bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective Alloreactive CD8 T Cell Function and Memory Response in Allograft Recipients in the Absence of CD4 Help
Authors: Zhai Yuan , Wang Yue , Wu Zheng , Kupiec-Weglinski Jerzy W.
Published: April 2014

We have shown that alloreactive CD8 T cell activation may proceed via CD4-dependent and CD4-independent pathways, and that CD8 T cell activation in Ag-primed animals is independent of CD154 costimulation. In this report, we further analyzed the activation and function of alloreactive CD8 CTL effectors in CD4 knockout (KO) skin/cardiac allograft recipients. FACS analysis showed that alloreactive CD8 T cells were activated at a significantly reduced level in CD4 KO mice. Importantly, these helpless CD8 T cells failed to develop CD154 blockade resistance following reactivation by the same alloantigen, indicative of defective memory formation. Only transient CD4 help was required, as short-term CD4 blockade at the time of first skin graft challenge only delayed alloreactive CD8 activation, without affecting the CD8 T cell memory response to a second skin graft. Moreover, postoperative CD4 blockade had no effect on alloreactive CD8 activation. Alloreactive CD8 cells generated in the absence of CD4 help exhibited decreased effector responses. Interestingly, intragraft induction of T cell-targeted chemokines early after transplant was also dependent on CD4 help, as the induction kinetics of CXCL9 and CCL5 in CD4 KO recipients was significantly delayed, coupled with similarly delayed infiltration by CD3/CD8 cells. Remarkably, helpless CD8 cells ultimately entering the graft still displayed significantly diminished T cell effector molecules (IFN-γ, granzyme B). Thus, CD4 help is critical for alloreactive CD8 activation, function, and memory formation.
Fyn Regulates the Duration of TCR Engagement Needed for Commitment to Effector Function
Authors: Filby Andrew , Seddon Benedict , Kleczkowska Joanna , Salmond Robert , Tomlinson Peter , Smida Michal , Lindquist Jonathan A. , Schraven Burkhart , Zamoyska Rose
Published: April 2014

In naive T cells, engagement of the TCR with agonist peptide:MHC molecules leads to phosphorylation of key intracellular signaling intermediates within seconds and this peaks within minutes. However, the cell does not commit to proliferation and IL-2 cytokine production unless receptor contact is sustained for several hours. The biochemical basis for this transition to full activation may underlie how T cells receive survival signals while maintaining tolerance, and is currently not well understood. We show here that for CD8 T cells commitment to proliferation and cytokine production requires sustained activation of the Src family kinase Lck and is opposed by the action of Fyn. Thus, in the absence of Fyn, commitment to activation occurs more rapidly, the cells produce more IL-2, and undergo more rounds of division. Our data demonstrate a role for Fyn in modulating the response to Ag in primary T cells.
Differential Role of TLR- and RLR-Signaling in the Immune Responses to Influenza A Virus Infection and Vaccination
Authors: Koyama Shohei , Ishii Ken J. , Kumar Himanshu , Tanimoto Takeshi , Coban Cevayir , Uematsu Satoshi , Kawai Taro , Akira Shizuo
Published: April 2014

The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-β promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.
Alternatively Activated Macrophages in Intestinal Helminth Infection: Effects on Concurrent Bacterial Colitis
Authors: Weng Meiqian , Huntley Deke , Huang I-Fei , Foye-Jackson Ondulla , Wang Lijian , Sarkissian Aliese , Zhou Qingping , Walker W. Allan , Cherayil Bobby J. , Shi Hai Ning
Published: April 2014

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host’s response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the Gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-α response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-α in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.
ASC/PYCARD and Caspase-1 Regulate the IL-18/IFN-γ Axis during Anaplasma phagocytophilum Infection
Authors: Pedra Joao H. F. , Sutterwala Fayyaz S. , Sukumaran Bindu , Ogura Yasunori , Qian Feng , Montgomery Ruth R. , Flavell Richard A. , Fikrig Erol
Published: April 2014

Anaplasma phagocytophilum is an obligate intracellular pathogen that resides within neutrophils and can cause fever, pancytopenia, or death. IFN-γ plays a critical role in the control of A. phagocytophilum; however, the mechanisms that regulate IFN-γ production remain unclear. In this study, we demonstrate that apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, a central adaptor molecule in the Nod-like receptor (NLR) pathway, regulates the IL-18/IFN-γ axis during A. phagocytophilum infection through its effect on caspase-1. Caspase-1- and asc-null mice were more susceptible than control animals to A. phagocytophilum infection due to the absence of IL-18 secretion and reduced IFN-γ levels in the peripheral blood. Moreover, caspase-1 and ASC deficiency reduced CD4+ T cell-mediated IFN-γ after in vitro restimulation with A. phagocytophilum. The NLR family member IPAF/NLRC4, but not NALP3/NLRP3, was partially required for IFN-γ production in response to A. phagocytophilum. Taken together, our data demonstrate that ASC and caspase-1 are critical for IFN-γ-mediated control of A. phagocytophilum infection.
Activation-Induced Cytidine Deaminase Expression in Follicular Dendritic Cell Networks and Interfollicular Large B Cells Supports Functionality of Ectopic Lymphoid Neogenesis in Autoimmune Sialoadenitis and MALT Lymphoma in Sjögren’s Syndrome
Authors: Bombardieri Michele , Barone Francesca , Humby Frances , Kelly Stephen , McGurk Mark , Morgan Peter , Challacombe Stephen , De Vita Salvatore , Valesini Guido , Spencer Jo , Pitzalis Costantino
Published: April 2014

Demonstration of ectopic germinal center-like structures (GC-LSs) in chronically inflamed tissues in patients with autoimmune disorders is a relatively common finding. However, to what extent ectopic lymphoid structures behave as true GC and are able to support class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes is still debated. In addition, no information is available on whether CSR and SHM can take place in the absence of GCs at extrafollicular sites in an ectopic lymphoid tissue. In this study, we show that in salivary glands (SGs) of Sjögren’s syndrome (SS) activation-induced cytidine deaminase (AID), the enzyme responsible for CSR and SHM is invariably expressed within follicular dendritic cell (FDC) networks but is not detectable in SGs in the absence of ectopic GC-LSs, suggesting that FDC networks play an essential role in sustaining the Ag-driven B cell proliferation within SS-SGs. We also show that the recently described population of interfollicular large B cells selectively expresses AID outside ectopic GC in the T cell-rich areas of periductal aggregates. Finally, we report that AID retains its exclusive association with numerous, residual GCs in parotid SS-MALT lymphomas, whereas neoplastic marginal zone-like B cells are consistently AID negative. These results strongly support the notion that ectopic lymphoid structures in SS-SGs express the molecular machinery to support local autoantibody production and B cell expansion and may play a crucial role toward lymphomagenesis.
The Role of Apoptosis in the Ameliorating Effects of a CDR1-Based Peptide on Lupus Manifestations in a Mouse Model
Authors: Sharabi Amir , Luger Dror , Ben-David Hava , Dayan Molly , Zinger Heidey , Mozes Edna
Published: April 2014

Experimental systemic lupus erythematosus (SLE) can be induced in mice following immunization with an anti-DNA mAb expressing a major Id, 16/6Id. Treatment with a peptide, designated human CDR1 (hCDR1; Edratide), that is based on the sequence of CDR1 of the 16/6Id ameliorated disease manifestations. In the present study, we investigated the roles of apoptosis and related molecules in BALB/c mice with induced experimental SLE following treatment with hCDR1. A higher state of activation and increased rate of apoptosis were found in lymphocytes of SLE-afflicted mice as compared with healthy controls. The latter effects were associated with up-regulated caspase-8 and caspase-3, and down-regulated Bcl-xL. The ameliorative effects of hCDR1 were associated with down-regulation of caspase-8 and caspase-3, up-regulation of Bcl-xL, and a reduced rate of apoptosis. Treatment of diseased mice with an apoptosis-reducing compound that inhibited caspases down-regulated the secretion of the pathogenic cytokine IFN-γ and lowered the intensity of glomerular immune complex deposits and the levels of proteinuria. Furthermore, coincubation of Bcl-xL inhibitors with hCDR1-treated cells abrogated the ability of hCDR1 to reduce the activation state of lymphocytes and to down-regulate the secretion of IL-10 and IFN-γ. Moreover, the Bcl-xL-expressing CD4+CD25+ cells from hCDR1-treated mice induced the expression of Bcl-xL in CFSE-labeled CD4+CD25− cells of the SLE-afflicted mice. Thus, the reduction of apoptosis and the up-regulation of Bcl-xL, which plays an apparent role in tolerance induction, contribute to at least part of the beneficial effects of hCDR1 on lupus manifestations.
Unique Features and Distribution of the Chicken CD83+ Cell
Authors: Hansell Chris , Zhu Xing Wu , Brooks Heather , Sheppard Mark , Withanage Sam , Maskell Duncan , McConnell Ian
Published: April 2014

The central importance of dendritic cells (DC) in both innate and acquired immunity is well recognized in the mammalian immune system. By contrast DC have yet to be characterized in avian species despite the fact that avian species such as the chicken have a well-developed immune system. CD83 has proven to be an excellent marker for DC in human and murine immune systems. In this study we identify chicken CD83 (chCD83) as the avian equivalent of the human and murine DC marker CD83. We demonstrate for the first time that unlike human and murine CD83, chCD83 is uniquely expressed in the B cell areas of secondary lymphoid organs and in organs with no human or murine equivalent such as the bursa and Harderian gland. Furthermore through multicolor immunofluorescence, we identify chCD83+ populations that have unique attributes akin to both DC and follicular DC. These attributes include colocalization with B cell microrenvironments, MHC class II expression, dendritic morphology, and distribution throughout peripheral and lymphoid tissues.
Hydrodynamic Vaccination with DNA Encoding an Immunologically Privileged Retinal Antigen Protects from Autoimmunity through Induction of Regulatory T Cells
Authors: Silver Phyllis B. , Agarwal Rajeev K. , Su Shao-Bo , Suffia Isabelle , Grajewski Rafael S. , Luger Dror , Chan Chi-Chao , Mahdi Rashid M. , Nickerson John M. , Caspi Rachel R.
Published: April 2014

The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 μg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8+ cells, IL-10, or TGF-β. In contrast, depletion of CD25+ cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4+CD25high T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3+CD4+CD25+ regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.
Enhanced Engagement of CTLA-4 Induces Antigen-Specific CD4+CD25+Foxp3+ and CD4+CD25− TGF-β1+ Adaptive Regulatory T Cells
Authors: Li Ruobing , Perez Nicolas , Karumuthil-Melethil Subha , Prabhakar Bellur S. , Holterman Mark J. , Vasu Chenthamarakshan
Published: April 2014

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4+CD25+Foxp3+ and CD4+CD25−TGF-β1+ adaptive Tregs. These cells were CD62Llow and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62Lhigh counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.
PDL1 Is Required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection
Authors: Tanaka Katsunori , Albin Monica J. , Yuan Xueli , Yamaura Kazuhiro , Habicht Antje , Murayama Takaya , Grimm Martin , Waaga Ana Maria , Ueno Takuya , Padera Robert F. , Yagita Hideo , Azuma Miyuki , Shin Tahiro , Blazar Bruce R. , Rothstein David M. , Sayegh Mohamed H. , Najafian Nader
Published: April 2014

The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-γ-producing alloreactive T cells and expansion of effector CD8+ T cells in the periphery, and a decline in the percentage of Foxp3+ graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.
Antibody-Mediated Rejection of Cardiac Allografts in CCR5-Deficient Recipients
Authors: Nozaki Taiji , Amano Hiroyuki , Bickerstaff Alice , Orosz Charles G. , Novick Andrew C. , Tanabe Kazunari , Fairchild Robert L.
Published: April 2014

Rejected MHC-mismatched cardiac allografts in CCR5−/− recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5−/− recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5−/− (H-2b) recipients of A/J (H-2a) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-γ, whereas CD4 T cells producing IL-4 were increased in CCR5−/− recipients. Numbers of donor-reactive CD8 T cells producing IFN-γ were reduced 60% in CCR5−/− recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5−/− allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1−/− recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5−/− recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12–60, mean 21 days) in μMT−/−/CCR5−/− recipients. These results indicate that the donor-specific Ab produced in CCR5−/− heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.
All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells
Authors: Chang Wei-Chiao , Di Capite Joseph , Nelson Charmaine , Parekh Anant B.
Published: April 2014

In nonexcitable cells, receptor stimulation evokes Ca2+ release from the endoplasmic reticulum stores followed by Ca2+ influx through store-operated Ca2+ channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca2+ release-activated Ca2+ (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca2+-dependent activation of protein kinase Cα and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca2+-dependent phospholipase A2 and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C4. In cell population studies, ERK activation, arachidonic acid release, and leukotriene C4 secretion were all graded with stimulus intensity. However, at a single cell level, Ca2+ influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca2+ influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca2+entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.
Dynamic Changes in Accessibility, Nuclear Positioning, Recombination, and Transcription at the Igκ Locus
Authors: Fitzsimmons Sean P. , Bernstein Ralph M. , Max Edward E. , Skok Jane A. , Shapiro Marjorie A.
Published: April 2014

The 3-megabase Igκ locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Igκ locus was determined in a murine pre-B cell line that can be induced to recombine at the Igκ locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Igκ locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific Vκ genes were observed. Histone modifications of the Vκ genes did not vary upon induction but the levels of modifications correlated with the levels of germline Vκ gene transcripts and recombination. Upon induction, but before VκJκ recombination, centromeric recruitment of single Igκ alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the Vκ genes was increased in the silencer regions upstream of Jκ1, within the Igκ sterile transcript, the κ constant region, the Eκi and Eκ3′ enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the Vκ genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.
The C/EBPβ Isoform 34-kDa LAP Is Responsible for NF-IL-6-Mediated Gene Induction in Activated Macrophages, but Is Not Essential for Intracellular Bacteria Killing
Authors: Uematsu Satoshi , Kaisho Tsuneyasu , Tanaka Takashi , Matsumoto Makoto , Yamakami Megumi , Omori Hiroko , Yamamoto Masahiro , Yoshimori Tamotsu , Akira Shizuo
Published: April 2014

The C/ebpb gene is translated into three different protein isoforms, two transcriptional activating proteins (38-kDa Full and 34-kDa liver-enriched transcriptional activation protein (LAP)) and one transcriptional inhibitory protein, by alternative use of different AUG initiation codons within the same open reading frame. The isoform 34-kDa LAP is thought to be the most transcriptionally active form of C/EBPβ in macrophages. To assess the function of the 34-kDa LAP in vivo, we generated knock-in mice, in which methionine 20 of C/EBPβ, the start site for the 34-kDa LAP is replaced with an alanine. The expression of the 34-kDa LAP was abolished in C/ebpbM20A/M20A mice. The induction of C/EBPβ target genes, such as inflammatory cytokines, chemokines, prostanoid synthetase, and antimicrobial peptides, was abolished in C/ebpbM20A/M20A macrophages, and C/ebpbM20A/M20A mice were susceptible to Listeria monocytogenes infection. Furthermore, the heat-killed Propionibacterium acnes-induced Th1 response, granuloma formation, and LPS shock were severely impaired. Nevertheless, impairment of intracellular bacteria killing, which is the most prominent phenotype in C/EBPβ-deficient mice, was not observed in C/ebpbM20A/M20A mice. Collectively, we demonstrated that 34-kDa LAP is responsible for NF-IL6-mediated gene induction, but not essential for intracellular bacteria killing in activated macrophages.
Gr-1high Polymorphonuclear Leukocytes and NK Cells Act via IL-15 to Clear Intracellular Haemophilus influenzae in Experimental Murine Peritonitis and Pneumonia
Authors: Miyazaki Shuichi , Ishikawa Fumio , Shimizu Kayo , Ubagai Tsuneyuki , Edelstein Paul H. , Yamaguchi Keizo
Published: April 2014

Polymorphonuclear leukocytes (PMNs) can be divided into Gr-1high and Gr-1low subpopulations, but the differences in the functions of these cells in the host are unknown. This study investigated the roles of these two cell populations in the clearance of an intracellular pathogen (Haemophilus influenzae) causing murine peritonitis and pneumonia. Microarray analysis and quantitative real-time PCR analysis of proteose peptone-elicited peritoneal murine PMNs showed that IL-15 mRNA levels were significantly higher in Gr-1high PMNs than in Gr-1low PMNs. In addition, IL-15 was produced only by Gr-1-positive PMNs, especially Gr-1high PMNs. IL-15 was required for efficient clearance of experimental murine H. influenzae pneumonia, as 4 days postinfection lungs from IL-15 knockout mice contained 50- to 100-fold more bacteria than did wild-type mouse lungs. Gr-1 PMN-depleted C57BL/6 mice were more susceptible to H. influenzae pneumonia than were Gr-1 PMN replete C57BL/6 mice or C57BL/6 nude mice, demonstrating that Gr-1 PMNs are important in the clearance of intracellular bacteria. IL-15-activated NK cells killed H. influenzae in PMNs. Flow cytometry confirmed the expression of CD69 on the cell membrane of IL-15-activated NK cells. Our results show that Gr-1high PMNs produce more IL-15 than Gr-1low PMNs, and that IL-15-activated NK cells protect against early infection by H. influenzae.
The IFN-Inducible GTPase LRG47 (Irgm1) Negatively Regulates TLR4-Triggered Proinflammatory Cytokine Production and Prevents Endotoxemia
Authors: Bafica Andre , Feng Carl G. , Santiago Helton C. , Aliberti Julio , Cheever Allen , Thomas Karen E. , Taylor Gregory A. , Vogel Stefanie N. , Sher Alan
Published: April 2014

LRG47/Irgm1, a 47-kDa IFN-inducible GTPase, plays a major role in regulating host resistance as well as the hemopoietic response to intracellular pathogens. LRG47 expression in macrophages has been shown previously to be stimulated in vitro by bacterial LPS, a TLR4 ligand. In this study, we demonstrate that induction of LRG47 by LPS is not dependent on MyD88 signaling, but rather, requires STAT-1 and IFN-β. In addition, LRG47-deficient mice are highly susceptible to LPS, but not TLR2 ligand-induced shock, an outcome that correlates with enhanced proinflammatory cytokine production in vitro and in vivo. Further analysis revealed that LPS-stimulated LRG47-deficient macrophages display enhanced phosphorylation of p38, a downstream response associated with TLR4/MyD88 rather than IFN-β/STAT-1 signaling. In contrast, LPS-induced phosphorylation of IFN regulatory factor-3 and expression of IFN-β or the type I IFN-regulated genes, CCL5 and CCL10, were unaltered in LRG47−/− cells. Together, these observations indicate that in LPS-stimulated murine macrophages LRG47 is induced by IFN-β and negatively regulates TLR4 signaling to prevent excess proinflammatory cytokine production and shock. Thus, our findings reveal a new host-protective function for this GTPase in the response to pathogenic encounter.
Dual T Cell Receptor Expressing CD8+ T Cells with Tumor- and Self-Specificity Can Inhibit Tumor Growth without Causing Severe Autoimmunity
Authors: Weinhold Monika , Sommermeyer Daniel , Uckert Wolfgang , Blankenstein Thomas
Published: April 2014

The engineering of Ag-specific T cells by expression of TCR genes is a convenient method for adoptive T cell immunotherapy. A potential problem is the TCR gene transfer into self-reactive T cells that survived tolerance mechanisms. We have developed an experimental system with T cells that express two TCRs with defined Ag-specificities, one recognizing a tumor-specific Ag (LCMV-gp33), the other recognizing a self-Ag in the pancreas (OVA). By using tumor cells expressing high and low amounts of Ag and mice expressing high and low levels of self-Ag in the pancreas (RIP-OVA-Hi and RIP-OVA-Lo), we show that 1) tumor rejection requires high amount of tumor Ag, 2) severe autoimmunity requires high amount of self-Ag, and 3) if Ag expression on tumor cells is sufficient and low in the pancreas, successful adoptive T cell therapy can be obtained in the absence of severe autoimmunity. These results are shown with T cells from dual TCR transgenic mice or T cells that were redirected by TCR gene transfer. Our data demonstrate that the approach of adoptively transferring TCR redirected T cells can be effective without severe side effects, even when high numbers of T cells with self-reactivity were transferred.
Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria
Authors: Pawluczkowycz Andrew W. , Lindorfer Margaret A. , Waitumbi John N. , Taylor Ronald P.
Published: April 2014

Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.
Impaired T Cell Protein Kinase Cδ Activation Decreases ERK Pathway Signaling in Idiopathic and Hydralazine-Induced Lupus
Authors: Gorelik Gabriela , Fang Jing Yuan , Wu Ailing , Sawalha Amr H. , Richardson Bruce
Published: April 2014

T cells from patients with lupus or treated with the lupus-inducing drug hydralazine have defective ERK phosphorylation. The reason for the impaired signal transduction is unknown but important to elucidate, because decreased T cell ERK pathway signaling causes a lupus-like disease in animal models by decreasing DNA methyltransferase expression, leading to DNA hypomethylation and overexpression of methylation-sensitive genes with subsequent autoreactivity and autoimmunity. We therefore analyzed the PMA stimulated ERK pathway phosphorylation cascade in CD4+ T cells from patients with lupus and in hydralazine-treated cells. The defect in these cells localized to protein kinase C (PKC)δ. Pharmacologic inhibition of PKCδ or transfection with a dominant negative PKCδ mutant caused demethylation of the TNFSF7 (CD70) promoter and CD70 overexpression similar to lupus and hydralazine-treated T cells. These results suggest that defective T cell PKCδ activation may contribute to the development of idiopathic and hydralazine-induced lupus through effects on T cell DNA methylation.
Calcineurin Inhibitors Block MHC-Restricted Antigen Presentation In Vivo
Authors: Lee Young-Hee , Lee Young-Ran , Im Sun-A , Park Sun-Im , Kim Ki-Hyang , Gerelchuluun Turmunkh , Song Sukgil , Kim Kyungjae , Lee Chong-Kil
Published: April 2014

APCs, like T cells, are affected by calcineurin inhibitors. In this study, we show that calcineurin inhibitors efficiently block MHC-restricted exogenous Ag presentation in vivo. Mice were injected with clinical doses of tacrolimus (FK-506) followed by soluble OVA, and dendritic cells (DCs) were isolated from lymph nodes and spleens. The efficacy of OVA peptide presentation by DCs was evaluated using OVA-specific CD8 and CD4 T cells. Tacrolimus inhibited both class I- and class II-restricted DC presentation of OVA to T cells. Tacrolimus also inhibited both class I- and class II-restricted presentation of OVA in peritoneal macrophages isolated from mice injected with tacrolimus followed by soluble OVA. Tacrolimus-treated peritoneal macrophages, however, were able to present synthetic OVA peptide, SIINFEKL. Inclusion of cyclosporine A to biodegradable microspheres containing OVA greatly reduced their capacity to induce OVA-specific CTL response in mice. These findings provide novel insight into the mode of action of calcineurin inhibitors and have important implications for clinical immunosuppression regimens.
Differential Regulation of the Nature and Functions of Dendritic Cells and Macrophages by Cathepsin E
Authors: Kakehashi Hiroe , Nishioku Tsuyoshi , Tsukuba Takayuki , Kadowaki Tomoko , Nakamura Seiji , Yamamoto Kenji
Published: April 2014

The aspartic proteinase cathepsin E is localized mainly in the endosomal structures of APCs and has been implicated in a variety of immune responses, however, the precise roles of cathepsin E in these cells remain speculative. In this study, we report the effect of disrupting the gene encoding cathepsin E on the nature and functions of dendritic cells (DCs) and macrophages derived from mouse bone marrow precursors, as well as mouse peritoneal macrophages. Whereas cathepsin E deficiency induced the accumulation of the lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and elevated the lysosomal pH in macrophages, it did not have these effects on DCs. Although cathepsin E deficiency also caused a marked decrease in degradation of phagocytosed OVA and chemotactic responses to MCP-1 and fMLP by macrophages, these abilities were little affected in DCs by the absence of cathepsin E. Interestingly, cathepsin E deficiency markedly decreased the ability of macrophages to present intact OVA, as well as an OVA-derived antigenic peptide (266–281), to cognate T cells, while that of DCs was inversely enhanced by the absence of this protein. This paradox was resolved, in part, by the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which amplify the response of T cells, in cathepsin E-deficient DCs compared with the wild-type cells. These results indicate that cathepsin E differentially regulates the nature and function of DCs and macrophages.
High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines
Authors: Zhao Yangbing , Bennett Alan D. , Zheng Zhili , Wang Qiong J. , Robbins Paul F. , Yu Lawrence Y. L. , Li Yi , Molloy Peter E. , Dunn Steven M. , Jakobsen Bent K. , Rosenberg Steven A. , Morgan Richard A.
Published: April 2014

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.
Differential Type I IFN-Inducing Abilities of Wild-Type versus Vaccine Strains of Measles Virus
Authors: Shingai Masashi , Ebihara Takashi , Begum Nasim A. , Kato Atsushi , Honma Toshiki , Matsumoto Kenji , Saito Hirohisa , Ogura Hisashi , Matsumoto Misako , Seya Tsukasa
Published: April 2014

Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-β production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-β mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-β mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.
SalmonellaFlagellin Induces Bystander Activation of Splenic Dendritic Cells and Hinders Bacterial Replication In Vivo
Authors: Salazar-Gonzalez Rosa-Maria , Srinivasan Aparna , Griffin Amanda , Muralimohan Guruprasaadh , Ertelt James M. , Ravindran Rajesh , Vella Anthony T. , McSorley Stephen J.
Published: April 2014

Bacterial flagellin is a target of innate and adaptive immune responses during Salmonella infection. Intravenous injection of Salmonella flagellin into C57BL/6 mice induced rapid IL-6 production and increased expression of activation markers by splenic dendritic cells. CD11b+, CD8α+, and plasmacytoid dendritic cells each increased expression of CD86 and CD40 in response to flagellin stimulation, although CD11b+ dendritic cells were more sensitive than the other subsets. In addition, flagellin caused the rapid redistribution of dendritic cells from the red pulp and marginal zone of the spleen into the T cell area of the white pulp. Purified splenic dendritic cells did not respond directly to flagellin, indicating that flagellin-mediated activation of splenic dendritic cells occurs via bystander activation. IL-6 production, increased expression of activation markers, and dendritic cell redistribution in the spleen were dependent on MyD88 expression by bone marrow-derived cells. Avoiding this innate immune response to flagellin is important for bacterial survival, because Salmonella-overexpressing recombinant flagellin was highly attenuated in vivo. These data indicate that flagellin-mediated activation of dendritic cells is rapid, mediated by bystander activation, and highly deleterious to bacterial survival.
Prostaglandin I2-IP Signaling Blocks Allergic Pulmonary Inflammation by Preventing Recruitment of CD4+ Th2 Cells into the Airways in a Mouse Model of Asthma
Authors: Jaffar Zeina , Ferrini Maria E. , Buford Mary C. , FitzGerald Garret A. , Roberts Kevan
Published: April 2014

PGI2 plays a key role in limiting Th2-mediated airway inflammation. In studies to investigate the mechanism underlying such regulation, we found that the PGI2 receptor, IP, is preferentially expressed by effector CD4+ Th2 cells, when compared with Th1 cells. Adoptive transfer of DO11.10 Th2 cells pretreated with PGI2 resulted in considerably attenuated pulmonary inflammation and airway hyperreactivity in BALB/c recipient mice in response to OVA inhalation. This suppression was independent of increased cAMP levels, because pretreatment of Th2 cells with dibutyryl cAMP before transfer had no effect on airway inflammation. Moreover, PGI2 pretreatment of Th2 cells suppressed the ability of the cells to infiltrate the lungs but not the spleen. In vitro studies showed that PGI2 did not affect IL-4 and IL-5 production or the level of IFN-γ by the T cells. However, the prostanoid strongly inhibited CCL17-induced chemotaxis of CD4+ Th2 but not Th1 cells. The IP was implicated in this process since migration of wild-type Th2 cells in response to CCL17 was markedly reduced following treatment with PGI2, whereas IP-deficient Th2 cells were unaffected and migrated effectively. Collectively, these experiments suggest that PGI2, which is generated by endothelial cells during lung inflammatory response, serves to limit the influx of Th2 cells to the airways. Our results identify PGI2-IP as an important pathway for inhibiting allergic pulmonary inflammation by controlling recruitment of CD4+ Th2 cells into the inflammatory site.
Urocortin Modulates Inflammatory Response and Neurotoxicity Induced by Microglial Activation
Authors: Wang Mei-Jen , Lin Shinn-Zong , Kuo Jon-Son , Huang Hsin-Yi , Tzeng Shih-Fang , Liao Chia-Hsin , Chen Der-Cherng , Chen Wu-Fu
Published: April 2014

Microglia are the major inflammatory cells in the brain. Recent studies have highlighted the reciprocal roles of other brain cells in modulating the microglial inflammatory responses. Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. In the present study, we demonstrated that expression of UCN in rat substantia nigra was found to be localized principally to dopaminergic neurons. In cell culture models, the CRH receptors were expressed in microglia, and CRHR expression was up-regulated by treatment with LPS. Thus, it might be proposed that UCN regulates cellular communication between dopaminergic neurons and microglia. We show that femtomolar concentrations of UCN could inhibit LPS-induced TNF-α production in cultured microglia. Investigation of the underlying signaling pathway that mediated the anti-inflammatory effect of UCN the involved PI3K/Akt and glycogen synthase kinase-3β pathway, but not cAMP pathway. Furthermore, UCN protected dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglial activation in LPS-treated mesencephalic neuron-glia cultures. These results suggest that endogenous UCN and its receptors might be involved in a complex network of paracrine interaction between dopaminergic neurons and glia.
Heat Shock Protein 90 Associates with Monarch-1 and Regulates Its Ability to Promote Degradation of NF-κB-Inducing Kinase
Authors: Arthur Janelle C. , Lich John D. , Aziz Ramy K. , Kotb Malak , Ting Jenny P.-Y.
Published: April 2014

Monarch-1/NLRP12 is expressed in myeloid cells and functions as a negative regulator of inflammation by inducing proteasome-mediated degradation of NF-κB-inducing kinase. Monarch-1 is a member of the CATERPILLER gene family, also known as the nucleotide-binding domain leucine-rich repeat gene family. This family shares strong structural homology to major immune regulators expressed in lower organisms, including plants. In plants, these disease-resistance proteins (R proteins) sense pathogenic insult and initiate a protective response to limit pathogen growth. To perform this role, many R proteins require the highly conserved chaperone molecule, heat shock protein (Hsp) 90. Using a two-dimensional gel/mass spectrometry system, we detected the association of the nucleotide-binding domain leucine-rich repeat protein Monarch-1 with heat shock proteins. Further analysis indicates that analogous to plant R proteins, Hsp90 is required for Monarch-1 activity. In human monocytes, Monarch-1 associates with Hsp90, and these complexes are sensitive to treatment with specific Hsp90 inhibitors. Disruption of these complexes results in rapid degradation of Monarch-1 via the proteasome and prevents Monarch-1-induced proteolysis of NF-κB-inducing kinase. This demonstrates that Hsp90 is a critical regulator of Monarch-1 anti-inflammatory activity.
Cutting Edge: Rapid Accumulation of Epidermal CCL27 in Skin-Draining Lymph Nodes following Topical Application of a Contact Sensitizer Recruits CCR10-Expressing T Cells
Authors: Huang Victor , Lonsdorf Anke S. , Fang Lei , Kakinuma Takashi , Lee Vivian C. , Cha Emily , Zhang Hong , Nagao Keisuke , Zaleska Marzanna , Olszewski Waldemar L. , Hwang Sam T.
Published: April 2014

CC chemokine receptor 10 and its ligand, CCL27, are important components of T cell-mediated cutaneous immunity, but whether they influence lymph node (LN) homing by T cells is unknown. In this study, CCL27 protein was detected in skin-draining LN by Western blotting and ELISA although CCL27 mRNA transcripts were low. CCL27 protein was present at higher levels in skin-draining LN compared with gut-draining LN and spleen. A single topical treatment of mouse skin with the contact sensitizer 2,4-dinitro-1-fluorobenzene (DNFB) resulted in a 13-fold increase in CCL27 protein accumulation in skin-draining LN within 1 h and a 5-fold elevation in CCR10 mRNA (normalized to the T cell marker CD2) within 6 h. DNFB treatment also resulted in rapid depletion of ∼75% of CCL27 from the epidermis. In summary, we describe a novel mechanism for the recruitment of CCR10-positive T cells to skin-draining LN following the rapid release of preformed CCL27 from the epidermis.