bluuebunny/crossref_metadata_2025 huggingface dataset:
Lutzomyia longipalpisSalivary Peptide Maxadilan Alters Murine Dendritic Cell Expression of CD80/86, CCR7, and Cytokine Secretion and Reprograms Dendritic Cell-Mediated Cytokine Release from Cultures Containing Allogeneic T Cells
Authors: Wheat William H. , Pauken Kristen E. , Morris Robin V. , Titus Richard G.
Published: April 2014

Leishmania protozoan parasites, the etiologic agent of leishmaniasis, are transmitted exclusively by phlebotomine sand flies of the genera Phlebotomus and Lutzomyia. In addition to parasites, the infectious bite inoculum contains arthropod salivary components. One well-characterized salivary component from Lutzomyia longipalpis is maxadilan (MAX), a vasodilator acting via the type I receptor for the pituitary cyclic AMP activating peptide. MAX has been shown to elicit immunomodulatory effects potentially dictating immune responses to Leishmania parasites. When exposed to MAX, both resting and LPS-stimulated dendritic cells (DCs) show reduced CD80 and CD86 expression on most DCs in vitro. However, CD86 expression is increased significantly on a subpopulation of DCs. Furthermore, MAX treatment promoted secretion of type 2 cytokines (IL-6 and IL-10) while reducing production of type 1 cytokines (IL-12p40, TNF-α, and IFN-γ) by LPS-stimulated DCs. A similar trend was observed in cultures of MAX-treated DCs containing naive allogeneic CD4+ T cells: type 2 cytokines (IL-6 and IL-13) increased while type 1 cytokines (TNF-α and IFN-γ) decreased. Additionally, the proinflammatory cytokine IL-1β was increased in cultures containing MAX-treated mature DCs. MAX treatment of LPS-stimulated DCs also prevented optimal surface expression of CCR7 in vitro. These MAX-dependent effects were evident in DCs from both Leishmania major-susceptible (BALB/c) and -resistant (C3H/HeN) murine strains. These data suggest that modification of DC phenotype and function by MAX likely affects crucial cellular components that determine the pathological response to infection with Leishmania.
The Central Memory CD4+ T Cell Population Generated duringLeishmania majorInfection Requires IL-12 to Produce IFN-γ
Authors: Pakpour Nazzy , Zaph Colby , Scott Phillip
Published: April 2014

Central memory CD4+ T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4+ T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4+ T cells require IL-12 to produce IFN-γ, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-γ in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4+ T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4+ T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4+ T cells.
Role of Chicken TL1A on Inflammatory Responses and Partial Characterization of Its Receptor
Authors: Takimoto Tetsuya , Sato Kan , Akiba Yukio , Takahashi Kazuaki
Published: April 2014

The role of chicken TNF-like ligand 1A (ChTL1A) on inflammation and its receptor candidates was investigated to further understand its function as a proinflammatory cytokine. ChTL1A decreased the viability of CHO-K1 cells transfected with chicken TNFR2 or decoy receptor 3 and bound to TNFR2 and decoy receptor 3. ChTL1A was detected in chicken blood samples taken 4 h after LPS injection. Increased mRNA for inflammatory response-related factors such as IL-1β, IL-6, ChTL1A, IFN-γ, inducible NO synthase, and cyclooxygenase 2 were found in spleen samples following LPS injection. Ceruloplasmin and α1 acid glycoprotein (as positive acute phase proteins) were increased in chicken plasma 12 h after ChTL1A injection. The injection of anti-ChTL1A Ab was able to prevent typical increases in plasma nitrite plus nitrate, ceruloplasmin, and α1 acid glycoprotein concentrations following LPS injection. These results indicate that ChTL1A is a proinflammatory cytokine in chickens, animals that do not have TNF-α and lymphotoxin α orthologous genes, and that its proinflammatory action is, at least in part, expressed through binding to TNFR2.
CCL11 and GM-CSF Differentially Use the Rho GTPase Pathway to Regulate Motility of Human Eosinophils in a Three-Dimensional Microenvironment
Authors: Muessel Michelle J. , Scott Karen S. , Friedl Peter , Bradding Peter , Wardlaw Andrew J.
Published: April 2014

Asthma is a common disease that causes considerable morbidity. Increased numbers of airway eosinophils are a hallmark of asthma. Mechanisms controlling the entry of eosinophils into asthmatic lung have been intensively investigated, but factors regulating migration within the tissue microenvironment are less well understood. We modeled this by studying chemoattractant and growth factor-mediated human eosinophil migration within a three-dimensional collagen matrix. Stimulation with GM-CSF induced dose-dependent, random migration with a maximum of 77 ± 4.7% of cells migrating. In contrast, CCL11 and C5a caused a more modest although significant degree of migration (19 ± 1.8% and 20 ± 2.6%, respectively). Migration to GM-CSF was partially dependent on Ca2+ and αΜβ2 integrins. The Rho family of small GTPases regulates intracellular signaling of cell migration. GM-CSF-induced migration was only partially dependent on Rho kinase/Rho-associated kinase (ROCK) and was independent of RhoA activation. In contrast, CCL11-induced migration was fully dependent on both RhoA and ROCK. Activation of RhoA was therefore neither necessary nor sufficient to cause eosinophil migration in a three-dimensional collagen environment. This study suggests that eosinophil growth factors are likely to be required for eosinophil migration within the bronchial mucosa, and this involves signal transduction pathways distinct from those used by G protein-associated chemoattractants.
Defective Generation of a Humoral Immune Response Is Associated with a Reduced Incidence and Severity of Collagen-Induced Arthritis in Microsomal Prostaglandin E Synthase-1 Null Mice
Authors: Kojima Fumiaki , Kapoor Mohit , Yang Lihua , Fleishaker Erica L. , Ward Martin R. , Monrad Seetha U. , Kottangada Ponnappa C. , Pace Charles Q. , Clark James A. , Woodward Jerold G. , Crofford Leslie J.
Published: April 2014

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen, including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.
Ghrelin Protects against Experimental Sepsis by Inhibiting High-Mobility Group Box 1 Release and by Killing Bacteria
Authors: Chorny Alejo , Anderson Per , Gonzalez-Rey Elena , Delgado Mario
Published: April 2014

Sepsis, a life-threatening complication of infections and the most common cause of death in intensive care units, is characterized by a hyperactive and out-of-balance network of endogenous proinflammatory cytokines. None of the current therapies are entirely effective, illustrating the need for novel therapeutic approaches. Ghrelin (GHR) is an orexigenic peptide that has emerged as a potential endogenous anti-inflammatory factor. In this study, we show that the delayed administration of GHR protects against the mortality in various models of established endotoxemia and sepsis. The therapeutic effect of GHR is mainly mediated by decreasing the secretion of the high mobility box 1 (HMGB1), a DNA-binding factor that acts as a late inflammatory factor critical for sepsis progression. Macrophages seem to be the major cell targets in the inhibition of HMGB1 secretion, in which GHR blocked its cytoplasmic translocation. Interestingly, we also report that GHR shows a potent antibacterial activity in septic mice and in vitro. Remarkably, GHR also reduces the severity of experimental arthritis and the release of HMGB1 to serum. Therefore, by regulating crucial processes of sepsis, such as the production of early and late inflammatory mediators by macrophages and the microbial load, GHR represents a feasible therapeutic agent for this disease and other inflammatory disorders.
Transamidation by Transglutaminase 2 Transforms S100A11 Calgranulin into a Procatabolic Cytokine for Chondrocytes
Authors: Cecil Denise L. , Terkeltaub Robert
Published: April 2014

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1β in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1β responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.
Antigen Presentation by Human Microvascular Endothelial Cells Triggers ICAM-1-Dependent Transendothelial Protrusion by, and Fractalkine-Dependent Transendothelial Migration of, Effector Memory CD4+ T Cells
Authors: Manes Thomas D. , Pober Jordan S.
Published: April 2014

TCR engagement on adherent human effector memory CD4+ T cells by TNF-treated HUVECs under flow induces formation of a transendothelial protrusion (TEP) by the T cell but fails to induce transendothelial migration (TEM). In contrast, TCR engagement of the same T cell populations by TNF-treated human dermal microvascular cells (HDMEC) not only induces TEP formation, but triggers TEM at or near the interendothelial cell junctions via a process in which TEP formation appears to be the first step. Transduction of adhesion molecules in unactivated HDMEC and use of blocking Abs as conducted with TNF-activated HDMEC indicate that ICAM-1 plays a nonredundant role in TCR-driven TEP formation and TEM, and that TCR-driven TEM is also dependent upon fractalkine. TEP formation, dependence on ICAM-1, and dependence on fractalkine distinguish TCR-induced TEM from IP-10-induced TEM. These in vitro observations suggest that presentation of Ag by human microvascular endothelial cells to circulating CD4+ effector memory T cells may function to initiate recall responses in peripheral tissues.
In Situ Activation of Antigen-Specific CD8+ T Cells in the Presence of Antigen in Organotypic Brain Slices
Authors: Ling Changying , Verbny Yakov I. , Banks Matthew I. , Sandor Matyas , Fabry Zsuzsanna
Published: April 2014

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8+ T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c+ cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c+ cells stimulate Ag-specific naive CD8+ T cells locally in the CNS and may contribute to immune responses in the brain.
Regulation of TLR2 Expression by Prostaglandins in Brain Glia
Authors: Yoon Hee Jung , Jeon Sae-Bom , Kim In-Hoo , Park Eun Jung
Published: April 2014

TLR have emerged as important primary sensors for diverse stimuli and are increasingly implicated in various diseases. However, the molecular mechanisms underlying the regulation of the TLR system remain poorly understood. In this study, we report that some PGs may control TLR-mediated inflammatory events through modulation of TLR2 expression in brain immune cells. We first found that 15-deoxy-Δ12,14-PG J2 (15d-PGJ2) markedly altered the expression of TLR2 but not TLR4, TLR1, and TLR9 at the message and protein levels in activated glia. Down-regulation of TLR2 expression and downstream events of TLR2 activation, including phagocytosis by 15d-PGJ2, were also observed in cells treated with representative TLR2 ligands such as lipoteichoic acid and Pam3CSK4. We further revealed that certain 15d-PGJ2-related PGs such as 15d-PGD2 and PGD2 also suppressed the ligand-stimulated increase of TLR2 expression, whereas PGE2 and arachidonic acids did not. Interestingly, TLR2 expression was down-regulated even when such PGs were added at several hours after stimulator treatment. These findings appear to be independent of peroxisome proliferator-activated receptor γ and D prostanoid receptors (DPs) because potent synthetic peroxisome proliferator-activated receptor γ agonists, selective DP1 agonist, or DP2 agonist did not mimic the effects of such PGs on TLR2 expression. Taken together, our results suggest that 15d-PGJ2, 15d-PGD2, and PGD2 may play notable roles as modulators of the TLR2-mediated inflammatory events, and provide new insight into the resolution of inflammation in the brain.
A Key Role for Redox Signaling in Rapid P2X7 Receptor-Induced IL-1β Processing in Human Monocytes
Authors: Hewinson James , Moore Samantha F. , Glover Christian , Watts Andrew G. , MacKenzie Amanda B.
Published: April 2014

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that trigger caspase-1 activation in the presence of TLR ligands. Inflammatory caspase-1 is responsible for the proteolytic activation of IL-1β. However, the signaling events that couple P2X7Rs to caspase-1 activation remain undefined. In this study we demonstrate that ATP-induced cellular oxidation is critical for caspase-1 activation and subsequent IL-1β processing. Purinergic receptor stimulation, including P2X7Rs, of endotoxin-primed human monocytes augments NADPH oxidase activity whereas concurrent purinergic receptor stimulation triggers protein denitroyslation, leading to the formation of peroxynitrite. IL-1β cleavage is blocked under conditions where superoxide anion formation is blocked or monocytes are treated with antioxidants or a peroxynitrite scavenger. Nigericin, a K+/H+ antiporter, also increases NADPH oxidase activity, leading to IL-1β and caspase-1 processing that is blocked by a peroxynitrite scavenger or inhibition of NADPH oxidase. These data demonstrate that signaling via NADPH oxidase activity is fundamental for the processing of mature IL-1β induced by P2X7R stimulation.
Peripheral T Cells Are the Therapeutic Targets of Glucocorticoids in Experimental Autoimmune Encephalomyelitis
Authors: Wüst Simone , van den Brandt Jens , Tischner Denise , Kleiman Anna , Tuckermann Jan P. , Gold Ralf , Lühder Fred , Reichardt Holger M.
Published: April 2014

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral TH17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.
Hydrolysis of Coagulation Factors by Circulating IgG Is Associated with a Reduced Risk for Chronic Allograft Nephropathy in Renal Transplanted Patients
Authors: Wootla Bharath , Nicoletti Antonino , Patey Natacha , Dimitrov Jordan D. , Legendre Christophe , Christophe Olivier D. , Friboulet Alain , Kaveri Srinivas V. , Lacroix-Desmazes Sébastien , Thaunat Olivier
Published: April 2014

Chronic allograft nephropathy (CAN), a major cause of late allograft failure, is characterized by a progressive decline in graft function correlated with tissue destruction. Uncontrolled activation of the coagulation cascade by the stressed endothelium of the graft is thought to play an important role in the pathophysiology of CAN. In this study, we demonstrate that circulating IgG from renal-transplanted patients are endowed with hydrolytic properties toward coagulation factors VIII and IX, but fail to hydrolyze factor VII and prothrombin. The hydrolytic activity of IgG was reliably quantified by the measure of the hydrolysis of a fluorescent synthetic substrate for serine proteases: proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA). A retrospective case-control study indicated that an elevated hydrolysis rate of PFR-MCA by circulating IgG correlated with the absence of CAN lesions on protocol graft biopsy performed 2 years posttransplantation. We propose that circulating hydrolytic IgG may counterbalance the procoagulation state conferred by the activated endothelium by disrupting the amplification loop of thrombin generation which is dependent on factors VIII and IX. Interestingly, low rates of PFR-MCA hydrolysis, measured 3 mo posttransplantation, were predictive of CAN at 2 years down the lane. These data suggest that PFR-MCA hydrolysis may be used as a prognosis marker for CAN in renal-transplanted patients.
Novel Vaccination for Allergy through Gene Silencing of CD40 Using Small Interfering RNA
Authors: Suzuki Motohiko , Zheng Xiufen , Zhang Xusheng , Li Mu , Vladau Costin , Ichim Thomas E. , Sun Hongtao , Min Lisa R. , Garcia Bertha , Min Wei-Ping
Published: April 2014

Small interfering RNA (siRNA) is a potent means of inducing gene-specific silencing. Gene silencing strategies using siRNA have demonstrated therapeutic benefits in animal models of various diseases, and are currently in clinical trials. However, the utility of gene silencing as a treatment for allergic diseases has not yet been reported. In this study, we report a novel therapy for allergy through gene silencing of CD40, a critical costimulatory molecule and a key factor in allergic immune responses. Silencing CD40 resulted in generation of immunoregulatory dendritic cells (DCs). Administration of CD40 siRNA remarkably reduced nasal allergic symptoms and local eosinophil accumulation in the OVA-induced allergic mice. The OVA-specific T cell response was inhibited after the CD40 siRNA treatment. Additionally, anti-OVA specific IgE and production of IL-4 and IL-5 of T cells stimulated by OVA were significantly decreased in CD40 siRNA-treated mice. Furthermore, we demonstrated that the therapeutic effects by CD40 siRNA were associated with impaired Ag-presenting functions of DCs and B cells, and generation of regulatory T cells. The present study highlights a therapeutic potential of siRNA-based treatment for allergic diseases.
Implications of the Near-Planar Solution Structure of Human Myeloma Dimeric IgA1 for Mucosal Immunity and IgA Nephropathy
Authors: Bonner Alexandra , Furtado Patricia B. , Almogren Adel , Kerr Michael A. , Perkins Stephen J.
Published: April 2014

IgA is unique in being able to form a diverse range of polymeric structures. Increases in the levels of dimeric IgA1 (dIgA1) in serum have been implicated in diseases such as IgA nephropathy. We have determined the solution structure for dIgA1 by synchrotron x-ray and neutron scattering and analytical ultracentrifugation. The Guinier radius of gyration (RG) of 7.60–8.65 nm indicated that the two monomers within dIgA1 are arranged in an extended conformation. The distance distribution curve P(r) gave an overall length (L) of 22–26 nm. These results were confirmed by the sedimentation coefficient and frictional ratio of dIgA1. Constrained scattering modeling starting from the IgA1 monomer solution structure revealed a near-planar dimer structure for dIgA1. The two Fc regions form a slightly bent arrangement in which they form end-to-end contacts, and the J chain was located at this interface. This structure was refined by optimizing the position of the four Fab regions. From this, the best-fit solution structures show that the four Fab Ag-binding sites are independent of one another, and the two Fc regions are accessible to receptor binding. This arrangement allows dIgA1 to initiate specific immune responses by binding to FcαRI receptors, while still retaining Ag-binding ability, and to be selectively transported to mucosal surfaces by binding to the polymeric Ig receptor to form secretory IgA. A mechanism for the involvement of dIgA1 oligomers in the pathology of IgA nephropathy is discussed in the light of this near-planar structure.
Requirement ofN-Myristoyltransferase 1 in the Development of Monocytic Lineage
Authors: Shrivastav Anuraag , Varma Shailly , Lawman Zoe , Yang Shao H. , Ritchie Shawn A. , Bonham Keith , Singh Sukh M. , Saxena Anurag , Sharma Rajendra K.
Published: April 2014

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/–) Nmt1-deficient mice. Homozygous (−/−) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c−Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.
Public T Cell Receptor β-Chains Are Not Advantaged during Positive Selection
Authors: Furmanski Anna L. , Ferreira Cristina , Bartok Istvan , Dimakou Sofia , Rice Jason , Stevenson Freda K. , Millrain Maggie M. , Simpson Elizabeth , Dyson Julian
Published: April 2014

Studies of human and murine T cells have shown that public TCR β-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vβ8.2-Jβ2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such β-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vβ8.2-Jβ2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR β-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public β-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that β-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public β-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.
The NF-κB Canonical Pathway Is Involved in the Control of the Exonucleolytic Processing of Coding Ends during V(D)J Recombination
Authors: Souto-Carneiro M. Margarida , Fritsch Ruth , Sepúlveda Nuno , Lagareiro M. João , Morgado Nuno , Longo Nancy S. , Lipsky Peter E.
Published: April 2014

V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-κB-binding sites are present in the human and mouse IgH, Igκ, and Igλ enhancer modules and RAG expression is controlled by NF-κB, it is not known whether NF-κB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-κB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-κB essential modulator (NEMO/Ikkγ). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkγ-mediated canonical NF-κB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3H from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-κB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-κB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.
Polymorphisms in theCD3ZGene Influence TCRζ Expression in Systemic Lupus Erythematosus Patients and Healthy Controls
Authors: Gorman Claire L. , Russell Andrew I. , Zhang Zhuoli , Cunninghame Graham Deborah , Cope Andrew P. , Vyse Timothy J.
Published: April 2014

TCRζ (CD247) functions as an amplification module in the TCR signaling cascade and is essential for assembly and surface expression of the TCR/CD3 complex. The TCRζ-chain is down-regulated in many chronic infectious and inflammatory diseases, including systemic lupus erythematosus (SLE). It is unclear whether reduced TCRζ expression is a cause or a consequence of chronic inflammatory responses. We have addressed this question by adopting a combined genetic and functional approach. We analyzed TCRζ protein expression using a FACS-based expression index and documented considerable, but longitudinally stable, variation in TCRζ expression in healthy individuals. The variation in TCRζ expression was associated with polymorphisms in the CD3Z 3′-untranslated region (UTR) in SLE patients and healthy controls. Detailed mapping of the 3′-UTR revealed that the minor alleles of two single nucleotide polymorphisms (SNPs) in strong disequilibrium (rs1052230 and rs1052231) were the causal variants associated with low TCRζ expression (p = 0.015). Using allelic imbalance analysis, the minor alleles of these 3′-UTR SNPs were associated with one-third of the level of mRNA compared with the major allele. A family-based association analysis showed that the haplotype carrying the low-expression variants predisposes to SLE (p = 0.033). This suggests that a genetically determined reduction in TCRζ expression has functional consequences manifested by systemic autoimmunity.
CXCL9 and CXCL10 Expression Are Critical for Control of Genital Herpes Simplex Virus Type 2 Infection through Mobilization of HSV-Specific CTL and NK Cells to the Nervous System
Authors: Thapa Manoj , Welner Robert S. , Pelayo Rosana , Carr Daniel J. J.
Published: April 2014

CXCL9 and CXCL10 mediate the recruitment of T lymphocytes and NK cells known to be important in viral surveillance. The relevance of CXCL10 in comparison to CXCL9 in response to genital HSV-2 infection was determined using mice deficient in CXCL9 (CXCL9−/−) and deficient in CXCL10 (CXCL10−/−) along with wild-type (WT) C57BL/6 mice. An increased sensitivity to infection was found in CXCL10−/− mice in comparison to CXCL9−/− or WT mice as determined by detection of HSV-2 in the CNS at day 3 postinfection. However, by day 7 postinfection both CXCL9−/− and CXCL10−/− mice possessed significantly higher viral titers in the CNS in comparison to WT mice consistent with mortality (18–35%) of these mice within the first 7 days after infection. Even though CXCL9−/− and CXCL10−/− mice expressed elevated levels of CCL2, CCL3, CCL5, and CXCL1 in the spinal cord in comparison to WT mice, there was a reduction in NK cell and virus-specific CD8+ T cell mobilization to this tissue, suggesting CXCL9 and CXCL10 are critical for recruitment of these effector cells to the spinal cord following genital HSV-2 infection. Moreover, leukocytes from the spinal cord but not from draining lymph nodes or spleens of infected CXCL9−/− or CXCL10−/− mice displayed reduced CTL activity in comparison to effector cells from WT mice. Thus, the absence of CXCL9 or CXCL10 expression significantly alters the ability of the host to control genital HSV-2 infection through the mobilization of effector cells to sites of infection.
CTL-Mediated Selective Pressure Influences Dynamic Evolution and Pathogenic Functions of HIV-1 Nef
Authors: Ueno Takamasa , Motozono Chihiro , Dohki Sachi , Mwimanzi Philip , Rauch Susanne , Fackler Oliver T. , Oka Shinichi , Takiguchi Masafumi
Published: April 2014

HIV-1 Nef plays multiple roles in modulating immune responses, even though it is a dominant CTL target itself. How Nef accomplishes the balance between such conflicting selective pressures remains elusive. By genetic and functional studies, we found that Arg75Thr and Tyr85Phe mutations, located in a well-conserved proline-rich region in Nef, were differently associated with escape from CTL responses specific for two overlapping HLA-B35-restricted epitopes. CTLs specific for an epitope, that selected Tyr85Phe, were elicited earlier and had more potent functional avidities than did those that selected Arg75Thr. Although the double mutant could escape from both CTLs, the mutations are rarely observed in combination naturally. Introduction of both mutations reduced Nef’s HLA class I down-regulation activity and increased the susceptibility of virus-infected cells to recognition by CTLs targeting other epitopes. Moreover, the mutant Nef was impaired in the association with activated cellular kinases and in the enhancement of viral replication. These results highlight CTL immunosurveillance as important modulators of Nef’s biological activity in the infected host.
The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation
Authors: Srivastava Raghvendra M. , Varalakshmi Chavvakula , Khar Ashok
Published: April 2014

Tumor recognition and killing, the uptake of released immunogenic substrate, and the generation of immunity are crucial aspects of dendritic cell (DC)-mediated antitumor immune response. In the context of direct tumoricidal activity, we have recently shown NK cell receptor protein-2 (NKR-P2)/NK group 2 member D (NKG2D) as a potent activation receptor on rat DCs. The activation of DCs with agonistic anti-NKR-P2 mAb, the binding of soluble NKR-P2 to the AK-5 tumor, and DC maturation with fixed AK-5 cells led us to identify a putative NKR-P2 ligand on the AK-5 cell surface. In this study we have shown that the AK-5 tumor-derived ischemia-responsive protein-94 (Irp94, a 110 kDa Hsp family member) acts as a functional ligand for NKR-P2 on DCs and enhances Irp94-NKR-P2 interaction-dependent tumor cell apoptosis via NO. Surface expression of Irp94 was also found on tumors of diverse origin in addition to AK-5. Furthermore, the Th1-polarizing cytokine IL-12, produced from Irp94-ligated BMDCs, augments NK cell cytotoxicity. Irp94-NKR-P2 interaction drives the maturation of BMDCs by up-regulating MHC class II, CD86, and CD1a and also induces autologous T cell proliferation, which displays a crucial state of DCs for adaptive antitumor immune response. These functional properties of Irp94 reside in the COOH terminus subdomain but not in the NH2 terminus ATPase domain of Irp94. We also show the involvement of PI3K, ERK, protein kinase C, phosphatases, and NF-κB translocation as downstream mediators of DCs activation upon NKR-P2 ligation with Irp94. Our studies demonstrate for the first time a novel role of a 110-kDa heat shock protein (Irp94) as a ligand for NKR-P2 on DCs, which in turn executes both innate and adaptive immunity.
CD8 T Cell Expansion and Memory Differentiation Are Facilitated by Simultaneous and Sustained Exposure to Antigenic and Inflammatory Milieu
Authors: Shaulov Angela , Murali-Krishna Kaja
Published: April 2014

Understanding the factors contributing to the generation of immune memory is important for rational vaccine design. In this study, we addressed the individual and combined roles of Ag and inflammation in sustaining the ability of primed CD8 T cells to clonally expand and differentiate into memory cells. We transferred CD8 T cells that were primed for a brief period into naive mice, mice infected with a pathogen not carrying the specific Ag (inflammation only), mice infected with a pathogen carrying the donor cell-specific Ag (inflammation plus Ag), or into mice exposed to soluble Ag (Ag only). We found that the donor CD8 T cells continued to proliferate in all the four conditions, but their ability to clonally expand and differentiate into memory cells was ∼1000-fold higher when transferred into mice acutely infected with pathogen carrying the relevant Ag. Memory cells generated under conditions of sustained exposure to inflammation and Ag during the priming phase were superior in their ability to elicit recall responses on a per cell basis. Thus, simultaneous and sustained exposure of donor CD8 T cells to inflammatory and antigenic stimuli, following the initial priming phase, leads to the greatest expansion of CD8 T cells at the peak of the immune response and induces an optimal memory differentiation program. These results suggest that vaccination strategies should attempt to provide sustained exposure to Ag plus inflammation but not either alone following the initial priming.
Control of Memory CD8+ T Cell Differentiation by CD80/CD86-CD28 Costimulation and Restoration by IL-2 during the Recall Response
Authors: Fuse Shinichiro , Zhang Weijun , Usherwood Edward J.
Published: April 2014

Memory CD8+ T cell responses have been considered to be independent of CD80/CD86-CD28 costimulation. However, recall responses are often severely blunted in CD28−/− mice. Whether this impairment represents a requirement for CD28 costimulation for proper memory CD8+ T cell development or a requirement during the recall response is unknown. Furthermore, how CD28 costimulation affects the phenotype and function of memory CD8+ T cells has not been characterized in detail. In this study, we investigate these questions by studying the role of the CD28 costimulatory pathway in memory CD8+ T cell responses to acute and persistent DNA virus infections. Memory CD8+ T cells against vaccinia virus (VV) infection which develop without CD28 costimulation exhibit lower expression of differentiation markers CD27 and CD122 (IL-15Rβ). These memory CD8+ T cells also fail to produce IL-2. Our data indicate that for an optimal recall response, CD28 costimulation is required both for T cell priming and also during the recall response. Similar requirements were observed for memory CD8+ T cell responses during persistent infection with murine gammaherpesvirus 68 (MHV-68) infection, indicating CD28 may play the same role in both acute and persistent infections. Finally, we show deficits in the recall response are restored by IL-2 signaling during recall, but not during priming. The data presented show that CD28 costimulation not only controls the magnitude of the primary response but also affects development of memory CD8+ T cells and is required during the recall response in addition to initial T cell priming.
Cigarette Smoke-Induced Pulmonary Inflammation Is TLR4/MyD88 and IL-1R1/MyD88 Signaling Dependent
Authors: Doz Emilie , Noulin Nicolas , Boichot Elisabeth , Guénon Isabelle , Fick Lizette , Le Bert Marc , Lagente Vincent , Ryffel Bernhard , Schnyder Bruno , Quesniaux Valérie F. J. , Couillin Isabelle
Published: April 2014

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1β only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1α and pro-IL-1β implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.
Cyclin-Dependent Kinase 4 Hyperactivity Promotes Autoreactivity in the Immune System but Protects Pancreatic β Cell Mass from Autoimmune Destruction in the Nonobese Diabetic Mouse Model
Authors: Marzo Nuria , Ortega Sagrario , Stratmann Thomas , García Ainhoa , Ríos Martín , Giménez América , Gomis Ramon , Mora Conchi
Published: April 2014

Cyclin-dependent kinase 4 (Cdk4) plays a central role in perinatal pancreatic β cell replication, thus becoming a potential target for therapeutics in autoimmune diabetes. Its hyperactive form, Cdk4R24C, causes β cell hyperplasia without promoting hypoglycemia in a nonautoimmune-prone mouse strain. In this study, we explore whether β cell hyperproliferation induced by the Cdk4R24C mutation balances the autoimmune attack against β cells inherent to the NOD genetic background. To this end, we backcrossed the Cdk4R24C knockin mice, which have the Cdk4 gene replaced by the Cdk4R24C mutated form, onto the NOD genetic background. In this study, we show that NOD/Cdk4R24C knockin mice exhibit exacerbated diabetes and insulitis, and that this exacerbated diabetic phenotype is solely due to the hyperactivity of the NOD/Cdk4R24C immune repertoire. Thus, NOD/Cdk4R24C splenocytes confer exacerbated diabetes when adoptively transferred into NOD/SCID recipients, compared with NOD/wild-type (WT) donor splenocytes. Accordingly, NOD/Cdk4R24C splenocytes show increased basal proliferation and higher activation markers expression compared with NOD/WT splenocytes. However, to eliminate the effect of the Cdk4R24C mutation specifically in the lymphocyte compartment, we introduced this mutation into NOD/SCID mice. NOD/SCID/Cdk4R24C knockin mice develop β cell hyperplasia spontaneously. Furthermore, NOD/SCID/Cdk4R24C knockin females that have been adoptively transferred with NOD/WT splenocytes are more resistant to autoimmunity than NOD/SCID WT female. Thus, the Cdk4R24C mutation opens two avenues in the NOD model: when expressed specifically in β cells, it provides a new potential strategy for β cell regeneration in autoimmune diabetes, but its expression in the immune repertoire exacerbates autoimmunity.
The IL-3/IL-5/GM-CSF Common β Receptor Plays a Pivotal Role in the Regulation of Th2 Immunity and Allergic Airway Inflammation
Authors: Asquith Kelly L. , Ramshaw Hayley S. , Hansbro Philip M. , Beagley Kenneth W. , Lopez Angel F. , Foster Paul S.
Published: April 2014

The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils are regulated by the CD4 Th2 cytokines IL-3, IL-5, and GM-CSF, which all signal through a common β receptor subunit (βc). Recent therapeutic approaches targeting IL-5 alone have not ablated tissue accumulation of eosinophils and have had limited effects on disease progression, suggesting important roles for IL-3 and GM-CSF. By using a mouse model of allergic airways inflammation, we show that allergen-induced expansion and accumulation of eosinophils in the lung are abolished in βc-deficient (βc−/−) mice. Moreover, βc deficiency resulted in inhibition of hallmark features of asthma, including airways hypersensitivity, mucus hypersecretion, and production of Ag-specific IgE. Surprisingly, we also identified a critical role for this receptor in regulating type 2 immunity. Th2 cells in the lung of allergen-challenged βc−/− mice were limited in their ability to proliferate, produce cytokines, and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus, the βc plays a critical role in allergen-induced eosinophil expansion and infiltration and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy.
Chemokine Gene Expression during Fatal Murine Cerebral Malaria and Protection Due to CXCR3 Deficiency
Authors: Miu Jenny , Mitchell Andrew J. , Müller Marcus , Carter Sally L. , Manders Peter M. , McQuillan James A. , Saunders Bernadette M. , Ball Helen J. , Lu Bao , Campbell Iain L. , Hunt Nicholas H.
Published: April 2014

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. Using murine models of malaria, we found much greater up-regulation of a number of chemokine mRNAs, including those for CXCR3 and its ligands, in the brain during fatal murine CM (FMCM) than in a model of non-CM. Expression of CXCL9 and CXCL10 RNA was localized predominantly to the cerebral microvessels and in adjacent glial cells, while expression of CCL5 was restricted mainly to infiltrating lymphocytes. The majority of mice deficient in CXCR3 were found to be protected from FMCM, and this protection was associated with a reduction in the number of CD8+ T cells in brain vessels as well as reduced expression of perforin and FasL mRNA. Adoptive transfer of CD8+ cells from C57BL/6 mice with FMCM abrogated this protection in CXCR3−/− mice. Moreover, there were decreased mRNA levels for the proinflammatory cytokines IFN-γ and lymphotoxin-α in the brains of mice protected from FMCM. These data suggest a role for CXCR3 in the pathogenesis of FMCM through the recruitment and activation of pathogenic CD8+ T cells.
Low-Dose Targeted Complement Inhibition Protects against Renal Disease and Other Manifestations of Autoimmune Disease in MRL/lpr Mice
Authors: Atkinson Carl , Qiao Fei , Song Hongbin , Gilkeson Gary S. , Tomlinson Stephen
Published: April 2014

Complement appears to play a dual role in the progression of systemic lupus erythematosus, serving a beneficial role in enhancing immune complex clearance, while serving a pathogenic role in inducing local inflammation. To investigate these different roles of complement in a therapeutic setting, MRL/lpr mice were treated with the targeted murine C3 complement inhibitor, CR2-Crry, from 16 to 24 wk of age (after the development of proteinuria). The targeting moiety, CR2, binds to C3 breakdown products deposited at sites of complement activation and has the potential to provide complement inhibition locally without causing systemic inhibition. Administration of CR2-Crry i.v., at a dose of 0.25 mg once a week, was associated with a significant survival benefit, improved kidney function, and a significant reduction in glomerulonephritis and renal vasculitis. The presence of skin lesions and lung bronchiolar and vascular inflammation was also dramatically reduced by CR2-Crry treatment. CR2-Crry treatment also resulted in a significant reduction in autoantibody production, as measured by anti-dsDNA Ab levels, and did not cause an increase in circulating immune complex levels. These effects on autoimmunity and circulating immune complexes represent significant potential advantages over the use of Crry-Ig in MRL/lpr mice, a systemic counterpart of CR2-Crry. CR2-Crry localized preferentially to the kidneys in 16-wk MRL/lpr mice with a kidney-localized half-life of ∼24 h. Thus, targeted complement inhibition at the C3 level is an effective treatment in murine lupus, even beginning after onset of disease.