the consequent need for its coordinated regulation by multiple death-signaling pathways.
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http://dx.doi.org/10.1002/eji.201040577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334341PMC
November 2010

Evidence that CD8 T-cell homeostasis and function remain intact during murine pregnancy.

Immunology 2010 Nov;131(3):426-37

University of Vermont College of Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, Burlington, VT 05405, USA.

Evolving models of immune tolerance have challenged the view that the response of the maternal immune system to environmental or fetal antigens must be suppressed or deviated. CD8 T cells play a central role in the immune response to viruses and intracellular pathogens so the maintenance of both the number and function of these cells is critical to protect both the mother and fetus. We show that the numbers of maternal CD8 T cells in both the spleen and the uterine draining lymph nodes are transiently increased at mid-gestation and this correlates with enhanced CD8 T-cell proliferation and an increased relative expression of both pro-survival and pro-apoptotic molecules. In transgenic mice bearing T-cell antigen receptors specific for the male HY or allo-antigens, the transgenic CD8 T cells retain the ability to proliferate and function during pregnancy. Moreover, anti-HY T-cell receptor transgenic mice have normal numbers of male pups despite the presence of CD8 T cells at the maternal-fetal interface. These data suggest that pregnancy is a dynamic state in which CD8 T-cell turnover is increased while the function and ending size of the CD8 T-cell compartment are maintained.
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http://dx.doi.org/10.1111/j.1365-2567.2010.03316.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996563PMC
November 2010

Pregnancy alters the proliferation and apoptosis of mouse splenic erythroid lineage cells and leukocytes.

Biol Reprod 2009 Sep 15;81(3):457-64. Epub 2009 Apr 15.

Department of Obstetrics, Gynecology, and Reproductive Sciences, The University of Vermont College of Medicine, Burlington, Vermont 05405, USA.

Pregnancy induces dynamic changes in the maternal environment that include reversible modifications in response to systemic mediators and local signals. The spleen can be used to determine the effects of pregnancy on multiple cellular populations, including those of the erythroid lineage and the immune system. Current evidence suggests that the transient increase in the size of the spleen during pregnancy is due to the expansion of erythroid precursors. However, it is unclear what factors contribute to this increase. Moreover, the additional erythroid cells may compete with neighboring leukocytes for growth factors or space, and this may in turn alter the function of these populations. Therefore, we assessed proliferation and apoptosis throughout gestation using in vivo bromodeoxyuridine incorporation and the TUNEL assay, respectively. Here, we show that erythroid-lineage TER-119(+) cells expanded significantly in midgestation because of enhanced proliferation and diminished apoptosis. This correlated with increased expression of the erythropoietin receptor (Epor) and decreased expression of the death receptor Fas, respectively. Leukocytes demonstrated population-specific responses. Natural killer cells proliferated in early pregnancy. Both lymphocytes and CD11B(+) cells underwent enhanced proliferation during midgestation. In contrast, neutrophils exhibited augmented proliferation throughout pregnancy. These subset-specific alterations in proliferation and death in the spleen suggest that complex regulation of population dynamics exists during pregnancy.
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http://dx.doi.org/10.1095/biolreprod.109.076976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731983PMC
September 2009

Activation of gamma delta T cells by Borrelia burgdorferi is indirect via a TLR- and caspase-dependent pathway.

J Immunol 2008 Aug;181(4):2392-8

Immunobiology Program, Department of Medicine, The University of Vermont College of Medicine, Burlington, VT 50405, USA.

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are gammadelta T cells. gammadelta T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human gammadelta T cells of the Vdelta1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine gammadelta T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of gammadelta T cells, are both caspase-dependent.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832482PMC
http://dx.doi.org/10.4049/jimmunol.181.4.2392DOI Listing
August 2008

Apoptosis regulators Fas and Bim cooperate in shutdown of chronic immune responses and prevention of autoimmunity.

Immunity 2008 Feb;28(2):197-205

Molecular Genetics of Cancer, The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia.

Apoptotic death of T lymphocytes is critical for shutdown of immune responses and hemopoietic cell homeostasis. Both death receptor (Fas) activation and mitochondrial apoptosis triggered by the BH3-only protein Bim have been implicated in the killing of antigen-stimulated T cells. We examined mice lacking the gene encoding Bim (Bcl2l11) and with the inactivating lpr mutation in the gene encoding Fas (Fas), designated Bcl2l11(-/-)Fas(lpr/lpr) mice. Shutdown of an acute T cell response to herpes simplex virus involved only Bim with no contribution by Fas, whereas both pathways synergized in killing antigen-stimulated T cells in chronic infection with murine gamma-herpesvirus. Bcl2l11(-/-)Fas(lpr/lpr) mice developed remarkably enhanced and accelerated fatal lymphadenopathy and autoimmunity compared to mice lacking only one of these apoptosis inducers. These results identify critical overlapping roles for Fas and Bim in T cell death in immune response shutdown and prevention of immunopathology and thereby resolve a long-standing controversy.
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http://dx.doi.org/10.1016/j.immuni.2007.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2270348PMC
February 2008

c-FLIP(S) reduces activation of caspase and NF-kappaB pathways and decreases T cell survival.

Eur J Immunol 2008 Jan;38(1):54-63

Immunobiology Program, Department of Medicine, The University of Vermont College of Medicine, Burlington, VT 05405-0068, USA.

Effective stimulation of NF-kappaB in T cells following TCR ligation requires the activity of caspase-8. The active caspase-8 complex includes the paracaspase, MALT1, and Bcl-10, which connect to the NF-kappaB pathway. It has been less clear what regulates the level of caspase-8 activity during T cell activation. A likely candidate is cellular FLIP (c-FLIP), an enzymatically inert caspase-8 homologue. Two alternatively spliced forms of c-FLIP exist, a long form (c-FLIP(L)) and a short-form (c-FLIP(S)). The latter lacks the C-terminal caspase-like domain. c-FLIP(L) can heterodimerize with and activate caspase-8 through an activation loop in the C terminus of c-FLIP(L). Here we show that, in contrast to c-FLIP(L), c-FLIP(S) inhibits activation of caspase-8 in T cells, and consequently reduces recruitment of MALT1 and Bcl-10 to the active caspase complex. This results in reduced activity of NF-kappaB. Consequently, T cells from c-FLIP(S)-transgenic mice undergo more rapid cell death both spontaneously and after activation. The findings suggest that c-FLIP(S) functions to reduce the expansion of T cells during an immune response.
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http://dx.doi.org/10.1002/eji.200636956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756655PMC
January 2008

The death receptor Fas (CD95/APO-1) mediates the deletion of T lymphocytes undergoing homeostatic proliferation.

J Immunol 2005 Oct;175(7):4374-82

Immunobiology Division, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405, USA.

Murine T cells adoptively transferred into syngeneic lymphopenic recipients undergo proliferation. Despite continued cell division, this lymphopenia-induced or homeostatic proliferation of a limited number of transferred T cells does not fill the T cell compartment. The continued expansion of the transferred T cells, even after stable T cell numbers have been reached, suggests that active cell death prevents further increase in T cell number. In this study, we show that wild-type T cells undergoing homeostatic proliferation are sensitive to Fas-mediated cell death. In the absence of Fas, T cells accumulate to significantly higher levels after transfer into lymphopenic recipients. Fas is, thus, a principal regulator of the expansion of peripheral T cells in response to self-peptide/MHC during T cell homeostasis. As Fas-deficient lpr mice manifest no significant abnormalities in thymic negative selection or in foreign Ag-induced peripheral T cell deletion, their lymphadenopathy may result from unrestrained homeostatic proliferation.
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http://dx.doi.org/10.4049/jimmunol.175.7.4374DOI Listing
October 2005

Cellular FLIP long form-transgenic mice manifest a Th2 cytokine bias and enhanced allergic airway inflammation.

J Immunol 2004 Apr;172(8):4724-32

Immunobiology Program, Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05405, USA.

Cellular FLIP long form (c-FLIP(L)) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIP(L) in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIP(L) with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIP(L)-transgenic mice. In this study we show that activated CD4(+) T cells from c-FLIP(L)-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIP(L)-transgenic CD4(+) T cells parallels impaired NF-kappa B activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-gamma and increased Th2 cytokines. The Th2 bias of c-FLIP(L)-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIP(L) can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.
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http://dx.doi.org/10.4049/jimmunol.172.8.4724DOI Listing
April 2004

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

Mol Cell Biol 2002 Aug;22(15):5419-33

Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC133948PMC
http://dx.doi.org/10.1128/MCB.22.15.5419-5433.2002DOI Listing
August 2002
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