bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Emergence of CD127high Functionally Competent Memory T Cells Is Compromised by High Viral Loads and Inadequate T Cell Help
Authors: Fuller Michael J. , Hildeman David A. , Sabbaj Steffanie , Gaddis Dalia E. , Tebo Anne E. , Shang Liang , Goepfert Paul A. , Zajac Allan J.
Published: April 2014

In this report we have inspected whether difficulties in controlling viral infections negatively impacts the generation of CD127high memory T cells. Using both MHC class I and II tetramers we reveal that CD127low T cells are not necessarily rapidly deleted but can persist in a pseudoeffector state in which they display the hallmarks of activated effector cells but are functionally inferior. CD127high cells can, however, emerge if the infection is contained. We also show that in the absence of CD4 T cell help significant populations of CD127high CD8 T cells fail to emerge. Analyses of cytokine-producing activities by both mouse and human CD8 T cells further document that the extended maintenance of T cells in a CD127low state has functional consequences which manifest as an impairment of IL-2 production.
Cutting Edge: The SLAM Family Receptor Ly108 Controls T Cell and Neutrophil Functions
Authors: Howie Duncan , Laroux F. Stephen , Morra Massimo , Satoskar Abhay R. , Rosas Lucia E. , Faubion William A. , Julien Aimee , Rietdijk Svend , Coyle Anthony J. , Fraser Christopher , Terhorst Cox
Published: April 2014

Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4+ T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108ΔE2+3) produce significantly less IL-4 than wild-type CD4+ cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108ΔE2+3 mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108ΔE2+3 neutrophils is defective, and their production of IL-6, IL-12, and TNF-α is increased. The aberrant bactericidal activity by Ly108ΔE2+3 neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.
Cutting Edge: Re-evaluating the In Vivo Cytokine Responses of CD8+ T Cells during Primary and Secondary Viral Infections
Authors: Liu Fei , Whitton J. Lindsay
Published: April 2014

Virus-specific CD8+ T cells produce IFN-γ after Ag contact and, in the absence of this cytokine, the host often cannot eradicate infection. However, our ability to identify cells that are actively expressing this critical effector function in vivo is limited, because the protein is rapidly secreted. In this study, we describe a simple approach that circumvents the need for ex vivo Ag stimulation and allows the enumeration of CD8+ T cells that are actively synthesizing IFN-γ in vivo during primary and secondary virus infections. The proportion of Ag-specific primary CD8+ T cells producing IFN-γ peaks at 5 days postinfection, when the T cell population is still expanding exponentially. In vivo IFN-γ synthesis by memory cells is explosive, peaking at ∼12 h after secondary infection and terminating hours thereafter. This technique will be useful when evaluating in vivo immune cell activity in many situations, including a variety of noninfectious (e.g., autoimmune) diseases.
CD4+CD25+ Regulatory T Cells Attenuate the Phosphatidylinositol 3-Kinase/Akt Pathway in Antigen-Primed Immature CD8+ CTLs during Functional Maturation
Authors: Kojima Hidefumi , Kanno Yumiko , Hase Hidenori , Kobata Tetsuji
Published: April 2014

This study was designed to determine the role of CD25+CD4+ regulatory T (Tr) cells in CTL maturation and effector functions using a murine CTL line and in vitro MLC. Tr cells inhibited CTL functional maturation, but had no effect on CTL effector functions. In CD4+ responder T cell-depleted MLC supplemented with IL-2, Tr cells suppressed mature CTL generation only when added within the first 2 days of culture. Tr cells down-regulated levels of active Akt, but not STAT5 or ZAP70 in Ag-primed immature CTLs. Down-regulation of active Akt was accompanied by a reduction in CTL cell size and IL-2Rα expression. In Tr cell-depleted MLC, CTLs were generated that exhibited high levels of nonspecific cytotoxicity. Our in vitro findings suggest that Tr cells regulate functional CTL maturation to generate optimal Ag-specific immune responses through the control of the PI3K/Akt pathway.
Multiple Costimulatory Modalities Enhance CTL Avidity
Authors: Hodge James W. , Chakraborty Mala , Kudo-Saito Chie , Garnett Charlie T. , Schlom Jeffrey
Published: April 2014

Recent studies in both animal models and clinical trials have demonstrated that the avidity of T cells is a major determinant of antitumor and antiviral immunity. In this study, we evaluated several different vaccine strategies for their ability to enhance both the quantity and avidity of CTL responses. CD8+ T cell quantity was measured by tetramer binding precursor frequency, and avidity was measured by both tetramer dissociation and quantitative cytolytic function. We have evaluated a peptide, a viral vector expressing the Ag transgene alone, with one costimulatory molecule (B7-1), and with three costimulatory molecules (B7-1, ICAM-1, and LFA-3), with anti-CTLA-4 mAb, with GM-CSF, and combinations of the above. We have evaluated these strategies in both a foreign Ag model using β-galactosidase as immunogen, and in a “self” Ag model, using carcinoembryonic Ag as immunogen in carcinoembryonic Ag transgenic mice. The combined use of several of these strategies was shown to enhance not only the quantity, but, to a greater magnitude, the avidity of T cells generated; a combination strategy is also shown to enhance antitumor effects. The results reported in this study thus demonstrate multiple strategies that can be used in both antitumor and antiviral vaccine settings to generate higher avidity host T cell responses.
IFN Regulatory Factor-2 Deficiency Revealed a Novel Checkpoint Critical for the Generation of Peripheral NK Cells
Authors: Taki Shinsuke , Nakajima Shinsuke , Ichikawa Eri , Saito Takashi , Hida Shigeaki
Published: April 2014

NK cell development is far less understood compared with that of T and B cells despite the critical importance of NK cells in innate immunity. Mice lacking the transcription factor IFN regulatory factor-2 (IRF-2) are known to exhibit NK cell deficiency. However, the role of IRF-2 in NK cell development has remained unclear. In this study we found that NK cell deficiency in the periphery in IRF-2-deficient mice was due to selective loss of mature NK cells, but not to maturation arrest, and NK cells in these mice exhibited very immature surface phenotypes (CD11blowDx5low) with highly compromised NK receptor expression. In contrast, IRF-2-deficient NK cells in bone marrow (BM) showed relatively mature phenotypes (CD11blowDx5high) with less compromised NK receptor repertoire. Furthermore, BM NK cells in IRF-2-deficient mice were found to proliferate almost normally, but underwent accelerated apoptosis. These observations indicated that NK cell maturation could advance up to a late, but not the final, stage in the BM, whereas these cells were incapable of contributing to the peripheral NK cell pool due to premature death in the absence of IRF-2. In contrast, NK cell numbers and Ly49 expression were much more severely reduced in BM in IL-15-deficient mice than in IRF-2−/− mice. The differential peripheral and central NK cell deficiencies in IRF-2−/− mice thus revealed a novel late checkpoint for NK cell maturation, distinct from the early IL-15-dependent expansion stage.
CD40 Ligation Activates Murine Macrophages via an IFN-γ-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro
Authors: Buhtoiarov Ilia N. , Lum Hillary , Berke Gideon , Paulnock Donna M. , Sondel Paul M. , Rakhmilevich Alexander L.
Published: April 2014

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-γ because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-γ mAb, as well as in IFN-γ-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-γ for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-γ. We confirmed this hypothesis by detecting intracytoplasmic IFN-γ in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-γ production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-γ and mediate tumor cell destruction.
Cyclophilin A-Deficient Mice Are Resistant to Immunosuppression by Cyclosporine
Authors: Colgan John , Asmal Mohammed , Yu Bin , Luban Jeremy
Published: April 2014

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia−/− CD4+ T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia−/− mice to allogeneic challenge. Rag2−/− mice reconstituted with Ppia−/− splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia−/− immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine.
Spontaneous and Homeostatic Proliferation of CD4 T Cells Are Regulated by Different Mechanisms
Authors: Min Booki , Yamane Hidehiro , Hu-Li Jane , Paul William E.
Published: April 2014

Transfer of naive CD4 T cells into lymphopenic mice initiates a proliferative response of the transferred cells, often referred to as homeostatic proliferation. Careful analysis reveals that some of the transferred cells proliferate rapidly and undergo robust differentiation to memory cells, a process we have designated spontaneous proliferation, and other cells proliferate relatively slowly and show more limited evidence of differentiation. In this study we report that spontaneous proliferation is IL-7 independent, whereas the slow proliferation (referred to as homeostatic proliferation) is IL-7 dependent. Administration of IL-7 induces homeostatic proliferation of naive CD4 T cells even within wild-type recipients. Moreover, the activation/differentiation pattern of the two responses are clearly distinguishable, indicating that different activation mechanisms may be involved. Our results reveal the complexity and heterogeneity of lymphopenia-driven T cell proliferation and suggest that they may have fundamentally distinct roles in the maintenance of CD4 T cell homeostasis.
Preferential Cell Death of CD8+ Effector Memory (CCR7−CD45RA−) T Cells by Hydrogen Peroxide-Induced Oxidative Stress
Authors: Takahashi Akihiro , Hanson Mikael G. V. , Norell Håkan R. , Havelka Aleksandra Mandic , Kono Koji , Malmberg Karl-Johan , Kiessling Rolf V. R.
Published: April 2014

T cells are used in many cell-based cancer treatments. However, oxidative stress that is induced during various chronic inflammatory conditions, such as cancer, can impair the immune system and have detrimental effects on T cell function. In this study, we have investigated the sensitivity of different human T cell subsets to H2O2-induced oxidative stress. We showed that central memory (CD45RA−CCR7+) and effector memory (CD45RA−CCR7−) T cells are more sensitive to H2O2 as compared with naive (CD45RA+CCR7+) T cells. Furthermore, the study showed that CD8+ effector memory T cells are more sensitive to low levels of H2O2 (5 μM) compared with other types of T cells investigated. H2O2-exposed CD45RO+ T cells showed mitochondrial depolarization prior to caspase 3 activity. Moreover, the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone rescued cells from death. These experiments suggest that H2O2-induced cell death of CD45RO+ T cells acts via the mitochondrial pathway and that caspase involvement is needed. This study suggests that oxidative stress in cancer patients can be disadvantageous for T cell-based adoptive cell transfer therapies, since effector memory T cells are the primary phenotype of the cells administered.
TGF-β Inhibits Fas-Mediated Apoptosis of a Follicular Dendritic Cell Line by Down-Regulating the Expression of Fas and Caspase-8: Counteracting Role of TGF-β on TNF Sensitization of Fas-Mediated Apoptosis
Authors: Park Sun-Mi , Kim Sunshin , Choi Jin-Suk , Hur Dae-Young , Lee Wang-Jae , Lee Myung-Shik , Choe Jongseon , Lee Tae H.
Published: April 2014

Follicular dendritic cells (FDC) constitute the framework of germinal center (GC) in secondary lymphoid follicles, and the integrity of FDC networks is critically affected by cytokines present in the GC. We have previously shown that TNF promotes Fas-mediated apoptosis of HK cells, an established FDC-like cell line, by up-regulating Fas expression. However, in the developing GC, FDC death is not a hallmark of GC despite the presence of TNF and FasL. In this study, we report that TGF-β inhibits Fas-mediated apoptosis of HK cells by down-regulating the expression of surface Fas and caspase-8. The inhibitory effect of TGF-β can be observed when HK cells were simultaneously treated with TNF and TGF-β, indicating that TGF-β counteracts the effect of TNF in sensitizing cells to Fas-mediated apoptosis. Furthermore, the deprivation of TGF-β by injecting neutralizing TGF-β Abs to the SRBC-immunized mice resulted in the sporadic appearance of FDC undergoing apoptosis in the lymphoid follicles, suggesting that TGF-β functions as a naturally occurring inhibitor that rescues FDCs which are predisposed to apoptosis. Our study documents a novel function of TGF-β in the maintenance of FDC networks.
Interallelic Class Switch Recombination Contributes Significantly to Class Switching in Mouse B Cells
Authors: Reynaud Stéphane , Delpy Laurent , Fleury Laurence , Dougier Hei-Lanne , Sirac Christophe , Cogné Michel
Published: April 2014

Except for the expression of IgM and IgD, DNA recombination is constantly needed for the expression of other Ig classes and subclasses. The predominant path of class switch recombination (CSR) is intrachromosomal, and the looping-out and deletion model has been abundantly documented. However, switch regions also occasionally constitute convenient substrates for interchromosomal recombination, since it is noticeably the case in a number of chromosomal translocations causing oncogene deregulation in the course of lymphoma and myeloma. Although asymmetric accessibility of Ig alleles should theoretically limit its occurrence, interallelic CSR was shown to occur at low levels during IgA switching in rabbit, where the definition of allotypes within both V and C regions helped identify interchromosomally derived Ig. Thus, we wished to evaluate precisely interallelic CSR frequency in mouse B cells, by using a system in which only one allele (of b allotype) could express a functional VDJ region, whereas only interallelic CSR could restore expression of an excluded (a allotype) allele. In our study, we show that interchromosomal recombination of VH and Cγ or Cα occurs in vivo in B cells at a frequency that makes a significant contribution to physiological class switching: trans-association of VH and CH genes accounted for 7% of all α mRNA, and this frequency was about twice higher for the γ3 transcripts, despite the much shorter distance between the JH region and the Cγ3 gene, thus confirming that this phenomenon corresponded to site-specific switching and not to random recombination between long homologous loci.
Demethylation of the Same Promoter Sequence Increases CD70 Expression in Lupus T Cells and T Cells Treated with Lupus-Inducing Drugs
Authors: Lu Qianjin , Wu Ailing , Richardson Bruce C.
Published: April 2014

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4+ lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.
Beyond the 12/23 Rule of VDJ Recombination Independent of the Rag Proteins
Authors: Olaru Alexandru , Petrie Howard T. , Livák Ferenc
Published: April 2014

The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRβ, and TCRδ chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRβ and TCRδ loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms “beyond the 12/23 rule.” In the TCRβ locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRδ locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Dδ genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.
A New Tyrosine Phosphorylation Site in PLCγ1: The Role of Tyrosine 775 in Immune Receptor Signaling
Authors: Serrano Carmen J. , Graham Laurie , DeBell Karen , Rawat Rashmi , Veri Maria Concetta , Bonvini Ezio , Rellahan Barbara L. , Reischl Ilona G.
Published: April 2014

Phospholipase Cγ (PLCγ) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCγ is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCγ1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCγ1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCγ1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCγ2 indicated homology of PLCγ1 tyrosine residue 775 (Y775) with PLCγ2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCγ1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCγ1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCγ1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCγ1 activation and confirm the functional importance of Y783.
Mouse Cathelin-Related Antimicrobial Peptide Chemoattracts Leukocytes Using Formyl Peptide Receptor-Like 1/Mouse Formyl Peptide Receptor-Like 2 as the Receptor and Acts as an Immune Adjuvant
Authors: Kurosaka Kahori , Chen Qian , Yarovinsky Felix , Oppenheim Joost J. , Yang De
Published: April 2014

Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.
Protective Immunity to Genital Herpes Simpex Virus Type 2 Infection Is Mediated by T-bet
Authors: Svensson Alexandra , Nordström Inger , Sun Jia-Bin , Eriksson Kristina
Published: April 2014

We show, for the first time, that the transcription factor T-bet, which is implicated in IFN-γ production, is required for the induction of vaccine-induced antiviral immune protection. T-bet was found to be important in both the innate and acquired immune protection against genital HSV-2 infection. T-bet−/− and T-bet+/+ mice were infected vaginally with HSV-2 and examined daily for disease and mortality. T-bet−/− mice had significantly higher virus titers than T-bet+/+ mice following a primary HSV-2 infection, and succumbed significantly earlier to the infection. This result was associated with an impaired NK cell cytotoxic capacity and NK cell-mediated IFN-γ production in the T-bet−/− mice. To assess the induction of acquired antiviral immune protection, mice were vaccinated with an attenuated virus before infection. Vaccinated T-bet−/− mice could not control viral replication following an HSV-2 challenge and had significantly higher virus titers and mortality rates than vaccinated T-bet+/+ mice that remained healthy. The impaired acquired immune protection in T-bet−/− mice was associated with a significantly decreased HSV-2-specific delayed-type hypersensitivity response and a significantly reduced HSV-2-specific IFN-γ production from CD4+ T cells. However, T-bet deficiency did not impair either the IFN-γ production or the cytotoxic capacity of HSV-2-specific CD8+ T cells. We conclude that T-bet plays a crucial role in both the innate defense and the generation of vaccine-induced immunity against genital HSV-2 infection in mice.
Analysis of the 5q31-q33 Locus Shows an Association between IL13-1055C/T IL-13-591A/G Polymorphisms and Schistosoma haematobium Infections
Authors: Kouriba Bourema , Chevillard Christophe , Bream Jay H. , Argiro Laurent , Dessein Helia , Arnaud Violaine , Sangare Lansana , Dabo Abdoulaye , Beavogui Abdou Habib , Arama Charles , Traoré Hamar A. , Doumbo Ogobara , Dessein Alain
Published: April 2014

Millions of humans are exposed to schistosome infections, which cause severe kidney and liver disease and 280,000 deaths annually. Th2-mediated immunity is critical to human defenses against this pathogen and susceptibility to infection is controlled by a major genetic locus that includes IL4, IL5, and IL13 genes. These observations led us to evaluate whether certain polymorphisms in IL4, IL5, or IL13 determine schistosome infection. The study was performed in two Dogon villages where Schistosoma haematobium is endemic. Schistosome infections were evaluated by counting eggs and measuring worm Ags in urine. Genetic polymorphisms were determined by restriction enzyme analysis or by primer extension and denaturing high-performance liquid chromatography analysis. Associations were tested using family-based association tests and logistical regression analysis. The alleles IL13-1055C (p = 0.05) and IL13-591A (p = 0.01) are shown, by family-based association test, to be preferentially transmitted to children with the 10% highest infections. A logistic regression analysis that included IL13-1055 G/G, G/T and T/T genotypes, age, gender, and village of residency, applied to the whole study population, showed that subjects bearing the IL13-1055T/T genotype were on average much less infected than individuals with other genotypes. Previous studies on asthma indicated that the IL13-1055T allele increased gene transcription, which is in agreement with the fact that this cytokine enhances resistance to infection by schistosome in humans.
The Mucosal Adjuvant Macrophage-Activating Lipopeptide-2 Directly Stimulates B Lymphocytes via the TLR2 without the Need of Accessory Cells
Authors: Borsutzky Stefan , Kretschmer Karsten , Becker Pablo D. , Mühlradt Peter F. , Kirschning Carsten J. , Weiss Siegfried , Guzmán Carlos A.
Published: April 2014

The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity as mucosal adjuvant, promoting strong humoral and cellular responses. Although B cells expressing TLR2/6 are potential targets, very little is known about the effect of MALP-2 on B cells. Studies were performed using total spleen cells or purified B cells from WT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2 promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) via TLR2. MALP-2 also increased the frequency of IgM- and IgG-secreting cells, but bystander cells were required for IgA secretion. Activated B cells exhibited increased expression of activation markers and ligands that are critical for cross-talk with T cells (CD19, CD25, CD80, CD86, MHC I, MHC II, and CD40). Immunization of mice lacking T cells showed that MALP-2-mediated stimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunization of mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve the immunogenicity of vaccine formulations.
Protein Kinase R Mediates Intestinal Epithelial Gene Remodeling in Response to Double-Stranded RNA and Live Rotavirus
Authors: Vijay-Kumar Matam , Gentsch Jon R. , Kaiser William J. , Borregaard Niels , Offermann Margaret K. , Neish Andrew S. , Gewirtz Andrew T.
Published: April 2014

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2–100 μg/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET50 ∼24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.
Control of Infection withLeishmania majorin Susceptible BALB/c Mice Lacking the Common γ-Chain for FcR Is Associated with Reduced Production of IL-10 and TGF-β by Parasitized Cells
Authors: Padigel Udaikumar M. , Farrell Jay P.
Published: April 2014

Previous studies have shown that the in vitro ligation of FcγRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common γ-chain of Fc receptors (FcγR−/−) or mice genetically altered to lack circulating Ab (JHD) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcγR−/− mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcγR−/− mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-γ-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-β within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcγRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.
Role of IFN-γ in Regulating T2 Immunity and the Development of Alternatively Activated Macrophages during Allergic Bronchopulmonary Mycosis
Authors: Arora Shikha , Hernandez Yadira , Erb-Downward John R. , McDonald Roderick A. , Toews Galen B. , Huffnagle Gary B.
Published: April 2014

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an “allergic” response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-γ plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-γ knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-γ was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-γ. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-γ KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-γ KO mice by 5 wk postinfection. IFN-γ production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-γ KO mice. Thus, in the absence of IFN-γ, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.
Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils
Authors: Borjesson Dori L. , Kobayashi Scott D. , Whitney Adeline R. , Voyich Jovanka M. , Argue Cynthia M. , DeLeo Frank R.
Published: April 2014

Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phagocytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial survival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis.
Gastrointestinal Ischemia-Reperfusion Injury Is Lectin Complement Pathway Dependent without Involving C1q
Authors: Hart Melanie L. , Ceonzo Kathleen A. , Shaffer Lisa A. , Takahashi Kazue , Rother Russell P. , Reenstra Wende R. , Buras Jon A. , Stahl Gregory L.
Published: April 2014

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.
Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils
Authors: Pederzoli Magali , Kantari Chahrazade , Gausson Valérie , Moriceau Sandra , Witko-Sarsat Véronique
Published: April 2014

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.
A Synthetic TLR4 Antagonist Has Anti-Inflammatory Effects in Two Murine Models of Inflammatory Bowel Disease
Authors: Fort Madeline M. , Mozaffarian Afsaneh , Stöver Axel G. , Correia Jean da Silva , Johnson David A. , Crane R. Thomas , Ulevitch Richard J. , Persing David H. , Bielefeldt-Ohmann Helle , Probst Peter , Jeffery Eric , Fling Steven P. , Hershberg Robert M.
Published: April 2014

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-α release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.
Regulation of c-Jun Phosphorylation by the IκB Kinase-ε Complex in Fibroblast-Like Synoviocytes
Authors: Sweeney Susan E. , Hammaker Deepa , Boyle David L. , Firestein Gary S.
Published: April 2014

Rheumatoid arthritis (RA) causes a symmetric, inflammatory polyarthritis that results in joint destruction and significant disability. Signaling pathways that regulate the production of cytokines and destructive enzymes have been implicated in its pathogenesis and represent potential therapeutic targets. The IκB kinase (IKK)-related kinase, IKKε/IKKi, which plays a pivotal role in regulating antiviral gene transcription, is constitutively expressed by cultured fibroblast-like synoviocytes (FLS) and could participate in the pathogenesis of RA. In the current studies we demonstrate that IKKε protein is expressed in RA and osteoarthritis synovium and that the protein is found primarily in the synovial intimal lining. Functional studies in cultured FLS showed that IKKε kinase activity is rapidly induced by cytokines, although IκB phosphorylation is significantly less compared with IKK2. Because NF-κB activation is similar in wild-type and IKKε knockout murine FLS, studies were performed to identify an alternative substrate for IKKε. Interestingly, c-Jun is a more efficient substrate for IKKε immunocomplexes in human FLS and this activity appears to be independent of JNK. The functional relevance of IKKε was examined using murine IKKε−/− cultured FLS. IL-1-, TNF-α-, and LPS-mediated induction of matrix metalloproteinases, MMP3 and MMP13, is significantly decreased in the IKKε−/− cells. These data suggest a novel role for the IKKε complex in synovial inflammation, extracellular matrix destruction, and activation of the viral program and innate immune response in RA.
Role of Lipoteichoic Acid in the Phagocyte Response to Group BStreptococcus
Authors: Henneke Philipp , Morath Siegfried , Uematsu Satoshi , Weichert Stefan , Pfitzenmaier Markus , Takeuchi Osamu , Müller Andrea , Poyart Claire , Akira Shizuo , Berner Reinhard , Teti Giuseppe , Geyer Armin , Hartung Thomas , Trieu-Cuot Patrick , Kasper Dennis L. , Golenbock Douglas T.
Published: April 2014

Group B Streptococcus (GBS) cell walls potently activate phagocytes by a largely TLR2-independent mechanism. In contrast, the cell wall component lipoteichoic acid (LTA) from diverse Gram-positive bacterial species has been shown to engage TLR2. In this study we examined the role of LTA from GBS in phagocyte activation and the requirements for TLR-LTA interaction. Using cells from knockout mice and genetic complementation in epithelial cells we found that highly pure LTA from both GBS and Staphylococcus aureus interact with TLR2 and TLR6, but not TLR1, in contrast to previous reports. Furthermore, NF-κB activation by LTA required the integrity of two putative PI3K binding domains within TLR2 and was inhibited by wortmannin, indicating an essential role for PI3K in cellular activation by LTA. However, LTA from GBS proved to be a relatively weak stimulus of phagocytes containing ∼20% of the activity observed with LTA from Staphylococcus aureus. Structural analysis by nuclear magnetic resonance spectrometry revealed important differences between LTA from GBS and S. aureus, specifically differences in glycosyl linkage, in the glycolipid anchor and a lack of N-acetylglucosamine substituents of the glycerophosphate backbone. Furthermore, GBS expressing LTA devoid of d-alanine residues, that are essential within immune activation by LTA, exhibited similar inflammatory potency as GBS with alanylated LTA. In conclusion, LTA from GBS is a TLR2/TLR6 ligand that might contribute to secreted GBS activity, but does not contribute significantly to GBS cell wall mediated macrophage activation.