bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Essential Role of Hypoxia Inducible Factor-1α in Development of Lipopolysaccharide-Induced Sepsis
Authors: Peyssonnaux Carole , Cejudo-Martin Pilar , Doedens Andrew , Zinkernagel Annelies S. , Johnson Randall S. , Nizet Victor
Published: April 2014

Sepsis, the leading cause of death in intensive care units, reflects a detrimental host response to infection in which bacteria or LPS act as potent activators of immune cells, including monocytes and macrophages. In this report, we show that LPS raises the level of the transcriptional regulator hypoxia-inducible factor-1α (HIF-1α) in macrophages, increasing HIF-1α and decreasing prolyl hydroxylase mRNA production in a TLR4-dependent fashion. Using murine conditional gene targeting of HIF-1α in the myeloid lineage, we demonstrate that HIF-1α is a critical determinant of the sepsis phenotype. HIF-1α promotes the production of inflammatory cytokines, including TNF-α, IL-1, IL-4, IL-6, and IL-12, that reach harmful levels in the host during early sepsis. HIF-1α deletion in macrophages is protective against LPS-induced mortality and blocks the development of clinical markers including hypotension and hypothermia. Inhibition of HIF-1α activity may thus represent a novel therapeutic target for LPS-induced sepsis.
Cutting Edge: Human Th17 Cells Are Identified as Bearing CCR2+CCR5− Phenotype
Authors: Sato Wakiro , Aranami Toshimasa , Yamamura Takashi
Published: April 2014

Recent reports have shown that IL-17-producing CD4+ T cells (Th17 cells) belong to a distinct helper T cell lineage and are critically involved in the pathogenesis of autoimmune diseases and allergies. However, the chemokine receptor profile of Th17 cells remains to be clarified. In this study, we report that human Th17 cells are identified as CCR2+CCR5− memory CD4+ T cells. Analysis of PBMC from healthy donors showed that CCR2+ cells produce much larger amounts of IL-17 than CCR2− cells, indicating the preferential expression of CCR2 on Th17 cells. Notably, CCR2+CCR5− memory CD4+ T cells produced a large amount of IL-17 and little IFN-γ, whereas CCR2+CCR5+ cells reciprocally produced an enormous amount of IFN-γ but little IL-17. Moreover, a higher expression of T-bet was seen in the CCR5+ memory T cells. These results indicate that absence of CCR5 distinguishes human Th17 cells from Th1 cells.
Type I IFN Contributes to NK Cell Homeostasis, Activation, and Antitumor Function
Authors: Swann Jeremy B. , Hayakawa Yoshihiro , Zerafa Nadeen , Sheehan Kathleen C. F. , Scott Bernadette , Schreiber Robert D. , Hertzog Paul , Smyth Mark J.
Published: April 2014

This study demonstrates that type I IFNs are an early and critical regulator of NK cell numbers, activation, and antitumor activity. Using both IFNAR1- and IFNAR2-deficient mice, as well as an IFNAR1-blocking Ab, we demonstrate that endogenous type I IFN is critical for controlling NK cell-mediated antitumor responses in many experimental tumor models, including protection from methylcholanthrene-induced sarcomas, resistance to the NK cell-sensitive RMA-S tumor and cytokine immunotherapy of lung metastases. Protection from RMA-S afforded by endogenous type I IFN is more potent than that of other effector molecules such as IFN-γ, IL-12, IL-18, and perforin. Furthermore, cytokine immunotherapy using IL-12, IL-18, or IL-21 was effective in the absence of endogenous type I IFN, however the antimetastatic activity of IL-2 was abrogated in IFNAR-deficient mice, primarily due to a defect in IL-2-induced cytotoxic activity. This study demonstrates that endogenous type I IFN is a central mediator of NK cell antitumor responses.
Agonist-Driven Development of CD4+CD25+Foxp3+ Regulatory T Cells Requires a Second Signal Mediated by Stat6
Authors: Sanchez-Guajardo Vanesa , Tanchot Corinne , O’Malley John T. , Kaplan Mark H. , Garcia Sylvie , Freitas Antonio A.
Published: April 2014

The factors that induce Foxp3 expression and regulatory T (Treg) cell development remain unknown. In this study, we investigated the role of STAT4 and STAT6 in agonist-driven generation of Ag-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3 expression and development of Ag-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by STAT6. Indeed, by comparing the development of wild-type and STAT4- and STAT6-deficient hemagglutinin-specific T cells in the presence of hemagglutinin Ag, we found that the absence of STAT6 impaired the generation of Ag-specific CD4+CD25+Foxp3+ cells. Moreover, in transgenic mice expressing a constitutively active form of STAT6, we found that the fraction of CD4+Foxp3+ cells exceeds that of control wild-type littermates. Overall these findings support a role for the STAT6 pathway in Treg cell development and maintenance.
A Novel Role of Complement Factor C1q in Augmenting the Presentation of Antigen Captured in Immune Complexes to CD8+ T Lymphocytes
Authors: van Montfoort Nadine , de Jong Judith M. H. , Schuurhuis Danita H. , van der Voort Ellen I. H. , Camps Marcel G. M. , Huizinga Tom W. J. , van Kooten Cees , Daha Mohamed R. , Verbeek J. Sjef , Ossendorp Ferry , Toes René E. M.
Published: April 2014

Ag-IgG immune complexes (IC) are efficiently taken up, and Ag-derived peptides are subsequently processed and presented by APC. In vitro experiments indicate that IgG Fc Receptors (FcγR) facilitate the efficient uptake of IC by dendritic cells. Previous experiments showed that the cross-presentation of Ag-derived peptides after s.c. administration of IC is FcγR-dependent. To study the role of different FcγR and complement in MHC class I Ag presentation after i.v. administration, we used mice deficient for FcγRs and complement components. These mice were injected with CFSE-labeled OVA-specific CD8+ T cells followed by administration of IC composed of OVA and rabbit anti-OVA IgG i.v. to measure MHC class I presentation of OVA-derived peptides. The Ag presentation was partly reduced in FcRγ-chain-deficient mice, but not affected in FcγRI/II/III-deficient mice, complement factor C3-deficient mice, or FcγRI/II/III × C3-deficient mice. Importantly, CD8+ T cell proliferation was significantly reduced in mice deficient for C1q. This proliferation could be restored when IC were incubated with purified human C1q before injection. Likewise, purified C1q could strongly enhance the uptake and presentation of IC by dendritic cells in vitro. Heat inactivation abrogated the C1q-mediated uptake of IC. In addition, in vivo uptake of OVA-IC in the spleen was significantly reduced in C1q-deficient mice compared with wild-type mice. Together, these results indicate a novel function of C1q, which is present in high levels in the bloodstream, by directly enhancing the uptake and MHC class I presentation of Ag captured in IC by APC to CD8+ T cells.
Uptake of Human Papillomavirus Virus-Like Particles by Dendritic Cells Is Mediated by Fcγ Receptors and Contributes to Acquisition of T Cell Immunity
Authors: Da Silva Diane M. , Fausch Steven C. , Verbeek J. Sjef , Kast W. Martin
Published: April 2014

Chimeric human papillomavirus virus-like particles (HPV cVLP) are immunogens able to elicit potent CTL responses in mice against HPV16-transformed tumors; however, the mechanism of T cell priming has remained elusive. HPV VLP bind to human MHC class II-positive APCs through interaction with FcγRIII, and immature dendritic cells (DC) become activated after incubation with HPV VLP; however, it is unclear whether FcγR on DC are involved. In mice, FcγRII and FcγRIII are homologous and bind similar ligands. In this study, we show that binding and uptake of VLP by DC from FcγRII, FcγRIII, and FcγRII/III-deficient mice are reduced by up to 50% compared with wild-type mice. Additionally, maturation of murine DC from FcγRII/III-deficient mice by VLP is also reduced, indicating that DC maturation, and thus Ag presentation, is diminished in the absence of expression of FcγR. To investigate the in vivo contribution of FcγR in the induction of cellular immunity, FcγR single- and double-knockout mice were immunized with HPV16 L1/L2-E7 cVLP, and the frequency of E7-specific T cells was analyzed by tetramer binding, IFN-γ ELISPOT, and cytotoxicity assays. All readouts indicated that the frequency of E7-specific CD4+ and CD8+ T cells induced in all FcγR-deficient mice after immunization with cVLP was significantly diminished. Based on these results, we propose that the low-affinity FcγR contribute to the high immunogenicity of HPV VLP during T cell priming by targeting VLP to DC and inducing a maturation state of the DC that facilitates Ag presentation to and activation of naive T cells.
IL-27 Synthesis Induced by TLR Ligation Critically Depends on IFN Regulatory Factor 3
Authors: Molle Céline , Nguyen Muriel , Flamand Véronique , Renneson Joelle , Trottein François , De Wit Dominique , Willems Fabienne , Goldman Michel , Goriely Stanislas
Published: April 2014

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-β and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3−/− upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-β-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.
Disregulated Influenza A Virus-Specific CD8+ T Cell Homeostasis in the Absence of IFN-γ Signaling
Authors: Turner Stephen J. , Olivas Elvia , Gutierrez Astrid , Diaz Gabriela , Doherty Peter C.
Published: April 2014

Recent studies indicate that IFN-γ may influence both the expansion and the trafficking of virus-specific CD8+ CTL, though the effects are not necessarily consistent for different models of viral and bacterial disease. Influenza A virus infection of mice deficient for IFN-γ (IFN-γ−/−) or deficient for the IFN-γ receptor 1 (IFNGR1−/−) was, when compared with the wild-type (WT) B6 controls, associated with increased Ag-specific CD8+ T cell counts in the spleen and mediastinal lymph nodes. At the same time, fewer of these CTL effectors were found in the bronchoalveolar lavage population recovered from the IFN-γ−/− mice. Comparable effects were observed for WT mice treated with a neutralizing IFN-γ-specific mAb. Transfer of WT memory Thy1.1+ CD8+ populations into Thy1.2+ B6 IFN-γ−/− or IFNGR1−/− mice followed by intranasal virus challenge demonstrated both that IFN-γ produced by the host was important for the regulation of Ag-specific CTL numbers and that IFN-γ was likely to act directly on the T cells themselves. In addition, the prevalence of CTLs undergoing apoptosis in spleen was lower when measured directly ex vivo for IFN-γ−/− vs WT B6 mice. The present analysis is the first comprehensive demonstration that IFN-γ signaling can differentially regulate both Ag-specific CTL homeostasis in secondary lymphoid organs and trafficking to a site of virus-induced pathology.
Immunomodulatory Mediators from Pollen Enhance the Migratory Capacity of Dendritic Cells and License Them for Th2 Attraction
Authors: Mariani Valentina , Gilles Stefanie , Jakob Thilo , Thiel Martina , Mueller Martin J. , Ring Johannes , Behrendt Heidrun , Traidl-Hoffmann Claudia
Published: April 2014

The immune response of atopic individuals against allergens is characterized by increased levels of Th2 cytokines and chemokines. However, the way in which the cytokine/chemokine profile is matched to the type of invading allergen, and why these profiles sometimes derail and lead to disease, is not well understood. We recently demonstrated that pollen modulates dendritic cell (DC) function in a way that results in an enhanced capacity to initiate Th2 responses in vitro. Here, we examined the effects of aqueous birch pollen extracts (Bet.-APE) on chemokine receptor expression and chemokine production by human monocyte-derived DCs. Bet.-APE strongly induced expression and function of CXCR4 and reduced CCR1 and CCR5 expression on immature DCs. In addition, DC treatment with Bet.-APE significantly reduced LPS-induced production of CXCL10/IP-10, CCL5/RANTES; induced CCL22/macrophage-derived chemokine; and did not significantly change release of CCL17/thymus and activation-regulated chemokine. At a functional level, Bet.-APE increased the capacity of LPS-stimulated DCs to attract Th2 cells, whereas the capacity to recruit Th1 cells was reduced. Bet.-APE significantly and dose-dependently enhanced intracellular cAMP, suggesting that water-soluble factors from pollen grains bind a Gαs-protein-coupled receptor. E1-Phytoprostanes were identified to be one player in the Th2-polarizing potential of aqueous pollen extracts. In summary, our results demonstrate that pollen itself releases regulatory mediators which generate a Th2-promoting micromilieu with preferential recruitment of Th2 cells to the site of pollen exposure.
IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype
Authors: Casey Kerry A. , Mescher Matthew F.
Published: April 2014

IL-21, the most recently described member of the common γ-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-α have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-γ or IL-4 production and can partially block IL-12 induction of IFN-γ production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44high, PD-1low, CD25low, CD134low, and CD137low. Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-γ.
Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells
Authors: Alter Galit , Suscovich Todd J. , Teigen Nickolas , Meier Angela , Streeck Hendrik , Brander Christian , Altfeld Marcus
Published: April 2014

Persistent immune activation is a hallmark of chronic viremic HIV-1 infection. Activation of cells of the innate immune system, such as NK cells, occurs rapidly upon infection, and is sustained throughout the course of the disease. However, the precise underlying mechanism accounting for the persistent HIV-1-induced activation of NK cells is poorly understood. In this study, we assessed the role of uridine-rich ssRNA derived from the HIV-1 long terminal repeat (ssRNA40) on activation of NK cells via TLR7/8. Although dramatic activation of NK cells was observed following stimulation of PBMC with ssRNA40, negligible activation was observed following stimulation of purified NK cells despite their expression of TLR8 mRNA and protein. The functional activation of NK cells by this HIV-1-encoded TLR7/8 ligand could not be reconstituted with exogenous IL-12, IFN-α, or TNF-α, but was critically dependent on the direct contact of NK cells with plasmacytoid dendritic cells or CD14+ monocytes, indicating an important level of NK cell cross-talk and regulation by accessory cells during TLR-mediated activation. Coincubation of monocyte/plasmacytoid dendritic cells, NK cells, and ssRNA40 potentiated NK cell IFN-γ secretion in response to MHC-devoid target cells. Studies using NK cells derived from individuals with chronic HIV-1 infection demonstrated a reduction of NK cell responsiveness following stimulation with TLR ligands in viremic HIV-1 infection. These data demonstrate that HIV-1-derived TLR ligands can contribute to the immune activation of NK cells and may play an important role in HIV-1-associated immunopathogenesis and NK cell dysfunction observed during acute and chronic viremic HIV-1 infection.
OX40-Mediated Differentiation to Effector Function Requires IL-2 Receptor Signaling but Not CD28, CD40, IL-12Rβ2, or T-bet
Authors: Williams Cortny A. , Murray Susan E. , Weinberg Andrew D. , Parker David C.
Published: April 2014

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-γ. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-γ in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-γ secretion.
Tumor-Reactive CD4+ T Cell Responses to the Melanoma-Associated Chondroitin Sulphate Proteoglycan in Melanoma Patients and Healthy Individuals in the Absence of Autoimmunity
Authors: Erfurt Cornelia , Sun Zhaojun , Haendle Ina , Schuler-Thurner Beatrice , Heirman Carlo , Thielemans Kris , van der Bruggen Pierre , Schuler Gerold , Schultz Erwin S.
Published: April 2014

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4+ T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4+ Th cells from the blood of a healthy donor-recognizing peptide MCSP693–709 on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4+ T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.
CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys Produce More IL-2 Than Cells from Disease-Susceptible Species: Involvement of p300 and CREB at the Proximal IL-2 Promoter in IL-2 Up-Regulation
Authors: Bostik Pavel , Noble Erika S. , Stephenson Susan T. , Villinger Francois , Ansari Aftab A.
Published: April 2014

IL-2 is an important cytokine required for the physiological function of CD4+ T cells. Immunological unresponsiveness—anergy— of CD4+ T cells is characterized by the inability of these cells to synthesize IL-2. Both progressive HIV infection leading to AIDS in humans and SIV infection in rhesus macaques (RM) are associated with dysregulation of IL-2 synthesis. In certain nonhuman primate species, such as sooty mangabeys (SM), SIV infection does not lead to AIDS. We have shown that this is associated with the resistance of the CD4+ T cells from SM to undergo anergy in vitro. In this study, we show that CD4+ T cells from SM spontaneously synthesize 2- to 3-fold higher levels of IL-2 than corresponding cells from RM. Proximal IL-2 promoter constructs derived from SM show significantly higher activity than the RM-derived constructs in primary CD4+ T cells, which is associated with an element at approximately nt −200. Activity of both constructs was up-regulated by p300 and down-regulated by CREB to a similar degree. Chromatin immunoprecipitation analysis showed significantly higher binding of p300 and lower binding of CREB to the SM promoter in vivo. Two single nucleotide substitutions present in the SM sequence around position −200 and −180 seem to increase the affinity of these sites for the binding of transcription factors, one of which was identified as Oct-1. These unique characteristics of the proximal IL-2 promoter in SM therefore can represent one of the mechanisms contributing to the resistance of these cells to undergo anergy.
Availability of a Diversely Avid CD8+ T Cell Repertoire Specific for the Subdominant HLA-A2-Restricted HIV-1 Gag p2419–27 Epitope
Authors: Schaubert Keri L. , Price David A. , Frahm Nicole , Li Jinzhu , Ng Hwee L. , Joseph Aviva , Paul Elyse , Majumder Biswanath , Ayyavoo Velpandi , Gostick Emma , Adams Sharon , Marincola Francesco M. , Sewell Andrew K. , Altfeld Marcus , Brenchley Jason M. , Douek Daniel C. , Yang Otto O. , Brander Christian , Goldstein Harris , Kan-Mitchell June
Published: April 2014

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p2419–27, TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.
Small Rho GTPases Mediate Tumor-Induced Inhibition of Endocytic Activity of Dendritic Cells
Authors: Tourkova Irina L. , Shurin Galina V. , Wei Sheng , Shurin Michael R.
Published: April 2014

The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines.
Exposure of Myeloid Dendritic Cells to Exogenous or Endogenous IL-10 during Maturation Determines Their Longevity
Authors: Chang W. L. William , Baumgarth Nicole , Eberhardt Meghan K. , Lee C. Y. Daniel , Baron Colin A. , Gregg Jeff P. , Barry Peter A.
Published: April 2014

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-xL expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-xL in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.
Immunomodulatory Effects of Viral TLR Ligands on Experimental Asthma Depend on the Additive Effects of IL-12 and IL-10
Authors: Sel Serdar , Wegmann Michael , Sel Sarper , Bauer Stefan , Garn Holger , Alber Gottfried , Renz Harald
Published: April 2014

Based on epidemiological data, the hygiene hypothesis associates poor hygienic living conditions during childhood with a lower risk for the development of allergic diseases such as bronchial asthma. The role of viral infections, and especially of viral TLR ligands, within this context remains to be clarified. Viral TLR ligands involve dsRNA and ssRNA which are recognized by TLR-3 or TLR-7, respectively. In this study, we evaluated the impact of TLR-3 or TLR-7 activation on experimental asthma in mice. Systemic application of the synthetic TLR-3 or TLR-7 ligands polycytidylic-polyinosinic acid (p(I:C)) or R-848, respectively, during the sensitization phase prevented the production of OVA-specific IgE and IgG1 Abs and subsequently abolished all features of experimental asthma including airway hyperresponsiveness and allergic airway inflammation. Furthermore, administration of p(I:C) or R-848 to animals with already established primary allergic responses revealed a markedly reduced secondary response following allergen aerosol rechallenges. In contrast to wild-type animals, application of p(I:C) or R-848 to IL-12p35−/− mice had no effect on airway inflammation, goblet cell hyperplasia, and airway hyperresponsiveness. However, in the absence of IL-12, the numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluids were still significantly reduced. These partial effects could also be abolished by neutralizing anti-IL-10 Abs in IL-12p35−/− mice. These data indicate that TLR-3 or TLR-7 activation by viral TLR ligands has both preventive as well as suppressive effects on experimental asthma which is mediated by the additive effects of IL-12 and IL-10.
Identification and Characterization of Novel Bone Marrow Myeloid DEC205+Gr-1+ Cell Subsets That Differentially Express Chemokine and TLRs
Authors: Lamb Roberta J. , Capocasale Renold J. , Duffy Karen E. , Sarisky Robert T. , Mbow M. Lamine
Published: April 2014

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205+CD11c−B220−Gr1+CD8α−CD11b+ cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205+CD11b+Gr-1+ bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205+CD11b+Gr-1low cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-α, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205+CD11b+Gr-1high cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205+CD11+Gr-1high cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.
Low-Dose Peptide Tolerance Therapy of Lupus Generates Plasmacytoid Dendritic Cells That Cause Expansion of Autoantigen-Specific Regulatory T Cells and Contraction of Inflammatory Th17 Cells
Authors: Kang Hee-Kap , Liu Michael , Datta Syamal K.
Published: April 2014

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H471–94, when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-β. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H471–94 peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-β, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H471–94 was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H471–94 peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.
Novel Suppressive Function of Transitional 2 B Cells in Experimental Arthritis
Authors: Evans Jamie G. , Chavez-Rueda Karina A. , Eddaoudi Ayad , Meyer-Bahlburg Almut , Rawlings David J. , Ehrenstein Michael R. , Mauri Claudia
Published: April 2014

The immune system contains natural regulatory cells important in the maintenance of tolerance. Although this suppressive function is usually attributed to CD4 regulatory T cells, recent reports have revealed an immunoregulatory role for IL-10-producing B cells in the context of several autoimmune diseases including collagen-induced arthritis. In the present study, we attribute this suppressive function to a B cell subset expressing high levels of CD21, CD23, and IgM, previously identified as transitional 2-marginal zone precursor (T2-MZP) B cells. T2-MZP B cells are present in the spleens of naive mice and increase during the remission phase of arthritis. Following adoptive transfer to immunized DBA/1 mice, T2-MZP B cells significantly prevented new disease and ameliorated established disease. The suppressive effect on arthritis was paralleled by an inhibition of Ag-specific T cell activation and a reduction in cells exhibiting Th1-type functional responses. We also provide evidence that this regulatory subset mediates its suppression through the secretion of suppressive cytokines and not by cell-to-cell contact. The ability to regulate an established immune response by T2-MZP B cells endows this subset of B cells with a striking and previously unrecognized immunoregulatory potential.
Coexpression of IL-5 and Eotaxin-2 in Mice Creates an Eosinophil-Dependent Model of Respiratory Inflammation with Characteristics of Severe Asthma
Authors: Ochkur Sergei I. , Jacobsen Elizabeth A. , Protheroe Cheryl A. , Biechele Travis L. , Pero Ralph S. , McGarry Michael P. , Wang Huiying , O’Neill Katie R. , Colbert Dana C. , Colby Thomas V. , Shen Huahao , Blackburn Michael R. , Irvin Charles C. , Lee James J. , Lee Nancy A.
Published: April 2014

Mouse models of allergen provocation and/or transgenic gene expression have provided significant insights regarding the cellular, molecular, and immune responses linked to the pathologies occurring as a result of allergic respiratory inflammation. Nonetheless, the inability to replicate the eosinophil activities occurring in patients with asthma has limited their usefulness to understand the larger role(s) of eosinophils in disease pathologies. These limitations have led us to develop an allergen-naive double transgenic mouse model that expresses IL-5 systemically from mature T cells and eotaxin-2 locally from lung epithelial cells. We show that these mice develop several pulmonary pathologies representative of severe asthma, including structural remodeling events such as epithelial desquamation and mucus hypersecretion leading to airway obstruction, subepithelial fibrosis, airway smooth muscle hyperplasia, and pathophysiological changes exemplified by exacerbated methacholine-induced airway hyperresponsiveness. More importantly, and similar to human patients, the pulmonary pathologies observed are accompanied by extensive eosinophil degranulation. Genetic ablation of all eosinophils from this double transgenic model abolished the induced pulmonary pathologies, demonstrating that these pathologies are a consequence of one or more eosinophil effector functions.
Paratope Plasticity in Diverse Modes Facilitates Molecular Mimicry in Antibody Response
Authors: Krishnan Lavanya , Lomash Suvendu , Raj Beena Patricia Jeevan , Kaur Kanwal J. , Salunke Dinakar M.
Published: April 2014

The immune response against methyl-α-d-mannopyranoside mimicking 12-mer peptide (DVFYPYPYASGS) was analyzed at the molecular level towards understanding the equivalence of these otherwise disparate Ags. The Ab 7C4 recognized the immunizing peptide and its mimicking carbohydrate Ag with comparable affinities. Thermodynamic analyses of the binding interactions of both molecules suggested that the mAb 7C4 paratope lacks substantial conformational flexibility, an obvious possibility for facilitating binding to chemically dissimilar Ags. Favorable changes in entropy during binding indicated the importance of hydrophobic interactions in recognition of the mimicking carbohydrate Ag. Indeed, the topology of the Ag-combining site was dominated by a cluster of aromatic residues, contributed primarily by the specificity defining CDR H3. Epitope-mapping analysis demonstrated the critical role of three aromatic residues of the 12-mer in binding to the Ab. Our studies delineate a mechanism by which mimicry is manifested in the absence of either structural similarity of the epitopes or conformational flexibility in the paratope. An alternate mode of recognition of dissimilar yet mimicking Ags by the anti-peptide Ab involves plasticity associated with aromatic/hydrophobic and van der Waals interactions. Thus, antigenic mimicry may be a consequence of paratope-specific modulations rather than being dependent only on the properties of the epitope. Such modulations may have evolved toward minimizing the consequences of antigenic variation by invading pathogens.
Lung Dendritic Cells Rapidly Mediate Anthrax Spore Entry through the Pulmonary Route
Authors: Cleret Aurélie , Quesnel-Hellmann Anne , Vallon-Eberhard Alexandra , Verrier Bernard , Jung Steffen , Vidal Dominique , Mathieu Jacques , Tournier Jean-Nicolas
Published: April 2014

Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially because anthrax is an emerging bioterrorism agent. The exact mechanisms leading to a severe clinical form through the inhalational route are still unclear, particularly how immobile spores are captured in the alveoli and transported to the lymph nodes in the early steps of infection. We investigated the roles of alveolar macrophages and lung dendritic cells (LDC) in spore migration. We demonstrate that alveolar macrophages are the first cells to phagocytose alveolar spores, and do so within 10 min. However, interstitial LDCs capture spores present in the alveoli within 30 min without crossing the epithelial barrier suggesting a specific mechanism for rapid alveolus sampling by transepithelial extension. We show that interstitial LDCs constitute the cell population that transports spores into the thoracic lymph nodes from within 30 min to 72 h after intranasal infection. Our results demonstrate that LDCs are central to spore transport immediately after infection. The rapid kinetics of pathogen transport may contribute to the clinical features of inhalational anthrax.
Depletion of CD8+ Cells in Sooty Mangabey Monkeys Naturally Infected with Simian Immunodeficiency Virus Reveals Limited Role for Immune Control of Virus Replication in a Natural Host Species
Authors: Barry Ashley P. , Silvestri Guido , Safrit Jeffrey T. , Sumpter Beth , Kozyr Natalia , McClure Harold M. , Staprans Silvija I. , Feinberg Mark B.
Published: April 2014

SIV infection of sooty mangabeys (SMs), a natural host species, does not cause AIDS despite high-level virus replication. In contrast, SIV infection of nonnatural hosts such as rhesus macaques (RMs) induces an AIDS-like disease. The depletion of CD8+ T cells during SIV infection of RMs results in marked increases in plasma viremia, suggesting a key role for CD8+ T cells in controlling levels of SIV replication. To assess the role that CD8+ T cells play in determining the virologic and immunologic features of nonpathogenic SIV infection in SMs, we transiently depleted CD8+ T cells in SIV-infected and uninfected SMs using a CD8α-specific Ab (OKT8F) previously used in studies of SIV-infected RMs. Treatment of SMs with the OKT8F Ab resulted in the prompt and profound depletion of CD8+ T cells. However, in contrast to CD8+ cell depleted, SIV-infected RMs, only minor changes in the levels of plasma viremia were observed in most SIV-infected SMs during the period of CD8+ cell deficiency. Those SMs demonstrating greater increases in SIV replication following CD8+ cell depletion also displayed higher levels of CD4+ T cell activation and/or evidence of CMV reactivation, suggesting that an expanded target cell pool rather than the absence of CD8+ T cell control may have been primarily responsible for transient increases in viremia. These data indicate that CD8+ T cells exert a limited influence in determining the levels of SIV replication in SMs and provide additional evidence demonstrating that the absence of AIDS in SIV-infected SMs is not due to the effective control of viral replication by cellular immune responses.
Macrophage-Inflammatory Protein-3α Mediates Epidermal Growth Factor Receptor Transactivation and ERK1/2 MAPK Signaling in Caco-2 Colonic Epithelial Cells via Metalloproteinase-Dependent Release of Amphiregulin
Authors: Keates Sarah , Han Xinbing , Kelly Ciarán P. , Keates Andrew C.
Published: April 2014

Previously, we reported that normal colonocytes produce the memory CD4+ T cell-directed chemokine MIP-3α, and that epithelial MIP-3α levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3α, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3α may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3α can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3α can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3α-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3α-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3α can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.
IL-10 Underlies Distinct Susceptibility of BALB/c and C57BL/6 Mice to Mycobacterium avium Infection and Influences Efficacy of Antibiotic Therapy
Authors: Roque Susana , Nobrega Claudia , Appelberg Rui , Correia-Neves Margarida
Published: April 2014

Increased production of IL-10 has been frequently associated with augmented susceptibility to infection. However, the correlation between IL-10 activity and susceptibility to mycobacterial infection is still uncertain. Although studies using transgenic mice overexpressing IL-10 consistently showed an increased susceptibility to mycobacterial infection, experimental approaches in which IL-10 activity was reduced or abrogated originated inconclusive data. We show here that this controversy might be due to the mouse strains used in the various experimental procedures. Our results show that BALB/c mice are more susceptible than C57BL/6 to Mycobacterium avium infection. This increased susceptibility of BALB/c mice is, to a great extent, due to distinct activity of IL-10 between the two mouse strains. In accordance, reduction of IL-10 activity through the administration of anti-IL-10R mAb, or the absence of IL-10 as studied in IL-10 knockout mice, clearly decreased the susceptibility of BALB/c mice to M. avium but had a less obvious effect in C57BL/6 mice. Moreover, abrogation of IL-10 activity in infected BALB/c mice increased the efficacy of antimycobacterial therapy, whereas for the C57BL/6 mice it produced no effect. These observations show that the activity of IL-10 in response to the same mycobacterial stimulus influences not only the susceptibility to infection but also the efficacy of antimycobacterial therapy. This should now be considered in the context of human response to mycobacterial infection, particularly as a possible strategy to improve treatment against infections by mycobacteria.
Leukotriene B4 Triggers the In Vitro and In Vivo Release of Potent Antimicrobial Agents
Authors: Flamand Louis , Tremblay Michel J. , Borgeat Pierre
Published: April 2014

Leukotriene B4 (LTB4) is a bioactive lipid derived from the metabolism of arachidonic acid. Mainly produced by polymorphonuclear leukocytes (PMN) and macrophages, LTB4 triggers several functional responses important in host defense, including the secretion of lysosomal enzymes, the activation of NADPH oxidase activity, NO formation, and phagocytosis. We report that LTB4, but not structural analogs thereof, stimulates primed human PMN to release molecules having potent antimicrobial activities. Exposure of bacteria (Escherichia coli and Staphylococcus aureus) or viruses (herpes simplex virus type 1 and HIV type 1) to supernatants of LTB4-activated PMN lead to ≥90% reduction in infectivity. ELISA and mass spectroscopy analysis of proteins released from LTB4-activated PMN have identified several antimicrobial proteins, including α-defensins, cathepsin G, elastase, lysozyme C, and LL-37, that are likely to participate in the killing of microorganisms. In addition to these in vitro observations, i.v. injections of LTB4 (50 μg/kg) to monkeys led to an increase in α-defensin plasmatic levels and enhanced ex vivo antimicrobial activities of plasma. These results demonstrate the ability of LTB4 to cause the release of potent antimicrobial agents from PMN in vitro as well as in vivo and add further support to the important role of LTB4 in host defense.
Human Neutrophil Defensins Increase Neutrophil Uptake of Influenza A Virus and Bacteria and Modify Virus-Induced Respiratory Burst Responses
Authors: Tecle Tesfaldet , White Mitchell R. , Gantz Don , Crouch Erika C. , Hartshorn Kevan L.
Published: April 2014

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.