Publications by authors named "Ralph C Budd"

54 Publications

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

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

Thrombo-inflammation response to Tocilizumab in COVID-19.

Res Pract Thromb Haemost 2020 Sep 25. Epub 2020 Sep 25.

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

Background: Coronavirus disease-19 (COVID-19) spans a wide spectrum of illness. Severe cases of COVID-19 can manifest inflammation in organs other than the lung, in tissues not known to support viral replication, and also in a hypercoagulable state. These observations have suggested that SARS-CoV-2 can provoke a hyperimmune response in some cases that could lead to secondary organ damage.

Methods: With evidence of elevated levels of interleuking-6 (IL-6) in patients with severe COVID-19, we conducted a small pilot off-label compassionate care study of the IL-6 receptor inhibitor tocilizumab patients with severe COVID-19.

Results: Following a single infusion of tocilizumab in patients with severe manifested rapid declines in C-reactive protein (CRP), D-Dimer, and gradual rises in lymphocyte and platelet counts.

Conclusions: These findings suggest both pathophysiological mechanisms as well as clinical benefit that might be seen with IL-6 inhibition in severe COVID-19.
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http://dx.doi.org/10.1002/rth2.12436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537313PMC
September 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

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

J Immunol 2019 11 23;203(9):2369-2376. Epub 2019 Sep 23.

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

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.
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http://dx.doi.org/10.4049/jimmunol.1900451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804759PMC
November 2019

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

Reducing protein oxidation reverses lung fibrosis.

Nat Med 2018 08 9;24(8):1128-1135. Epub 2018 Jul 9.

Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, VT, USA.

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death. Oxidative stress is believed to be critical in this disease pathogenesis, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX). It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.
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http://dx.doi.org/10.1038/s41591-018-0090-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204256PMC
August 2018

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

Reactive oxygen species induce virus-independent MAVS oligomerization in systemic lupus erythematosus.

Sci Signal 2016 11 29;9(456):ra115. Epub 2016 Nov 29.

Department of Pathology, Vermont Center for Immunology and Infectious Diseases, University of Vermont, Burlington, VT 05405, USA.

The increased expression of genes induced by type I interferon (IFN) is characteristic of viral infections and systemic lupus erythematosus (SLE). We showed that mitochondrial antiviral signaling (MAVS) protein, which normally forms a complex with retinoic acid gene I (RIG-I)-like helicases during viral infection, was activated by oxidative stress independently of RIG-I helicases. We found that chemically generated oxidative stress stimulated the formation of MAVS oligomers, which led to mitochondrial hyperpolarization and decreased adenosine triphosphate production and spare respiratory capacity, responses that were not observed in similarly treated cells lacking MAVS. Peripheral blood lymphocytes of SLE patients also showed spontaneous MAVS oligomerization that correlated with the increased secretion of type I IFN and mitochondrial oxidative stress. Furthermore, inhibition of mitochondrial reactive oxygen species (ROS) by the mitochondria-targeted antioxidant MitoQ prevented MAVS oligomerization and type I IFN production. ROS-dependent MAVS oligomerization and type I IFN production were reduced in cells expressing the MAVS-C79F variant, which occurs in 30% of sub-Saharan Africans and is linked with reduced type I IFN secretion and milder disease in SLE patients. Patients expressing the MAVS-C79F variant also had reduced amounts of oligomerized MAVS in their plasma compared to healthy controls. Together, our findings suggest that oxidative stress-induced MAVS oligomerization in SLE patients may contribute to the type I IFN signature that is characteristic of this syndrome.
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http://dx.doi.org/10.1126/scisignal.aaf1933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321043PMC
November 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

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione--transferase π.

JCI Insight 2016 Jun;1(8)

Department of Pathology and Laboratory Medicine, University of Vermont, Burlington, Vermont, USA.

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione--transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.
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http://dx.doi.org/10.1172/jci.insight.85717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922427PMC
June 2016

J. Donald Capra, M.D. (1937-2015).

J Immunol 2015 Jun;194(12):5575-6

Director, Vermont Center for Immunology & Infectious Diseases University of Vermont, Department of Medicine.

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http://dx.doi.org/10.4049/jimmunol.1590010DOI Listing
June 2015

γδ T Cells and dendritic cells in refractory Lyme arthritis.

J Leukoc Biol 2015 Apr 20;97(4):653-63. Epub 2015 Jan 20.

*Texas A&M Health Science, Temple, Texas, USA; and University of Vermont, Burlington, Vermont, USA

Lyme disease is a multisystem infection transmitted by tick vectors with an incidence of up to 300,000 individuals/yr in the United States. The primary treatments are oral or i.v. antibiotics. Despite treatment, some individuals do not recover and have prolonged symptoms affecting multiple organs, including the nervous system and connective tissues. Inflammatory arthritis is a common symptom associated with Lyme pathology. In the past decades, γδ T cells have emerged as candidates that contribute to the transition from innate to adaptive responses. These cells are also differentially regulated within the synovia of patients affected by RLA. Here, we review and discuss potential cellular mechanisms involving γδ T cells and DCs in RLA. TLR signaling and antigen processing and presentation will be the key concepts that we review in aid of understanding the impact of γδ T cells in RLA.
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http://dx.doi.org/10.1189/jlb.2RU0714-343RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763954PMC
April 2015

c-FLIP-Short reduces type I interferon production and increases viremia with coxsackievirus B3.

PLoS One 2014 9;9(5):e96156. Epub 2014 May 9.

Department of Medicine, Vermont Center for Immunology and Infectious Diseases, University of Vermont, Burlington, Vermont, United States of America.

Cellular FLIP (c-FLIP) is an enzymatically inactive paralogue of caspase-8 and as such can block death receptor-induced apoptosis. However, independent of death receptors, c-FLIP-Long (c-FLIPL) can heterodimerize with and activate caspase-8. This is critical for promoting the growth and survival of T lymphocytes as well as the regulation of the RIG-I helicase pathway for type I interferon production in response to viral infections. Truncated forms of FLIP also exist in mammalian cells (c-FLIPS) and certain viruses (v-FLIP), which lack the C-terminal domain that activates caspase-8. Thus, the ratio of c-FLIPL to these short forms of FLIP may greatly influence the outcome of an immune response. We examined this model in mice transgenically expressing c-FLIPS in T cells during infection with Coxsackievirus B3 (CVB3). In contrast to our earlier findings of reduced myocarditis and mortality with CVB3 infection of c-FLIPL-transgenic mice, c-FLIPS-transgenic mice were highly sensitive to CVB3 infection as manifested by increased cardiac virus titers, myocarditis score, and mortality compared to wild-type C57BL/6 mice. This observation was paralleled by a reduction in serum levels of IL-10 and IFN-α in CVB3-infected c-FLIPS mice. In vitro infection of c-FLIPS T cells with CVB3 confirmed these results. Furthermore, molecular studies revealed that following infection of cells with CVB3, c-FLIPL associates with mitochondrial antiviral signaling protein (MAVS), increases caspase-8 activity and type I IFN production, and reduces viral replication, whereas c-FLIPS promotes the opposite phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015977PMC
October 2015

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

Aging Cell 2014 Aug 13;13(4):765-8. Epub 2014 Mar 13.

MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Cambridge, CB2 0XY, UK.

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria-targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro-apoptotic and pro-inflammatory redox signaling pathways.
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http://dx.doi.org/10.1111/acel.12212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326952PMC
August 2014

The role of sex differences in autophagy in the heart during coxsackievirus B3-induced myocarditis.

J Cardiovasc Transl Res 2014 Mar 10;7(2):182-91. Epub 2013 Dec 10.

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

Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation, or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy, were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3)-induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3-infected, cardiac samples of both sexes, suggests sex differences in autophagy-related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.
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http://dx.doi.org/10.1007/s12265-013-9525-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115281PMC
March 2014

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

Caspase-8 and FLIP regulate RIG-I/MDA5-induced innate immune host responses to picornaviruses.

Future Virol 2012 Dec;7(12):1221-1236

Department of Pathology, Vermont Center for Immunology & Infectious Diseases, University of Vermont, Burlington, VT 05405, USA.

Picornaviruses are small, nonenveloped, positive-stranded RNA viruses, which cause a wide range of animal and human diseases, based on their distinct tissue and cell type tropisms. Myocarditis, poliomyelitis, hepatitis and the common cold are the most significant human illnesses caused by picornaviruses. The host response to picornaviruses is complex, and the damage to tissues occurs not only from direct viral replication within infected cells. Picornaviruses exhibit an exceptional ability to evade the early innate immune response, resulting in chronic infection and autoimmunity. This review discusses the detailed aspects of the early innate host response to picornaviruses infection mediated by RIG-I-like helicases, their adaptor, mitochondrial ant iviral signaling protein, innate immune-induced apoptosis, and the role of caspase-8 and its regulatory paralog, FLIP, in these processes.
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http://dx.doi.org/10.2217/fvl.12.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595017PMC
December 2012

Interleukin-1 receptor and caspase-1 are required for the Th17 response in nitrogen dioxide-promoted allergic airway disease.

Am J Respir Cell Mol Biol 2013 May;48(5):655-64

Vermont Lung Center, Division of Pulmonary Disease and Critical Care, Department of Medicine, University of Vermont, Burlington, VT 05405, USA.

Nitrogen dioxide (NO2) is an environmental pollutant and endogenously generated oxidant associated with the development, severity, and exacerbation of asthma. NO2 exposure is capable of allergically sensitizing mice to the innocuous inhaled antigen ovalbumin (OVA), promoting neutrophil and eosinophil recruitment, and a mixed Th2/Th17 response upon antigen challenge that is reminiscent of severe asthma. However, the identity of IL-17A-producing cells and the mechanisms governing their ontogeny in NO2-promoted allergic airway disease remain unstudied. We measured the kinetics of lung inflammation after antigen challenge in NO2-promoted allergic airway disease, including inflammatory cells in bronchoalveolar lavage and antigen-specific IL-17A production from the lung. We determined that IL-17A(+) cells were predominately CD4(+)T cell receptor (TCR)β(+) Th17 cells, and that a functional IL-1 receptor was required for Th17, but not Th2, cytokine production after in vitro antigen restimulation of lung cells. The absence of natural killer T cells, γδ T cells, or the inflammasome scaffold nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain (Nlrp)3 did not affect the development of NO2-promoted allergic inflammation or IL-17A production. Similarly, neutrophil depletion or the neutralization of IL-1α during sensitization exerted no effect on these parameters. However, the absence of caspase-1 significantly reduced IL-17A production from lung cells without affecting Th2 cytokines or lung inflammation. Finally, the intranasal administration of IL-1β and the inhalation of antigen promoted allergic sensitization that was reflected by neutrophilic airway inflammation and IL-17A production from CD4(+)TCRβ(+) Th17 cells subsequent to antigen challenge. These data implicate a role for caspase-1 and IL-1β in the IL-1 receptor-dependent Th17 response manifest in NO2-promoted allergic airway disease.
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http://dx.doi.org/10.1165/rcmb.2012-0423OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707382PMC
May 2013

Oxidative processing of latent Fas in the endoplasmic reticulum controls the strength of apoptosis.

Mol Cell Biol 2012 Sep 2;32(17):3464-78. Epub 2012 Jul 2.

Department of Pathology, University of Vermont, Burlington, Vermont, USA.

We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241-252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.
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http://dx.doi.org/10.1128/MCB.00125-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422013PMC
September 2012

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

Redox-based regulation of apoptosis: S-glutathionylation as a regulatory mechanism to control cell death.

Antioxid Redox Signal 2012 Mar 22;16(6):496-505. Epub 2011 Dec 22.

Department of Pathology, University of Vermont College of Medicine, Burlington, 05405, USA.

Significance: Redox-based signaling governs a number of important pathways in tissue homeostasis. Consequently, deregulation of redox-controlled processes has been linked to a number of human diseases. Among the biological processes regulated by redox signaling, apoptosis or programmed cell death is a highly conserved process important for tissue homeostasis. Apoptosis can be triggered by a wide variety of stimuli, including death receptor ligands, environmental agents, and cytotoxic drugs. Apoptosis has also been implicated in the etiology of many human diseases.

Recent Advances: Recent discoveries demonstrate that redox-based changes are required for efficient activation of apoptosis. Among these redox changes, alterations in the abundant thiol, glutathione (GSH), and the oxidative post-translational modification, protein S-glutathionylation (PSSG) have come to the forefront as critical regulators of apoptosis.

Critical Issues: Although redox-based changes have been documented in apoptosis and disease pathogenesis, the mechanistic details, whereby redox perturbations intersect with pathogenic processes, remain obscure.

Future Directions: Further research will be needed to understand the context in which of the members of the death receptor pathways undergo ligand dependent oxidative modifications. Additional investigation into the interplay between oxidative modifications, redox enzymes, and apoptosis pathway members are also critically needed to improve our understanding how redox-based control is achieved. Such analyses will be important in understanding the diverse chronic diseases. In this review we will discuss the emerging paradigms in our current understanding of redox-based regulation of apoptosis with an emphasis on S-glutathionylation of proteins and the enzymes involved in this important post-translational modification.
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http://dx.doi.org/10.1089/ars.2011.4281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3304251PMC
March 2012

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

Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease.

Ann N Y Acad Sci 2010 Aug;1203:23-8

Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA.

Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired reepithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor Fas, which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with profibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, such as S-glutathionylation (PSSG), is known to act as a regulatory event that affects protein function. This manuscript will discuss evidence that S-glutathionylation regulates death receptor induced apoptosis, and the potential implications for cysteine oxidations in the pathogenesis of in fibrotic lung disease.
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http://dx.doi.org/10.1111/j.1749-6632.2010.05553.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943339PMC
August 2010

Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.

J Cell Biol 2009 Jan;184(2):241-52

Department of Pathology, University of Vermont, Burlington, VT 05405, USA.

Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.
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http://dx.doi.org/10.1083/jcb.200807019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654302PMC
January 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
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