bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas-Associated Death Domain-Containing Protein-Mediated Antiviral Innate Immune Signaling Involves the Regulation of Irf7
Authors: Balachandran Siddharth , Venkataraman Thiagarajan , Fisher Paul B. , Barber Glen N.
Published: April 2014

The induction of type I (αβ) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-β and IFN-α4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-α subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-αs and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-α production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd−/− murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD’s antiviral activity involves regulation of IRF-7-dependent production of IFN-α subtypes and consequent induction of secondary antiviral genes.
Simvastatin Potentiates TNF-α-Induced Apoptosis through the Down-Regulation of NF-κB-Dependent Antiapoptotic Gene Products: Role of IκBα Kinase and TGF-β-Activated Kinase-1
Authors: Ahn Kwang Seok , Sethi Gautam , Aggarwal Bharat B.
Published: April 2014

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-α-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-α as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-xL, cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-κB. We found that simvastatin inhibited TNF-α-induced NF-κB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-κB activation. Simvastatin inhibited TNF-α-induced IκBα kinase activation, which led to inhibition of IκBα phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-κB-dependent reporter gene expression induced by TNF-α, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-β-activated kinase 1, receptor-interacting protein, NF-κB-inducing kinase, and IκB kinase β was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IκB kinase and NF-κB-regulated gene products.
Glucocorticoid Conditioning of Myeloid Progenitors Enhances TLR4 Signaling via Negative Regulation of the Phosphatidylinositol 3-Kinase-Akt Pathway
Authors: Zhang Tian Y. , Daynes Raymond A.
Published: April 2014

The immunomodulatory effects of glucocorticoids (GCs) have been described as bimodal, with high levels of GCs exerting immunosuppressive effects and low doses of GCs being immunopermissive. While the mechanisms used by GCs to achieve immunosuppression have been investigated intensely, the molecular mechanisms underlying the permissive effects of GCs remain uncharacterized. Herein, we demonstrate that GC conditioning during the differentiation of myeloid progenitors into macrophages (Mφs) results in their enhanced LPS responsiveness, demonstrated by an overexpression of the inflammatory cytokines TNF-α, IL-6, and IL-12. Inflammatory cytokine overexpression resulted from an increased activation of NF-κB and the MAPK signaling cascade and a reduced activation of the PI3K-Akt pathway following LPS stimulation. GC conditioning during Mφ differentiation induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway. Small interfering RNA-mediated knockdown of SHIP1 expression increased PI3K-dependent Akt activation and subsequently decreased inflammatory cytokine expression, suggesting GC-mediated up-regulation of SHIP1 expression is responsible for the augmentation in inflammatory cytokine production following LPS stimulation. We also show that splenic Mφs purified from normal mice that were implanted with timed-release GC pellets exhibited an enhanced LPS responsiveness and increased SHIP1 expression, indicating that GCs can regulate SHIP1 expression in vivo. Our results suggest that minor fluctuations in physiological levels of endogenous GCs can program endotoxin-responsive hemopoietic cells during their differentiation by regulating their sensitivity to stimulation.
Differential Biological Role of CD3 Chains Revealed by Human Immunodeficiencies
Authors: Recio María J. , Moreno-Pelayo Miguel Angel , Kiliç Sara S. , Guardo Alberto C. , Sanal Ozden , Allende Luis M. , Pérez-Flores Verónica , Mencía Angeles , Modamio-Høybjør Silvia , Seoane Elena , Regueiro José R.
Published: April 2014

The biological role in vivo of the homologous CD3γ and δ invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3γ deficiency and SCID symptoms and compared them with three CD3γ-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild αβ and γδ T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA+ αβ T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3δ (or CD3ε) deficiencies are in infants with life-threatening SCID and very severe αβ and γδ T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3γ deficiencies despite poor thymus output or clinical outcome. We propose a CD3δ ≫ CD3γ hierarchy for the relative impact of their absence on the signaling for T cell production in humans.
Expansion of CD4+CD25+and FOXP3+ Regulatory T Cells during the Follicular Phase of the Menstrual Cycle: Implications for Human Reproduction
Authors: Arruvito Lourdes , Sanz Marianela , Banham Alison H. , Fainboim Leonardo
Published: April 2014

Regulatory T cells (Tregs) are thought to affect the severity of various infectious and autoimmune diseases. The incidence of autoimmune disease is higher in fertile women than in men. Thus, we investigated whether Treg numbers were modulated during the menstrual cycle by sex hormones. In fertile nonpregnant women, we detected an expansion of CD4+CD25+FOXP3+ Tregs in the late follicular phase of the menstrual cycle. This increase was tightly correlated with serum levels of estradiol and was followed by a dramatic decrease in Treg numbers at the luteal phase. Women who have had recurrent spontaneous abortions (RSA) showed similarly low numbers of Tregs at both the follicular and luteal phases, comparable to numbers we observed in postmenopausal women. In addition to decreased numbers, Tregs from women with RSA were also functionally deficient, as higher numbers were required to exert a similar magnitude of suppression to CD4+CD25+FOXP3+ cells from fertile women. Consequently, reproductive failure might result from the inability of Tregs in women with RSA to expand during the preimplantatory phase combined with their lower functional capacity. Additionally, the modulation of Treg numbers we observed in fertile women suggests that the stage of the menstrual cycle should be taken into account when Treg numbers are investigated clinically.
Cutting Edge: TFII-I Controls B Cell Proliferation via Regulating NF-κB
Authors: Ashworth Todd , Roy Ananda L.
Published: April 2014

The multifunctional transcription factor TFII-I physically and functionally interacts with Bruton’s tyrosine kinase in murine B cells. However, the downstream functions of TFII-I in B cells are unknown. Toward achieving this goal, we established stable posttranscriptional silencing of TFII-I in WEHI-231 immature murine B cells, which undergoes growth arrest and apoptosis either upon anti-IgM or TGF-β signaling. In this study, we show that TFII-I promotes growth arrest of cells in a signal-dependent manner. Unlike control cells, B cells exhibiting loss of TFII-I function fail to undergo arrest upon signaling due to up-regulation of c-Myc expression and concomitant down-regulation of both p21 and p27. Loss of TFII-I is also associated with simultaneous increase in nuclear c-rel and decrease in p50 homodimer binding. Thus, besides controlling c-myc transcription, TFII-I controls B cell proliferation by regulating both nuclear translocation of c-rel and DNA-binding activity of p50 NF-κB.
Cutting Edge: Differential Inhibition of TLR Signaling Pathways by Cell-Permeable Peptides Representing BB Loops of TLRs
Authors: Toshchakov Vladimir Y. , Fenton Matthew J. , Vogel Stefanie N.
Published: April 2014

We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-κB translocation and early IL-1β mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys4-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-κB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins.
Virus-Specific CD8+ T Cells in the Liver: Armed and Ready to Kill
Authors: Keating Rachael , Yue Wen , Rutigliano John A. , So Jenny , Olivas Elvia , Thomas Paul G. , Doherty Peter C.
Published: April 2014

Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2DbNP366–374 (DbNP366) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-γ secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a “graveyard” for influenza virus-specific CD8+ T cells.
CD4+CD25+ T Cell-Dependent Inhibition of Autoimmunity in Transgenic Mice Overexpressing Human Bcl-2 in T Lymphocytes
Authors: González Jovanna , Tamayo Esther , Santiuste Inés , Marquina Regina , Buelta Luis , González-Gay Miguel A. , Izui Shozo , López-Hoyos Marcos , Merino Jesús , Merino Ramón
Published: April 2014

Regulation of lymphocyte survival is essential for the maintenance of lymphoid homeostasis preventing the development of autoimmune diseases. Recently, we described a systemic lupus erythematosus associated with an IgA nephropathy in autoimmune-prone (NZW × C57BL/6)F1 overexpressing human Bcl-2 (hBcl-2) in B cells (transgenic (Tg) 1). In the present study, we analyze in detail a second line of hBcl-2 Tg mice overexpressing the transgene in all B cells and in a fraction of CD4+ and CD8+ T cells (Tg2). We demonstrate here that the overexpression of hBcl-2 in T cells observed in Tg2 mice is associated with a resistance to the development of lupus disease and collagen type II-induced arthritis in both (NZW × C57BL/6)F1 and (DBA/1 × C57BL/6)F1 Tg2 mice, respectively. The disease-protective effect observed in autoimmune-prone Tg2 mice is accompanied by an increase of peripheral CD4+CD25+ hBcl-2+ regulatory T cells (Tregs), expressing glucocorticoid-induced TNFR, CTLA-4, and FoxP3. Furthermore, the in vivo depletion of CD4+CD25+ Tregs in (DBA/1 × C57BL/6)F1 Tg2 mice promotes the development of a severe collagen type II-induced arthritis. Taken together, our results indicate that the overexpression of hBcl-2 in CD4+ T cells alters the homeostatic mechanisms controlling the number of CD4+CD25+ Tregs resulting in the inhibition of autoimmune diseases.
CD40-CD40 Ligand Interaction between Dendritic Cells and CD8+ T Cells Is Needed to Stimulate Maximal T Cell Responses in the Absence of CD4+ T Cell Help
Authors: Hernandez Maria Genevive H. , Shen Lianjun , Rock Kenneth L.
Published: April 2014

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and “licenses” the APCs to prime CD8+ T cell responses. Although other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naive CD8+ T cells. We find an ∼50% reduction in expansion and cytokine production in TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+ T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of CD8+ T cell responses.
Involvement of Src and Syk Tyrosine Kinases in HIV-1 Transfer from Dendritic Cells to CD4+ T Lymphocytes
Authors: Gilbert Caroline , Barat Corinne , Cantin Réjean , Tremblay Michel J.
Published: April 2014

Dendritic cells (DCs) are considered as key mediators of the early events in HIV-1 infection at mucosal sites. Although several aspects of the complex interactions between DCs and HIV-1 have been elucidated, there are still basic questions that remain to be answered about DCs/HIV-1 interplay. In this study, we examined the contribution of nonreceptor TKs in the known ability of DCs to efficiently transfer HIV-1 to CD4+ T cells in trans. Experiments performed with specific inhibitors of Src and Syk family members indicate that these tyrosine kinases (TKs) are participating to HIV-1 transfer from immature monocyte-derived DCs (IM-MDDCs) to autologous CD4+ T cells. Experiments with IM-MDDCs transfected with small interfering RNAs targeting Lyn and Syk confirmed the importance of these nonreceptor TKs in HIV-1 transmission. The Src- and Syk-mediated effect on virus transfer was linked with infection of IM-MDDCs in cis-as monitored by quantifying integrated viral DNA and de novo virus production. The process of HIV-1 transmission from IM-MDDCs to CD4+ T cells was unaffected following treatment with protein kinase C and protein kinase A inhibitors. These data suggest that Src and Syk TKs play a functional role in productive HIV-1 infection of IM-MDDCs. Additional work is needed to facilitate our comprehension of the various mechanisms underlying the exact contribution of Src and Syk TKs to this phenomenon.
IL-21 and BAFF/BLyS Synergize in Stimulating Plasma Cell Differentiation from a Unique Population of Human Splenic Memory B Cells
Authors: Ettinger Rachel , Sims Gary P. , Robbins Rachel , Withers David , Fischer Randy T. , Grammer Amrie C. , Kuchen Stefan , Lipsky Peter E.
Published: April 2014

Both constitutive Ig secretion by long-lived plasma cells (PC) and the recurrent differentiation of memory (mem) B cells into PC contribute to the maintenance of serologic mem. However, the relative contribution of each is unknown. In this study, we describe a novel population of human postswitched mem B cells that rapidly differentiate into PC and thus contribute to serologic mem. These IgG+ B cells reside in the region of human spleen analogous to the murine marginal zone and have not previously been examined. These cells are highly responsive to IL-21 in the context of CD40 stimulation. Uniquely, IgG+ marginal zone analog B cells are exquisitely sensitive to the combination of IL-21 and B cell-activating factor belonging to the TNF family (BAFF/BLyS) that synergize in the absence of further costimulation to induce up-regulation of B lymphocyte-induced maturation protein-1 and drive PC differentiation. Other cytokine combinations are not active in this regard. This is the first demonstration that this unique population of mem B cells can respond specifically and exclusively to IL-21 and BAFF/BLyS by differentiating into IgG-secreting PC, and thus contributing to serologic mem in an Ag-independent manner.
HLA Transgenic Mice Provide Evidence for a Direct and Dominant Role of HLA Class II Variation in Modulating the Severity of Streptococcal Sepsis
Authors: Nooh Mohammed M. , El-Gengehi Nagala , Kansal Rita , David Chella S. , Kotb Malak
Published: April 2014

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.
Functional Synergy between CD40 Ligand and HIV-1 Tat Contributes to Inflammation: Implications in HIV Type 1 Dementia
Authors: Sui Ziye , Sniderhan Lynn F. , Schifitto Giovanni , Phipps Richard P. , Gelbard Harris A. , Dewhurst Stephen , Maggirwar Sanjay B.
Published: April 2014

HIV type 1 (HIV-1)-associated dementia (HAD) is believed to occur due to aberrant activation of monocyte-derived macrophages and brain-resident microglial cells by viral proteins as well as by the proinflammatory mediators released by infected cells. To investigate the inflammatory aspects of the disease, we examined the levels of soluble CD40L (sCD40L) in paired samples of plasma and cerebrospinal fluid obtained from 25 HIV-infected individuals. A significantly higher level of sCD40L was detected in both cerebrospinal fluid and plasma from HIV-infected patients with cognitive impairment, compared with their nonimpaired counterparts. The contribution of sCD40L to the pathogenesis of HAD was then examined by in vitro experiments. rCD40L synergized with HIV-1 Tat to increase TNF-α release from primary human monocytes and microglia, in an NF-κB-dependent manner. The mechanistic basis for this synergism was attributed to a Tat-mediated up-regulation of CD40 in monocytes and microglia. Finally, the CD40L-mediated increase in TNF-α production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-α was essential for the neurotoxic effects of conditioned medium recovered from Tat/CD40L-treated monocytes. Taken together, our results show that CD40 signaling in microglia and monocytes can synergize with the effects of Tat, further amplifying inflammatory processes within the CNS and influencing neuronal survival.
Cutting Edge: Antibody-Mediated TLR7-Dependent Recognition of Viral RNA
Authors: Wang Jennifer P. , Asher Damon R. , Chan Melvin , Kurt-Jones Evelyn A. , Finberg Robert W.
Published: April 2014

TLR7 recognizes the genome of ssRNA viruses such as Coxsackievirus B. Because TLR7 is expressed in intracellular compartments, viral RNA must be internalized before its recognition by TLR7. In this study, we define plasmacytoid dendritic cells (pDC) as peripheral blood mononuclear immune cells that respond to Coxsackievirus. pDC activation by Coxsackievirus B requires the presence of specific antiviral Abs. We show that Fc receptors mediate the recognition of virus-Ab complexes and that TLR7 is required for human and murine pDC production of cytokines. These data define a pathway by which intracellular TLR7 senses viral RNA and indicate a role for TLRs in association with Abs in sustaining virus-specific responses.
TLR Ligands Act Directly upon T Cells to Restore Proliferation in the Absence of Protein Kinase C-θ Signaling and Promote Autoimmune Myocarditis
Authors: Marsland Benjamin J. , Nembrini Chiara , Grün Katja , Reissmann Regina , Kurrer Michael , Leipner Carola , Kopf Manfred
Published: April 2014

The serine/threonine kinase, protein kinase C-θ (PKC-θ), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-θ may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-θ in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with α-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-θ-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-θ was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-θ in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-θ-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-xL, but exogenous IL-6 and TGF-β was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-θ signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.
Kinetic Proofreading of Ligand-FcεRI Interactions May Persist beyond LAT Phosphorylation
Authors: Torigoe Chikako , Faeder James R. , Oliver Janet M. , Goldstein Byron
Published: April 2014

Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal. This suggests that an assay for kinetic proofreading might be used to determine which activation events occur within the initial signaling complex. In signaling through the high affinity IgE receptor FcεRI, the transmembrane adaptor called linker for activation of T cells (LAT) is thought to nucleate a distinct secondary complex. Experiments in which the concentrations of two ligands with different dwell times are adjusted to equalize the level of LAT phosphorylation in rat basophilic leukemia 2H3 cells show that Erk2 phosphorylation, intracellular Ca2+, and degranulation exhibit kinetic proofreading downstream of LAT phosphorylation. These results suggest that ligand-bound FcεRI and LAT form a complex that is required for effective signal transmission.
Differential Regulation of Human NK Cell-Mediated Cytotoxicity by the Tyrosine Kinase Itk
Authors: Khurana Dianne , Arneson Laura N. , Schoon Renee A. , Dick Christopher J. , Leibson Paul J.
Published: April 2014

NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.
A Quantitative Study of NF-κB Activation by H2O2: Relevance in Inflammation and Synergy with TNF-α
Authors: de Oliveira-Marques Virgínia , Cyrne Luísa , Marinho H. Susana , Antunes Fernando
Published: April 2014

Although the germicide role of H2O2 released during inflammation is well established, a hypothetical regulatory function, either promoting or inhibiting inflammation, is still controversial. In particular, after 15 years of highly contradictory results it remains uncertain whether H2O2 by itself activates NF-κB or if it stimulates or inhibits the activation of NF-κB by proinflammatory mediators. We investigated the role of H2O2 in NF-κB activation using, for the first time, a calibrated and controlled method of H2O2 delivery—the steady-state titration—in which cells are exposed to constant, low, and known concentrations of H2O2. This technique contrasts with previously applied techniques, which disrupt cellular redox homeostasis and/or introduce uncertainties in the actual H2O2 concentration to which cells are exposed. In both MCF-7 and HeLa cells, H2O2 at extracellular concentrations up to 25 μM did not induce significantly per se NF-κB translocation to the nucleus, but it stimulated the translocation induced by TNF-α. For higher H2O2 doses this stimulatory role shifts to an inhibition, which may explain published contradictory results. The stimulatory role was confirmed by the observation that 12.5 μM H2O2, a concentration found during inflammation, increased the expression of several proinflammatory NF-κB-dependent genes induced by TNF-α (e.g., IL-8, MCP-1, TLR2, and TNF-α). The same low H2O2 concentration also induced the anti-inflammatory gene coding for heme oxygenase-1 (HO-1) and IL-6. We propose that H2O2 has a fine-tuning regulatory role, comprising both a proinflammatory control loop that increases pathogen removal and an anti-inflammatory control loop, which avoids an exacerbated harmful inflammatory response.
Natural and Synthetic TLR7 Ligands Inhibit CpG-A- and CpG-C-Oligodeoxynucleotide-Induced IFN-α Production
Authors: Berghöfer Beate , Haley Gabriela , Frommer Ture , Bein Gregor , Hackstein Holger
Published: April 2014

Plasmacytoid dendritic cells (pDCs) are unique with respect to their capacity to produce unsurpassed amounts of IFN-α and coexpress TLR7 and TLR9, mediating IFN-α production. Although TLRs are critical receptors of innate immunity, little is known about the immunological effects of TLR7/TLR9 costimulation. We have analyzed the effects of TLR7/TLR9 costimulation on IFN-α production by leukocytes and pDCs. Our experiments revealed that both synthetic (resiquimod and loxoribine) and natural (ssRNA40) TLR7 ligands abrogate CpG-A- and CpG-C-oligodeoxynucleotide (ODN)-induced IFN-α production by human leukocytes. Because TLR7 ligands themselves represent important IFN-α inducers, we demonstrated that substimulatory TLR7 ligand concentrations significantly inhibited CpG-A-induced IFN-α. Delayed addition of TLR7 ligands still resulted in complete suppression of CpG-A-ODN-induced IFN-α production, suggesting that the inhibition is unlikely to be caused by a kinetic uptake advantage. Unlike for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-ODN-induced IFN-α production. Experiments with purified human pDCs demonstrated that the inhibitory effects of TLR7/TLR9 costimulation were mediated directly by pDCs. Suppression of IFN-α production was not related to increased cell death and was also detectable in enriched mouse pDCs. Analyses of pDCs suggested that the TLR7 signal regulates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either inhibit (IFN-α) or promote (IL-8/CD40) cytokine and surface marker expression. Our data reveal for the first time a strong inhibitory effect of TLR7 stimulation on IFN-α production induced by CpG-A- and CpG-C-ODNs. These findings provide novel insight into the effects of TLR7/TLR9 costimulation and may support the development of novel TLR9 inhibitors.
Delivery of Antigen to CD40 Induces Protective Immune Responses against Tumors
Authors: Schjetne Karoline W. , Fredriksen Agnete B. , Bogen Bjarne
Published: April 2014

Ligation of CD40 induces maturation of dendritic cells (DC) and could be a useful target for vaccines. In this study, we have constructed two types of Ab-based vaccine constructs that target mouse CD40. One type is a recombinant Ab with V regions specific for CD40 and has defined T cell epitopes inserted into its C region. The other type is a homodimer, each chain of which is composed of a targeting unit (single-chain fragment variable targeting CD40), a dimerization motif, and an antigenic unit. Such proteins bound CD40, stimulated maturation of DC, and enhanced primary and memory T cell responses. When delivered i.m. as naked DNA followed by electroporation, the vaccines induced T cell responses against MHC class II-restricted epitopes, Ab responses, and protection in two tumor models (myeloma and lymphoma). Two factors apparently contributed to these results: 1) agonistic ligation of CD40 and induction of DC maturation, and 2) delivery of Ag to APC and presentation on MHC class II molecules. These results highlight the importance of agonistic targeting of Ag to CD40 for induction of long-lasting and protective immune responses.
Distinct Cytokine-Driven Responses of Activated Blood γδ T Cells: Insights into Unconventional T Cell Pleiotropy
Authors: Vermijlen David , Ellis Peter , Langford Cordelia , Klein Anne , Engel Rosel , Willimann Katharina , Jomaa Hassan , Hayday Adrian C. , Eberl Matthias
Published: April 2014

Human Vγ9/Vδ2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3+ cells. This “transitional response,” occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in αβ T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vγ9/Vδ2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic αβ T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate γδ T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of γδ T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vγ9/Vδ2 TCR agonists.
Factor H Binding and Function in Sialylated Pathogenic Neisseriae is Influenced by Gonococcal, but Not Meningococcal, Porin
Authors: Madico Guillermo , Ngampasutadol Jutamas , Gulati Sunita , Vogel Ulrich , Rice Peter A. , Ram Sanjay
Published: April 2014

Neisseria gonorrhoeae and Neisseria meningitidis both express the lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) molecule that can be sialylated. Although gonococcal LNT LOS sialylation enhances binding of the alternative pathway complement inhibitor factor H and renders otherwise serum-sensitive bacteria resistant to complement-dependent killing, the role of LOS sialylation in meningococcal serum resistance is less clear. We show that only gonococcal, but not meningococcal, LNT LOS sialylation enhanced factor H binding. Replacing the porin (Por) B molecule of a meningococcal strain (LOS sialylated) that did not bind factor H with gonococcal Por1B augmented factor H binding. Capsule expression did not alter factor H binding to meningococci that express gonococcal Por. Conversely, replacing gonococcal Por1B with meningococcal PorB abrogated factor H binding despite LNT LOS sialylation. Gonococcal Por1B introduced in the background of an unsialylated meningococcus itself bound small amounts of factor H, suggesting a direct factor H-Por1B interaction. Factor H binding to unsialylated meningococci transfected with gonococcal Por1B was similar to the sialylated counterpart only in the presence of higher (20 μg/ml) concentrations of factor H and decreased in a dose-responsive manner by ∼80% at 1.25 μg/ml. Factor H binding to the sialylated strain remained unchanged over this factor H concentration range however, suggesting that LOS sialylation facilitated optimal factor H-Por1B interactions. The functional counterpart of factor H binding showed that sialylated meningococcal mutants that possessed gonococcal Por1B were resistant to complement-mediated killing by normal human serum. Our data highlight the different mechanisms used by these two related species to evade complement.
IFN Mimetic as a Therapeutic for Lethal Vaccinia Virus Infection: Possible Effects on Innate and Adaptive Immune Responses
Authors: Ahmed Chulbul M. , Martin James P. , Johnson Howard M.
Published: April 2014

We have developed small peptide mimetics of IFN-γ that can bypass the poxvirus virulence factor B8R protein, which binds to intact IFN-γ and prevents its interaction with receptor extracellular domain. Thus, these peptides inhibit vaccinia virus replication in cell culture where intact IFN-γ is ineffective. We demonstrate here that the mouse IFN-γ-mimetic peptide, IFN-γ95–132, protects C57BL/6 mice against overwhelming lethal vaccinia virus infection. The mimetic peptide was synthesized with an attached lipophilic group for penetration of cell plasma membrane. Injection of mimetic i.p. before and at the time of intranasal (106 PFU) or i.p. (107 PFU) challenge with virus resulted in complete protection at 200 μg of mimetic and 40–60% protection at 5 μg of mimetic. Initiation of treatment of mice with IFN-γ mimetic up to 2 days postinfection resulted in complete protection against death, whereas initiation of treatment at 6 days postinfection resulted in 40% protection. Administration of mimetic by the oral route also completely protected mice against the intranasal route of a lethal dose of vaccinia virus challenge. In addition to its direct antiviral effect, the mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to vaccinia virus. The combination of antiviral and adjuvant effects by the IFN mimetic probably plays a role in its potent anti-vaccinia virus properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses.
The Chemokine Binding Protein M3 Prevents Diabetes Induced by Multiple Low Doses of Streptozotocin
Authors: Martin Andrea P. , Alexander-Brett Jennifer M. , Canasto-Chibuque Claudia , Garin Alexandre , Bromberg Jonathan S. , Fremont Daved H. , Lira Sergio A.
Published: April 2014

Multiple injections of low-dose streptozotocin (MLDS) induce lymphocytic insulitis and diabetes in rodents. To test whether the influx of inflammatory cells was associated with changes in the expression of chemokines, we measured the expression of all known chemokine ligands by real-time quantitative PCR in isolated islets. With the exception of CCL20 and CCL19, chemokines were not significantly expressed in islets from wild-type mice before MLDS treatment. Ten days after treatment, the expression of several chemokines, including CXCL9, CCL1, CXCL10, and CCL21, was dramatically up-regulated. The expression of CCL1, CXCL9, and CCL21 protein was confirmed by immunohistochemistry and was mostly associated with the infiltrating cells. The mouse herpesvirus 68-encoded chemokine decoy receptor M3 can broadly engage these chemokines with high affinity. To test whether a blockade of chemokine function would alter the onset or magnitude of insulitis and diabetes, we used transgenic mice expressing M3 in β cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction.
Grass Pollen Immunotherapy Induces an Allergen-Specific IgA2 Antibody Response Associated with Mucosal TGF-β Expression
Authors: Pilette Charles , Nouri-Aria Kayhan T. , Jacobson Mikila R. , Wilcock Louisa K. , Detry Bruno , Walker Samantha M. , Francis James N. , Durham Stephen R.
Published: April 2014

Allergen immunotherapy (IT) has long-term efficacy in IgE-mediated allergic rhinitis and asthma. IT has been shown to modify lymphocyte responses to allergen, inducing IL-10 production and IgG Abs. In contrast, a putative role for IgA and local TGF-β-producing cells remains to be determined. In 44 patients with seasonal rhinitis/asthma, serum IgA1, IgA2, and polymeric (J chain-containing) Abs to the major allergen Phl p 5 were determined by ELISA before and after a 2-year double-blind trial of grass pollen (Phleum pratense) injection IT. Nasal TGF-β expression was assessed by in situ hybridization. Sera from five IT patients were fractionated for functional analysis of the effects of IgA and IgG Abs on IL-10 production by blood monocytes and allergen-IgE binding to B cells. Serum Phl p 5-specific IgA2 Abs increased after a 2-year treatment (∼8-fold increase, p = 0.002) in contrast to IgA1. Increases in polymeric Abs to Phl p 5 (∼2-fold increase, p = 0.02) and in nasal TGF-β mRNA (p = 0.05) were also observed, and TGF-β mRNA correlated with serum Phl p 5 IgA2 (r = 0.61, p = 0.009). Post-IT IgA fractions triggered IL-10 secretion by monocytes while not inhibiting allergen-IgE binding to B cells as observed with IgG fractions. This study shows for the first time that the IgA response to IT is selective for IgA2, correlates with increased local TGF-β expression, and induces monocyte IL-10 expression, suggesting that IgA Abs could thereby contribute to the tolerance developed in IT-treated allergic patients.
Prothymosin α Lacking the Nuclear Localization Signal as an Effective Gene Therapeutic Strategy in Collagen-Induced Arthritis
Authors: Shiau Ai-Li , Chen Shih-Yao , Chang Meng-Ya , Su Chih-Hau , Chung Shih-Ye , Yo Yi-Te , Wang Chrong-Reen , Wu Chao-Liang
Published: April 2014

Prothymosin α (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTΔNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTΔNLS treatment abolished the up-regulation of the MIP-1α promoter activity induced by TNF-α in synovial fibroblasts. AdProTΔNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1α in macrophage chemotaxis. Administration of AdProTΔNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-α (mean ± SEM, 1261.9 ± 107.9 vs 2880.1 ± 561.4 pg/mg total protein; p < 0.05), IL-1β (56.8 ± 8.0 vs 109.2 ± 4.9 pg/mg total protein; p < 0.01), and MIP-1α (41.7 ± 3.6 vs 55.2 ± 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTΔNLS-treated rats with CIA than those in their control counterparts. In the AdProTΔNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTΔNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.
NK Cell Maturation and Peripheral Homeostasis Is Associated with KLRG1 Up-Regulation
Authors: Huntington Nicholas D. , Tabarias Hy , Fairfax Kirsten , Brady Jason , Hayakawa Yoshihiro , Degli-Esposti Mariapia A. , Smyth Mark J. , Tarlinton David M. , Nutt Stephen L.
Published: April 2014

NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1+ NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1− NK cells are precursors of KLRG1+ NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1+ NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45−/− mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.