bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Reciprocal Lmb1–4 Interval MRL-Fas
lpr and C57BL/6-Fas
lpr Congenic Mice Reveals Significant Effects from Lmb3
Authors: Santiago-Raber Marie-Laure , Haraldsson M. Katarina , Theofilopoulos Argyrios N. , Kono Dwight H.
Published: April 2014

Susceptibility to severe lupus in MRL-Faslpr mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Faslpr × B6-Faslpr)F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1–4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Faslpr with MRL.B6-Lmb3 Faslpr congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4+ and double-negative (CD4−CD8−) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-γ-producing CD4+ T cells were also reduced in MRL.B6-Lmb3 Faslpr mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4+ and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.
Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion
Authors: Rosenstiel Philip , Sina Christian , End Caroline , Renner Marcus , Lyer Stefan , Till Andreas , Hellmig Stephan , Nikolaus Susanna , Fölsch Ulrich R. , Helmke Burkhard , Autschbach Frank , Schirmacher Peter , Kioschis Petra , Hafner Mathias , Poustka Annemarie , Mollenhauer Jan , Schreiber Stefan
Published: April 2014

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.
Loss of Discrete Memory B Cell Subsets Is Associated with Impaired Immunization Responses in HIV-1 Infection and May Be a Risk Factor for Invasive Pneumococcal Disease
Authors: Hart Melanie , Steel Alan , Clark Sally A. , Moyle Graeme , Nelson Mark , Henderson Don C. , Wilson Robert , Gotch Frances , Gazzard Brian , Kelleher Peter
Published: April 2014

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.
Priming with Cold-Adapted Influenza A Does Not Prevent Infection but Elicits Long-Lived Protection against Supralethal Challenge with Heterosubtypic Virus
Authors: Powell Timothy J. , Strutt Tara , Reome Joyce , Hollenbaugh Joseph A. , Roberts Alan D. , Woodland David L. , Swain Susan L. , Dutton Richard W.
Published: April 2014

We show in this study several novel features of T cell-based heterosubtypic immunity against the influenza A virus in mice. First, T cell-mediated heterosubtypic protection against lethal challenge can be generated by a very low priming dose. Second, it becomes effective within 5–6 days. Third, it provides protection against a very high dose challenge for >70 days. Also novel is the finding that strong, long-lasting, heterosubtypic protection can be elicited by priming with attenuated cold-adapted strains. We demonstrate that priming does not prevent infection of the lungs following challenge, but leads to earlier clearance of the virus and 100% survival after otherwise lethal challenge. Protection is dependent on CD8 T cells, and we show that CD4 and CD8 T cells reactive to conserved epitopes of the core proteins of the challenge virus are present after priming. Our results suggest that intranasal vaccination with cold-adapted, attenuated live virus has the potential to provide effective emergency protection against emerging influenza strains for several months.
Activation of the MAPK, ERK, following Leishmania amazonensis Infection of Macrophages
Authors: Yang Ziyan , Mosser David M. , Zhang Xia
Published: April 2014

IL-10 is a critical cytokine in determining host susceptibility to Leishmania spp. We previously demonstrated that macrophage-derived IL-10 could contribute to disease exacerbation, but the mechanisms whereby Leishmania infections led to IL-10 induction were not fully understood. In this study, we demonstrated that infection of macrophages with Leishmania amazonensis amastigotes led to the activation of the MAPK, ERK1/2. This activation was required, but not sufficient for IL-10 induction. In addition to ERK activation, an inflammatory stimulus, such as low m.w. hyaluronic acid from the extracellular matrix, must also be present. The combination of these two signals resulted in the superinduction of IL-10. We also demonstrated that IgG on the surface of Leishmania amastigotes was required to achieve maximal IL-10 production from infected macrophages. Surface IgG engages macrophage FcγR to induce ERK activation. Macrophages lacking FcγR, or macrophages treated with an inhibitor of spleen tyrosine kinase, the tyrosine kinase that signals via FcγR, failed to activate ERK and consequently failed to produce IL-10 following infection with Leishmania amastigotes. We confirmed that ERK1/2 activation led to the phosphorylation of histone H3 at the IL-10 promoter, and this phosphorylation allowed for the binding of the transcription factor, Sp1, to the IL-10 promoter. Finally, the administration of U0126, an inhibitor of ERK activation, to infected mice resulted in decreased lesion progression with reduced numbers of parasites in them. Thus, our findings reveal an important role of MAPK, ERK signaling in the pathogenesis of Leishmania infection.
The Mechanism Underlying Defective Fcγ Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages
Authors: Leeansyah Edwin , Wines Bruce D. , Crowe Suzanne M. , Jaworowski Anthony
Published: April 2014

Clearance of IgG-opsonized erythrocytes is impaired in HIV-1-infected patients, suggesting defective FcγR-mediated phagocytosis in vivo. We have previously shown defective FcγR-mediated phagocytosis in HIV-1-infected human monocyte-derived macrophages (MDM), establishing an in vitro model for defective tissue macrophages. Inhibition was associated with decreased protein expression of FcR γ-chain, which transduces immune receptor signals via ITAM motifs. FcγRI and FcγRIIIa signal via γ-chain, whereas FcγRIIa does not. In this study, we showed that HIV-1 infection inhibited FcγRI-, but not FcγRIIa-dependent Syk activation in MDM, showing that inhibition was specific for γ-chain-dependent signaling. HIV-1 infection did not impair γ-chain mRNA levels measured by real-time PCR, suggesting a posttranscriptional mechanism of γ-chain depletion. HIV-1 infection did not affect γ-chain degradation (n = 7, p = 0.94) measured in metabolic labeling/chase experiments, whereas γ-chain biosynthesis was inhibited (n = 12, p = 0.0068). Using an enhanced GFP-expressing HIV-1 strain, we showed that FcγR-mediated phagocytosis inhibition is predominantly due to a bystander effect. Experiments in which MDM were infected in the presence of the antiretroviral drug 3TC suggest that active viral replication is required for inhibition of phagocytosis in MDM. These data suggest that HIV-1 infection may affect only γ-chain-dependent FcγR functions, but that this is not restricted to HIV-1-infected cells.
CD99 Is a Key Mediator of the Transendothelial Migration of Neutrophils
Authors: Lou Olivia , Alcaide Pilar , Luscinskas Francis W. , Muller William A.
Published: April 2014

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.
Human C-Reactive Protein Protects Mice fromStreptococcus pneumoniaeInfection without Binding to Pneumococcal C-Polysaccharide
Authors: Suresh Madathilparambil V. , Singh Sanjay K. , Ferguson Donald A. , Agrawal Alok
Published: April 2014

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcγ receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.
MyD88-Dependent and MyD88-Independent Pathways in Synergy, Priming, and Tolerance between TLR Agonists
Authors: Bagchi Aranya , Herrup Elizabeth A. , Warren H. Shaw , Trigilio James , Shin Hae-Sook , Valentine Catherine , Hellman Judith
Published: April 2014

TLRs sense components of microorganisms and are critical host mediators of inflammation during infection. Different TLR agonists can profoundly alter inflammatory effects of one another, and studies suggest that the sequence of exposure to TLR agonists may importantly impact on responses during infection. We tested the hypothesis that synergy, priming, and tolerance between TLR agonists follow a pattern that can be predicted based on differential engagement of the MyD88-dependent (D) and the MyD88-independent (I) intracellular signaling pathways. Inflammatory effects of combinations of D and I pathway agonists were quantified in vivo and in vitro. Experiments used several D-specific agonists, an I-specific agonist (poly(I:C)), and LPS, which acts through both the D and I pathways. D-specific agonists included: peptidoglycan-associated lipoprotein, Pam3Cys, flagellin, and CpG DNA, which act through TLR2 (peptidoglycan-associated lipoprotein and Pam3Cys), TLR5, and TLR9, respectively. D and I agonists were markedly synergistic in inducing cytokine production in vivo in mice. All of the D-specific agonists were synergistic with poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages. Pretreatment of bone marrow-derived macrophages with poly(I:C) led to a primed response to subsequent D-specific agonists and vice versa, as indicated by increased cytokine production, and increased NF-κB translocation. Pretreatment with a D-specific agonist augmented LPS-induced IFN-β production. All D-specific agonists induced tolerance to one another. Thus, under the conditions studied here, simultaneous and sequential activation of both the D and I pathways causes synergy and priming, respectively, and tolerance is induced by agonists that act through the same pathway.
A Defect of Regulatory T Cells in Patients with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy
Authors: Kekäläinen Eliisa , Tuovinen Heli , Joensuu Joonas , Gylling Mikhail , Franssila Rauli , Pöntynen Nora , Talvensaari Kimmo , Perheentupa Jaakko , Miettinen Aaro , Arstila T. Petteri
Published: April 2014

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic recessive disease characterized by autoimmunity against multiple tissues, offers a unique possibility to study the breakdown of self-tolerance in humans. It is caused by mutations in the autoimmune regulator gene (AIRE), which encodes a transcriptional regulator. Work using Aire−/− mice suggests that Aire induces ectopic expression of peripheral Ags and promotes their presentation in the thymus. We have explored reasons for the difference between the comparatively mild phenotype of Aire-deficient mice and human APECED patients. We provide evidence that, unlike in the Aire−/− mice, in the patients a key mediator of active tolerance, the CD4+CD25+ regulatory T (Treg) cell subset is impaired. This was shown by significantly decreased expression of FOXP3 mRNA and protein, decreased function, and alterations in TCR repertoire. Also, in the normal human thymus a concentric accumulation of AIRE+ cells was seen around thymic Hassall’s corpuscles, suggesting that in the patients these cells may be involved in the observed Treg cell failure. In Aire−/− mice the expression of FoxP3 was normal and even increased in target tissues in parallel with the lymphocyte infiltration process. Our results suggest that a Treg cell defect is involved in the pathogenesis of APECED and emphasize the importance of active tolerance mechanisms in preventing human autoimmunity.
The Varicellovirus-Encoded TAP Inhibitor UL49.5 Regulates the Presentation of CTL Epitopes by Qa-1b1
Authors: van Hall Thorbald , Laban Sandra , Koppers-Lalic Danijela , Koch Joachim , Precup Calin , Asmawidjaja Patrick , Offringa Rienk , Wiertz Emmanuel J. H. J.
Published: April 2014

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1b revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.
A Costimulatory Function for T Cell CD40
Authors: Munroe Melissa E. , Bishop Gail A.
Published: April 2014

CD40 plays a significant role in the pathogenesis of inflammation and autoimmunity. B cell CD40 directly activates cells, which can result in autoantibody production. T cells can also express CD40, with an increased frequency and amount of expression seen in CD4+ T lymphocytes of autoimmune mice, including T cells from mice with collagen-induced arthritis. However, the mechanisms of T cell CD40 function have not been clearly defined. To test the hypothesis that CD40 can serve as a costimulatory molecule on T lymphocytes, CD40+ T cells from collagen-induced arthritis mice were examined in parallel with mouse and human T cell lines transfected with CD40. CD40 served as effectively as CD28 in costimulating TCR-mediated activation, including induction of kinase and transcription factor activities and production of cytokines. An additional enhancement was seen when both CD40 and CD28 signals were combined with AgR stimulation. These findings reveal potent biologic functions for T cell CD40 and suggest an additional means for amplification of autoimmune responses.
Enforced Expression of Bcl-2 Partially Restores Cell Numbers but Not Functions of TCRγδ Intestinal Intraepithelial T Lymphocytes in IL-15-Deficient Mice
Authors: Nakazato Kenji , Yamada Hisakata , Yajima Toshiki , Kagimoto Yoshiko , Kuwano Hiroyuki , Yoshikai Yasunobu
Published: April 2014

IL-15 knockout (KO) mice have severely reduced numbers of TCRγδ intestinal intraepithelial T lymphocytes (i-IEL), suggesting requirements of IL-15 signaling in the development or maintenance of i-IEL. To determine an involvement of survival signals via Bcl-2 in IL-15-mediated homeostasis of TCRγδ i-IEL, we introduced a bcl-2 transgene into IL-15 KO mice. In situ apoptosis of TCRγδ i-IEL was decreased in Bcl-2 transgenic (Tg) × IL-15 KO mice compared with IL-15 KO mice. The enforced expression of Bcl-2 partially restored the numbers of TCRγδ i-IEL in IL-15 KO mice. However, effector functions of TCRγδ i-IEL, including cytokine production and cytotoxic activity, were not recovered in Bcl-2 Tg × IL-15 KO mice. Importantly, TCRγδ i-IEL in Bcl-2 Tg × IL-15 KO mice expressed a reduced level of eomesodermin, a transcription factor critical for effector functions of NK cells and CD8+ T cells. Similar to the case of TCRγδ i-IEL, enforced expression of Bcl-2 restored the numbers but not the functions of NK cells in IL-15 KO mice. These results suggest that Bcl-2-mediated survival signal is involved in the IL-15-mediated homeostasis of TCRγδ i-IEL and NK cells, but other signals from IL-15 are critical for inducing transcription factors, such as eomesodermin for their effector functions.
IL-10 Regulation by HIV-Tat in Primary Human Monocytic Cells: Involvement of Calmodulin/Calmodulin-Dependent Protein Kinase-Activated p38 MAPK and Sp-1 and CREB-1 Transcription Factors
Authors: Gee Katrina , Angel Jonathan B. , Mishra Sasmita , Blahoianu Maria A. , Kumar Ashok
Published: April 2014

The anti-inflammatory cytokine, IL-10 plays an important role in HIV immunopathogenesis. The HIV accessory protein, Tat is not only critical for viral replication, but affects the host immune system by influencing cytokine production including IL-10. During HIV infection, IL-10 production by monocytic cells is up-regulated, representing a critical pathway by which HIV may induce immunodeficiency. Herein, we show that extracellular Tat-induced IL-10 expression in normal human monocytes. To understand the signaling pathways underlying HIV-Tat induced IL-10 transcription, we investigated the involvement of MAPK as well as calcium signaling and the downstream transcription factor(s). Our results suggest that Tat-induced calcium influx regulated IL-10 transcription in monocytic cells. The experiments designed to further understand the molecules involved in the calcium signaling suggested that calmodulin and calmodulin-dependent protein kinase-II (CaMK-II)-activated p38 MAPK played a role in extracellular Tat-induced IL-10 expression in primary human monocytes. Furthermore, Tat-induced IL-10 expression was regulated by p38 MAPK- and CaMK II-activated CREB-1 as well as Sp-1 transcription factors. Taken together, our results suggest that extracellular HIV-Tat induced IL-10 transcription in primary human monocytes is regulated by CREB-1 and Sp-1 transcription factors through the activation of calmodulin/CaMK-II-dependent p38 MAPK.
SAP Regulation of Follicular Helper CD4 T Cell Development and Humoral Immunity Is Independent of SLAM and Fyn Kinase
Authors: McCausland Megan M. , Yusuf Isharat , Tran Hung , Ono Nobuyuki , Yanagi Yusuke , Crotty Shane
Published: April 2014

Mutations in SH2D1A resulting in lack of SLAM-associated protein (SAP) expression cause the human genetic immunodeficiency X-linked lymphoproliferative disease. A severe block in germinal center development and lack of long-term humoral immunity is one of the most prominent phenotypes of SAP− mice. We show, in this study, that the germinal center block is due to an essential requirement for SAP expression in Ag-specific CD4 T cells to develop appropriate follicular helper T cell functions. It is unknown what signaling molecules are involved in regulation of SAP-dependent CD4 T cell help functions. SAP binds to the cytoplasmic tail of SLAM, and we show that SLAM is expressed on resting and activated CD4 T cells, as well as germinal center B cells. In addition, SAP can recruit Fyn kinase to SLAM. We have now examined the role(s) of the SLAM-SAP-Fyn signaling axis in in vivo CD4 T cell function and germinal center development. We observed normal germinal center development, long-lived plasma cell development, and Ab responses in SLAM−/− mice after a viral infection (lymphocytic choriomeningitis virus). In a separate series of experiments, we show that SAP is absolutely required in CD4 T cells to drive germinal center development, and that requirement does not depend on SAP-Fyn interactions, because CD4 T cells expressing SAP R78A are capable of supporting normal germinal center development. Therefore, a distinct SAP signaling pathway regulates follicular helper CD4 T cell differentiation, separate from the SLAM-SAP-Fyn signaling pathway regulating Th1/Th2 differentiation.
Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells
Authors: Lomada Dakshayani , Liu Bigang , Coghlan Lezlee , Hu Yinling , Richie Ellen R.
Published: April 2014

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-κB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IκB kinase (IKK)α, a required intermediate in the noncanonical NF-κB signaling pathway. Introduction of an IKKα transgene driven by a K5 promoter restores the K5+K14+ mTEC subset in IKKα−/− mice. Unexpectedly, the K5-IKKα transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKα transgene ameliorates autoimmune symptoms in IKKα−/− mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-κB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.
HIV-1 Trans Activator of Transcription Protein Elicits Mitochondrial Hyperpolarization and Respiratory Deficit, with Dysregulation of Complex IV and Nicotinamide Adenine Dinucleotide Homeostasis in Cortical Neurons
Authors: Norman John P. , Perry Seth W. , Kasischke Karl A. , Volsky David J. , Gelbard Harris A.
Published: April 2014

HIV-1 causes a common, progressive neurological disorder known as HIV-associated dementia (HAD). The prevalence of this disorder has increased despite the use of highly active antiretroviral therapy, and its underlying pathogenesis remains poorly understood. However, evidence suggests that some aspects of HAD may be reversible. To model the reversible aspects of HAD, we have used the HIV-1 neurotoxin trans activator of transcription protein (Tat) to investigate nonlethal changes in cultured neurons. Exposure of rodent cortical neurons to sublethal concentrations of Tat elicits mitochondrial hyperpolarization. In this study, we used the cationic lipophilic dye rhodamine 123 to confirm this observation, and then performed follow-up studies to examine the mechanism involved. In intact neurons, we found Tat elicited a rapid drop in internal mitochondrial pH, and addition of Tat to purified mitochondrial extracts inhibited complex IV of the electron transport chain. To correlate enzyme activity in mitochondrial extracts with results in intact cells, we measured neuronal respiration following Tat exposure. Cortical neurons demonstrated decreased respiration upon Tat treatment, consistent with inhibition of complex IV. We examined mitochondrial Ca2+ homeostasis using a mitochondrial targeted enhanced yellow fluorescent protein-calmodulin construct. We detected a decrease in mitochondrial calcium concentration following exposure to Tat. Finally, we measured the energy intermediate NAD(P)H after Tat treatment, and found a 20% decrease in the autofluorescence. Based on these findings, we suggest that decreased NAD(P)H and calcium concentration contribute to subsequent respiratory decline after exposure to Tat, with detrimental effects on neuronal signaling.
CD27low CD4 T Lymphocytes That Accumulate in the Mouse Lungs during Mycobacterial Infection Differentiate from CD27high Precursors In Situ, Produce IFN-γ, and Protect the Host against Tuberculosis Infection
Authors: Kapina Marina A. , Shepelkova Galina S. , Mischenko Vladimir V. , Sayles Peter , Bogacheva Polina , Winslow Gary , Apt Alexander S. , Lyadova Irina V.
Published: April 2014

The generation of effector, IFN-γ producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44highCD62Llow effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27low subset exhibited a high capacity for IFN-γ secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27low IFN-γ-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27low CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27low CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27high effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27low CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.
Age-Associated Decline in Effective Immune Synapse Formation of CD4+ T Cells Is Reversed by Vitamin E Supplementation
Authors: Marko Melissa G. , Ahmed Tanvir , Bunnell Stephen C. , Wu Dayong , Chung Heekyung , Huber Brigitte T. , Meydani Simin Nikbin
Published: April 2014

Aging is associated with reduced IL-2 production and T cell proliferation. Vitamin E supplementation, in aged animals and humans, increases cell division and IL-2 production by naive T cells. The immune synapse forms at the site of contact between a T cell and an APC and participates in T cell activation. We evaluated whether vitamin E affects the redistribution of signaling proteins to the immune synapse. Purified CD4+ T cells, from the spleens of young and old mice, were treated with vitamin E before stimulation with a surrogate APC expressing anti-CD3. Using confocal fluorescent microscopy, we observed that CD4+ T cells from old mice were significantly less likely to recruit signaling proteins to the immune synapse than cells from young mice. Vitamin E increased the percentage of old CD4+ T cells capable of forming an effective immune synapse. Similar results were found following in vivo supplementation with vitamin E. When compared with memory cells, naive T cells from aged mice were more defective in immune synapse formation and were more responsive to vitamin E supplementation. These data show, for the first time, that vitamin E significantly improves age-related early T cell signaling events in naive CD4+ T cells.
F2L, a Peptide Derived from Heme-Binding Protein, Chemoattracts Mouse Neutrophils by Specifically Activating Fpr2, the Low-Affinity N-Formylpeptide Receptor
Authors: Gao Ji-Liang , Guillabert Aude , Hu Jinyue , Le Yingying , Urizar Eneko , Seligman Eva , Fang Kevin J. , Yuan Xiaoning , Imbault Virginie , Communi David , Wang Ji Ming , Parmentier Marc , Murphy Philip M. , Migeotte Isabelle
Published: April 2014

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC50 ∼400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC50 values similar to those found for Fpr2-expressing cell lines (∼500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.
Dendritic Cell-Independent B Cell Activation During Acute Virus Infection: A Role for Early CCR7-Driven B-T Helper Cell Collaboration
Authors: Scandella Elke , Fink Katja , Junt Tobias , Senn Beatrice M. , Lattmann Evelyn , Förster Reinhold , Hengartner Hans , Ludewig Burkhard
Published: April 2014

This study provides a detailed spatiotemporal interaction analysis between B cells, Th cells, and dendritic cells (DC) during the generation of protective antiviral B cell immunity. Following vesicular stomatitis virus (VSV) infection, conditional ablation of CD11c-positive DC at the time-point of infection did not impair extrafollicular plasma cell generation and Ig class switching. In contrast, the generation of Th and B cell responses following immunization with recombinant VSV-glycoprotein was DC-dependent. Furthermore, we show that the CCR7-dependent interplay of the three cell-types is crucial for virus-neutralizing B cell responses in the presence of limiting amounts of Ag. An immediate event following VSV infection was the CCR7-mediated interaction of VSV-specific B and Th cells at the T cell-B cell zone border that facilitated plasma cell differentiation and Th cell activation. Taken together, these experiments provide evidence for a direct, CCR7-orchestrated and largely DC-independent mutual activation of Th cells and Ag-specific B cells that is most likely a critical step during early immune responses against cytopathic viruses.
Expression Patterns of H2-O in Mouse B Cells and Dendritic Cells Correlate with Cell Function
Authors: Fallas Jennifer L. , Yi Woelsung , Draghi Nicole A. , O’Rourke Helen M. , Denzin Lisa K.
Published: April 2014

In the endosomes of APCs, the MHC class II-like molecule H2-M catalyzes the exchange of class II-associated invariant chain peptides (CLIP) for antigenic peptides. H2-O is another class II-like molecule that modulates the peptide exchange activity of H2-M. Although the expression pattern of H2-O in mice has not been fully evaluated, H2-O is expressed by thymic epithelial cells, B cells, and dendritic cells (DCs). In this study, we investigated H2-O, H2-M, and I-Ab-CLIP expression patterns in B cell subsets during B cell development and activation. H2-O was first detected in the transitional 1 B cell subset and high levels were maintained in marginal zone and follicular B cells. H2-O levels were down-regulated specifically in germinal center B cells. Unexpectedly, we found that mouse B cells may have a pool of H2-O that is not associated with H2-M. Additionally, we further evaluate H2-O and H2-M interactions in mouse DCs, as well as H2-O expression in bone marrow-derived DCs. We also evaluated H2-O, H2-M, I-Ab, and I-Ab-CLIP expression in splenic DC subsets, in which H2-O expression levels varied among the splenic DC subsets. Although it has previously been shown that H2-O modifies the peptide repertoire, H2-O expression did not alter DC presentation of a number of endogenous and exogenous Ags. Our further characterization of H2-O expression in DCs, as well as the identification of a potential free pool of H2-O in mouse splenic B cells, suggest that H2-O may have a yet to be elucidated role in immune responses.
Expression and Function of Synaptotagmin VII in CTLs
Authors: Fowler Kimberly T. , Andrews Norma W. , Huleatt James W.
Published: April 2014

The Ca2+ sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca2+-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII+/+ controls, Syt VII−/− animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII−/− mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII+/+ or Syt VII−/− CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.
Effects of Vascular Endothelial Growth Factor on the Lymphocyte-Endothelium Interactions: Identification of Caveolin-1 and Nitric Oxide as Control Points of Endothelial Cell Anergy
Authors: Bouzin Caroline , Brouet Agnès , De Vriese Joelle , DeWever Julie , Feron Olivier
Published: April 2014

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-α and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-α-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.
Necrotic Tumor Cell Death In Vivo Impairs Tumor-Specific Immune Responses
Authors: Gamrekelashvili Jaba , Krüger Christine , von Wasielewski Reinhard , Hoffmann Matthias , Huster Katharina M. , Busch Dirk H. , Manns Michael P. , Korangy Firouzeh , Greten Tim F.
Published: April 2014

The manner in which cells die is believed to have a major impact on the nature of immune responses to their released Ags. In this study, we present the first direct analysis of tumor-specific immune responses to in vivo occurring tumor cell death through apoptosis or necrosis. Mice bearing thymidine kinase-transfected tumors were treated either with ganciclovir to induce tumor cell apoptosis in vivo or a vascular targeting agent, ZD6126, to induce tumor cell necrosis in vivo. In contrast to tumor apoptosis, induction of necrosis reduced the frequency and impaired the function of tumor-specific CD8+ T cells. Adoptive transfer of lymphocytes from mice with apoptotic tumors into tumor-challenged mice resulted in a significant tumor protection, which was absent when splenocytes were transferred from mice with necrotic tumors. Anti-CD40 treatment reversed impaired Ag-specific CD8+ T cell responses in these mice. These observations have not only fundamental importance for the development of immunotherapy protocols but also help to understand the underlying mechanism of in vivo immune responses to tumor cell death.
Haplotype-Independent Costimulation of IL-10 Secretion by SDF-1/CXCL12 Proceeds via AP-1 Binding to the Human IL-10 Promoter
Authors: Kremer Kimberly N. , Kumar Ashok , Hedin Karen E.
Published: April 2014

Costimulation by the chemokine, stromal cell-derived factor-1 (SDF-1)/CXCL12, has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however, the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at −1082, −819, and −592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA− (“memory”) phenotype T cells that includes cells expressing the presumed regulatory T cell marker, Foxp3. To address the molecular mechanisms of this response, we showed that SDF-1 costimulates the transcriptional activities in normal human T cells of reporter plasmids containing 1.1 kb of all three of the common IL-10 promoter haplotypes. IL-10 promoter activity was ablated by mutating two nonpolymorphic binding sites for the AP-1 transcription factor, and chromatin immunoprecipitation assays of primary human T cells revealed that SDF-1 costimulation enhances AP-1 binding to both of these sites. Together, these results delineate the molecular mechanisms responsible for SDF-1 costimulation of T cell IL-10 secretion. Because it is preserved among several human haplotypes and in diverse T cell populations including Foxp3+ T cells, this pathway of IL-10 regulation may represent a key mechanism for modulating expression of this important immunoregulatory cytokine.
Dissociation of the Genetic Loci Leading to B1a and NKT Cell Expansions from Autoantibody Production and Renal Disease in B6 Mice with an Introgressed New Zealand Black Chromosome 4 Interval
Authors: Loh Christina , Cai Yong-Chun , Bonventi Gabriel , Lajoie Ginette , MacLeod Ralph , Wither Joan E.
Published: April 2014

Previous mapping studies have linked New Zealand Black (NZB) chromosome 4 to several lupus traits, including autoantibody production, splenomegaly, and glomerulonephritis. To confirm the presence of these traits, our laboratory introgressed homozygous NZB chromosome 4 intervals extending from either 114 to 149 Mb or 32 to 149 Mb onto the lupus-resistant C57BL/6 background (denoted B6.NZBc4S and B6.NZBc4L, respectively). Characterization of aged cohorts revealed that B6.NZBc4L mice exhibited a striking increase in splenic B1a and NKT cells in the absence of high titer autoantibody production and significant renal disease. Tissue-specific expansion of these subsets was also seen in the peritoneum and liver for B1a cells and in the bone marrow for NKT cells. Staining with CD1d tetramers loaded with an α-galactosylceramide analog (PBS57) demonstrated that the expanded NKT cell population was mainly CD1d-dependent NKT cells. The lack of both cellular phenotypes in B6.NZBc4S mice demonstrates that the genetic polymorphism(s) that result in these phenotypes are on the proximal region of NZB chromosome 4. This study confirms the presence of a locus that promotes the expansion of B1a cells and newly identifies a region that promotes CD1d-restricted NKT cell expansion on NZB chromosome 4. Taken together, the data indicate that neither an expansion of B1a cells and/nor NKT cells is sufficient to promote autoantibody production and ultimately, renal disease.