bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR2 and TLR9 Synergistically Control Herpes Simplex Virus Infection in the Brain
Authors: Sørensen Louise N. , Reinert Line S. , Malmgaard Lene , Bartholdy Christina , Thomsen Allan R. , Paludan Søren R.
Published: April 2014

Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few studies have defined essential roles for single TLRs in innate immune defense in vivo. This could suggest that PRRs act in concert to mount the first line of defense against virus infections. To test this hypothesis we have examined the host response of C57BL/6, TLR2−/−, TLR9−/−, and TLR2/9−/− mice toward HSV-2 infection. After a systemic infection, the cytokine serum response was markedly reduced in the double knockout mice, but only partly affected in either strain of the single knockout mice. This was supported by in vitro data showing that HSV-induced cytokine expression relayed on TLR2 and TLR9 in a cytokine- and cell type-dependent manner. With respect to the cellular response to infection, we found that recruitment but not activation of NK cells was impaired in TLR2/9−/− mice. Importantly, the viral load in the brain, but not liver, was significantly higher in the brain of TLR2/9−/− mice whereas the viral loads in organs of single knockout mice were statistically indistinguishable from C57BL/6 mice. In the brain we found that TNF-α and the IFN-stimulated gene CXCL9 were expressed during infection and were dependent on either TLR2 or TLR9. Thus, TLR2 and TLR9 synergistically stimulate innate antiviral activities, thereby protecting against HSV infection in the brain.
Rapid Appearance of Resolvin Precursors in Inflammatory Exudates: Novel Mechanisms in Resolution
Authors: Kasuga Kie , Yang Rong , Porter Timothy F. , Agrawal Nitin , Petasis Nicos A. , Irimia Daniel , Toner Mehmet , Serhan Charles N.
Published: April 2014

Resolution of inflammation is essential. Although supplementation of ω-3 fatty acids is widely used, their availability at sites of inflammation is not known. To this end, a multidisciplinary approach was taken to determine the relationship of circulating ω-3 to inflammatory exudates and the generation of resolution signals. In this study, we monitored resolvin precursors in evolving exudates, which initially paralleled increases in edema and infiltrating neutrophils. We also prepared novel microfluidic chambers to capture neutrophils from a drop of blood within minutes that permitted single-cell monitoring. In these, docosahexaenoic acid-derived resolvin D1 rapidly stopped neutrophil migration, whereas precursor docosahexaenoic acid did not. In second organ injury via ischemia-reperfusion, resolvin metabolically stable analogues were potent organ protectors reducing neutrophils. Together, these results indicate that circulating ω-3 fatty acids rapidly appear in inflammatory sites that require conversion to resolvins that control excessive neutrophil infiltration, protect organs, and foster resolution.
Lipoxin A4 Counterregulates GM-CSF Signaling in Eosinophilic Granulocytes
Authors: Starosta Vitaliy , Pazdrak Konrad , Boldogh Istvan , Svider Tetyana , Kurosky Alexander
Published: April 2014

Eosinophils are granulated leukocytes that are involved in many inflammation-associated pathologies including airway inflammation in asthma. Resolution of eosinophilic inflammation and return to homeostasis is in part due to endogenous chemical mediators, for example, lipoxins, resolvins, and protectins. Lipoxins are endogenous eicosanoids that demonstrate antiinflammatory activity and are synthesized locally at sites of inflammation. In view of the importance of lipoxins (LXs) in resolving inflammation, we investigated the molecular basis of LXA4 action on eosinophilic granulocytes stimulated with GM-CSF employing the eosinophilic leukemia cell line EoL-1 as well as peripheral blood eosinophils. We report herein that LXA4 (1–100 nM) decreased protein tyrosine phosphorylation in EoL-1 cells stimulated with GM-CSF. Additionally, the expression of a number of GM-CSF-induced cytokines was inhibited by LXA4 in a dose-dependent manner. Furthermore, using a proteomics approach involving mass spectrometry and immunoblot analysis we identified 11 proteins that were tyrosine phosphorylated after GM-CSF stimulation and whose phosphorylation was significantly inhibited by LXA4 pretreatment. Included among these 11 proteins were α-fodrin (nonerythroid spectrin) and actin. Microscopic imaging showed that treatment of EoL-1 cells or blood eosinophils with GM-CSF resulted in the reorganization of actin and the translocation of α-fodrin from the cytoplasm to the plasma membrane. Importantly, α-fodrin translocation was prevented by LXA4 but actin reorganization was not. Thus, the mechanism of LXA4 action likely involves prevention of activation of eosinophilic granulocytes by GM-CSF through inhibition of protein tyrosine phosphorylation and modification of some cytoskeletal components.
T Cell Responses to Mycobacterial Catalase-Peroxidase Profile a Pathogenic Antigen in Systemic Sarcoidosis
Authors: Chen Edward S. , Wahlström Jan , Song Zhimin , Willett Matthew H. , Wikén Maria , Yung Rex C. , West Erin E. , McDyer John F. , Zhang Ying , Eklund Anders , Grunewald Johan , Moller David R.
Published: April 2014

Sarcoidosis is a systemic granulomatous disease associated with local epithelioid granulomas, CD4+ T cells, and Th1 cytokines. The tissue Ags that drive this granulomatous inflammation are uncertain. In this study, we used IFN-γ-ELISPOT assays and flow cytometry to assess lung and blood T cell responses to the candidate pathogenic Ag, Mycobacterium tuberculosis catalase-peroxidase (mKatG) in patients with sarcoidosis from two centers. Despite differences in patient phenotypic, genetic, and prognostic characteristics, we report that T cell responses to mKatG were remarkably similar in these cohorts, with higher frequencies of mKatG-reactive, IFN-γ-expressing T cells in the blood of sarcoidosis patients compared with nontuberculosis sensitized healthy controls, and (in a subset) in greater numbers than T cells reactive to purified protein derivative. In sarcoidosis, mKatG-reactive CD4+ Th1 cells preferentially accumulated in the lung, indicating a compartmentalized response. Patients with or without Löfgren syndrome had similar frequencies of mKatG specific IFN-γ-expressing blood T cells. Circulating mKatG-reactive T cells were found in chronic active sarcoidosis but not in patients with inactive disease. Together, these results demonstrate that T cell responses to mKatG in sarcoidosis fit a profile expected for a pathogenic Ag, supporting an immunotherapeutic approach to this disease.
CD40 Ligand Protects from TRAIL-Induced Apoptosis in Follicular Lymphomas through NF-κB Activation and Up-Regulation of c-FLIP and Bcl-xL
Authors: Travert Marion , Ame-Thomas Patricia , Pangault Céline , Morizot Alexandre , Micheau Olivier , Semana Gilbert , Lamy Thierry , Fest Thierry , Tarte Karin , Guillaudeux Thierry
Published: April 2014

The TNF family member TRAIL is emerging as a promising cytotoxic molecule for antitumor therapy. However, its mechanism of action and the possible modulation of its effect by the microenvironment in follicular lymphomas (FL) remain unknown. We show here that TRAIL is cytotoxic only against FL B cells and not against normal B cells, and that DR4 is the main receptor involved in the initiation of the apoptotic cascade. However, the engagement of CD40 by its ligand, mainly expressed on a specific germinal center CD4+ T cell subpopulation, counteracts TRAIL-induced apoptosis in FL B cells. CD40 induces a rapid RNA and protein up-regulation of c-FLIP and Bcl-xL. The induction of these antiapoptotic molecules as well as the inhibition of TRAIL-induced apoptosis by CD40 is partially abolished when NF-κB activity is inhibited by a selective inhibitor, BAY 117085. Thus, the antiapoptotic signaling of CD40, which interferes with TRAIL-induced apoptosis in FL B cells, involves NF-κB-mediated induction of c-FLIP and Bcl-xL which can respectively interfere with caspase 8 activation or mitochondrial-mediated apoptosis. These findings suggest that a cotreatment with TRAIL and an inhibitor of NF-κB signaling or a blocking anti-CD40 Ab could be of great interest in FL therapy.
ICOS Costimulation Expands Th2 Immunity by Augmenting Migration of Lymphocytes to Draining Lymph Nodes
Authors: Tesciuba Amanda G. , Shilling Rebecca A. , Agarwal Monica D. , Bandukwala Hozefa S. , Clay Bryan S. , Moore Tamson V. , Weinstock Joel V. , Welcher Andrew A. , Sperling Anne I.
Published: April 2014

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS−/− mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.
Inhibition of CD26/Dipeptidyl Peptidase IV Enhances CCL11/Eotaxin-Mediated Recruitment of Eosinophils In Vivo
Authors: Forssmann Ulf , Stoetzer Carsten , Stephan Michael , Kruschinski Carsten , Skripuletz Thomas , Schade Jutta , Schmiedl Andreas , Pabst Reinhard , Wagner Leona , Hoffmann Torsten , Kehlen Astrid , Escher Sylvia E. , Forssmann Wolf-Georg , Elsner Jörn , von Hörsten Stephan
Published: April 2014

Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11(3–74). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.
Renal Urokinase-Type Plasminogen Activator (uPA) Receptor but not uPA Deficiency Strongly Attenuates Ischemia Reperfusion Injury and Acute Kidney Allograft Rejection
Authors: Gueler Faikah , Rong Song , Mengel Michael , Park Joon-Keun , Kiyan Julia , Kirsch Torsten , Dumler Inna , Haller Hermann , Shushakova Nelli
Published: April 2014

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inadequate ICAM-1 up-regulation in uPAR−/− primary aortic endothelial cells after C5a and TNF-α stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory responses mediating IR-injury and transplant rejection.
CpG Oligonucleotides Enhance Proliferative and Effector Responses of B Cells in HIV-Infected Individuals
Authors: Malaspina Angela , Moir Susan , DiPoto Angela C. , Ho Jason , Wang Wei , Roby Gregg , O'Shea Marie A. , Fauci Anthony S.
Published: April 2014

Stimulation through TLR represents a new therapeutic approach for enhancing Ab responses to vaccination. Considering that Ab responses are decreased in HIV disease and that B cells express TLR9 and respond to TLR9 agonists, we investigated the responsiveness of B cell subpopulations from HIV-infected and uninfected individuals to the TLR9 agonist CpG oligonucleotide type B (CpG-B) in the presence and absence of BCR ligation and T cell help (CD40L). CpG-B was equally effective in stimulating the proliferation of naive B cells of HIV-infected individuals and HIV-negative individuals, and, when combined with BCR and CD40 ligation, cytokine secretion by naive B cells was also comparable in HIV-infected and uninfected individuals. In contrast, CD27+ memory/activated B cells of HIV-infected individuals with active disease were less responsive to CpG-B in terms of proliferation and cytokine secretion when compared with CD27+ B cells of HIV-negative and HIV-infected individuals whose viremia was controlled by antiretroviral therapy. These findings suggest that despite abnormalities in memory B cells of HIV-infected individuals with active disease, naive B cells of HIV-infected individuals, irrespective of disease status, can respond to TLR9 agonists and that the incorporation of such agents in vaccine formulations may enhance their Ab responses to vaccination.
Critical Role of Effector Macrophages in Mediating CD4-Dependent Alloimmune Injury of Transplanted Liver Parenchymal Cells
Authors: Horne Phillip H. , Zimmerer Jason M. , Fisher Mason G. , Lunsford Keri E. , Nadasdy Gyongyi , Nadasdy Tibor , van Rooijen Nico , Bumgardner Ginny L.
Published: April 2014

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4+ T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4+ T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.
Demyelinating Myelin Oligodendrocyte Glycoprotein-Specific Autoantibody Response Is Focused on One Dominant Conformational Epitope Region in Rodents
Authors: Breithaupt Constanze , Schäfer Beatrix , Pellkofer Hannah , Huber Robert , Linington Christopher , Jacob Uwe
Published: April 2014

Conformational epitopes of myelin oligodendrocyte glycoprotein (MOG) provide a major target for demyelinating autoantibodies in experimental autoimmune encephalomyelitis and recent studies indicate that a similar situation may exist in multiple sclerosis. We recently solved the crystal structure of the extracellular domain of MOG (MOGex) in complex with a Fab derived from the demyelinating mAb 8-18C5 and identified the conformational 8-18C5 epitope on MOG that is dominated by the surface exposed FG loop of MOG. To determine the importance of this epitope with regard to the polyclonal Ab response to MOGex we investigated the effects of mutating His103 and Ser104, the two central amino acids of the FG loop, on Ab binding. Mutation of these two residues reduced binding of a panel of eight demyelinating conformation-dependent mAbs to <20% compared with binding to wild-type MOGex, whereas substitution of amino acids that do not contribute to the 8-18C5 epitope had only a minor effect on Ab binding. The same restriction was observed for the polyclonal MOG-specific Ab response of MOG DNA-vaccinated BALB/c and SJL/J mice. Our data demonstrate that the pathogenic anti-MOG Ab response primarily targets one immunodominant region centered at the FG loop of MOG. Comparison of the structure of MOGex with the structures of related IgV-like domains yields a possible explanation for the focused Ab response.
Type 1 TNF Receptor Forms a Complex with and Uses Jak2 and c-Src to Selectively Engage Signaling Pathways That Regulate Transcription Factor Activity
Authors: Pincheira Roxana , Castro Ariel F. , Ozes Osman Nidai , Idumalla Prema S. , Donner David B.
Published: April 2014

The type 1 TNFR (TNFR1) contains a death domain through which it interacts with other death-domain proteins to promote cellular responses. However, signaling through death-domain proteins does not explain how TNFR1 induces the tyrosine phosphorylation of intracellular proteins, which are important to cellular responses induced by TNFR1. In this study, we show that TNFR1 associates with Jak2, c-Src, and PI3K in various cell types. Jak2 and c-Src constitutively associate with and are constitutively active in the TNFR1 complex. Stimulation with TNF induces a time-dependent change in the level of Jak2, c-Src, and PI3K associated with TNFR1. The tyrosine kinase activity of the complex varies with the level of tyrosine kinase associated with TNFR1. TNFR1/c-Src plays a role in activating Akt, but not JNK or p38 MAPK, whereas TNFR1/Jak2 plays a role in activating p38 MAPK, JNK, and Akt. TNFR1/c-Src, but not TNFR1/Jak2, plays an obligate role in the activation of NF-κB by TNF, whereas TNFR1/Jak2, but not TNFR1/c-Src, plays an obligate role in the activation of STAT3. Activation of TNFR1 increased the expression of vascular endothelial growth factor, p21WAF1/CIP1, and manganese superoxide dismutase in MCF7 breast cancer cells, and increased the expression of CCl2/MCP-1 and IL-1β in THP-1 macrophages. Inhibitors of Jak2 and c-Src impaired the induction of each of these target proteins. These observations show that TNFR1 associates with and uses nonreceptor tyrosine kinases to engage signaling pathways, activate transcription factors, and modulate gene expression in cells.
Characterization of Ig Gene Somatic Hypermutation in the Absence of Activation-Induced Cytidine Deaminase
Authors: Longo Nancy S. , Satorius Colleen L. , Plebani Alessandro , Durandy Anne , Lipsky Peter E.
Published: April 2014

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase η (POL η). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL η-mediated mutations, 1470 H chain and 1313 κ- and λ-chain rearrangements from three AICDA−/− patients were analyzed. The Ig mutation frequency of all VH genes from AICDA−/− patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated VH sequences from AICDA−/− patients was 6.8-fold less than that of normal donors. AICDA−/− B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL η and uracil DNA-glycosylase activity. Similar results were observed in VL rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.
Selective Activation of Human Intestinal Mast Cells by Escherichia coli Hemolysin
Authors: Krämer Sigrid , Sellge Gernot , Lorentz Axel , Krueger Dagmar , Schemann Michael , Feilhauer Katharina , Gunzer Florian , Bischoff Stephan C.
Published: April 2014

Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that α-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly+ strains. Hly+ E. coli but not Hly− E. coli caused an increase in intracellular Ca2+ levels. Blocking of extracellular Ca2+ and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly+ E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly+ E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly+ E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.
Mechanisms Underlying Blockade of Allograft Acceptance by TLR Ligands
Authors: Porrett Paige M. , Yuan Xueli , LaRosa David F. , Walsh Patrick T. , Yang Jaeseok , Gao Wenda , Li Peiying , Zhang Jidong , Ansari Javeed M. , Hancock Wayne W. , Sayegh Mohamed H. , Koulmanda Maria , Strom Terry B. , Turka Laurence A.
Published: April 2014

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4+Foxp3+ T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4+Foxp3+ Tregs.
Macrophages Pulsed withStreptococcus pneumoniaeElicit a T Cell-Dependent Antibody Response upon Transfer into Naive Mice
Authors: Vasilevsky Sam , Colino Jesus , Puliaev Roman , Canaday David H. , Snapper Clifford M.
Published: April 2014

Macrophages are less effective than DC at priming naive CD4+ T cells, suggesting that DC are unique in initiating T cell-dependent Ab responses. We compared the ability of DC and macrophages, pulsed in vitro with Streptococcus pneumoniae, to elicit protein- and polysaccharide-specific Ig isotype production upon adoptive transfer into naive mice. S. pneumoniae-activated DC secreted more proinflammatory and anti-inflammatory cytokines, expressed higher levels of surface MHC class II and CD40, and presented S. pneumoniae or recombinant pneumococcal surface protein A (PspA) to a PspA-specific T hybridoma more efficiently than macrophages. However, upon adoptive transfer into naive mice, S. pneumoniae-pulsed macrophages elicited an IgM or IgG anti-PspA and anti-polysaccharide response comparable in serum titers and IgG isotype distribution to that induced by DC. The IgG anti-PspA response, in contrast to the IgG anti-polysaccharide, to S. pneumoniae-pulsed macrophages was T cell-dependent. S. pneumoniae-pulsed macrophages that were paraformaldehyde-fixed before transfer or lacking expression of MHC class II or CD40 were highly defective in eliciting an anti-PspA response, although the anti-polysaccharide response was largely unaffected. To our knowledge, these data are the first to indicate that macrophages can play an active role in the induction of a T cell-dependent humoral immune response in a naive host.
Polyclonal Adaptive Regulatory CD4 Cells That Can Reverse Type I Diabetes Become Oligoclonal Long-Term Protective Memory Cells
Authors: Godebu Elana , Summers-Torres Daphne , Lin Melissa M. , Baaten Bas J. G. , Bradley Linda M.
Published: April 2014

Type 1 diabetes is a CD4 cell-dependent disease that results from destruction of insulin-producing β cells in pancreatic islets. An ideal therapy would reverse diabetes shortly after onset when islet function in not yet fully ablated, and also prevent re-emergence of disease through the generation of memory cells that control the autoimmune response. In this study, we show that adaptive/induced polyclonal regulatory (TR) cells, which contain islet-reactive cells, fulfill these criteria in the NOD mouse model. CD4 cells induced to express FoxP3, IL-10, and TGF-β1 in response to TCR signaling and TGF-β1 can reverse diabetes with clinical restoration of prediabetic serum levels of IL-10. Unlike naturally occurring TR cells, these adaptive TR cells persist indefinitely (>1 year) as FoxP3+, CD25− memory cells that self-renew. Establishment of memory is accompanied by narrowing of the T cell repertoire to usage of a single TCR β-chain, Vβ11, implying selection by Ag. With islet-specific adaptive TR cells, we show that memory is functionally stable and transferable. Therefore, adaptive TR cells, which can be readily generated from normal CD4 populations and become focused by Ag with induction of memory, may provide a treatment and a vaccine for the long-term cure of diabetes making them attractive as immunotherapeutic agents.
Dual Signaling of MyD88 and TRIF Is Critical for Maximal TLR4-Induced Dendritic Cell Maturation
Authors: Shen Hua , Tesar Bethany M. , Walker Wendy E. , Goldstein Daniel R.
Published: April 2014

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.
Identification of an Evolutionarily Conserved Transcriptional Signature of CD8 Memory Differentiation That Is Shared by T and B Cells
Authors: Haining W. Nicholas , Ebert Benjamin L. , Subrmanian Aravind , Wherry E. John , Eichbaum Quentin , Evans John W. , Mak Raymond , Rivoli Stephen , Pretz Jennifer , Angelosanto Jill , Smutko John S. , Walker Bruce D. , Kaech Susan M. , Ahmed Rafi , Nadler Lee M. , Golub Todd R.
Published: April 2014

After Ag encounter, naive lymphocytes differentiate into populations of memory cells that share a common set of functions including faster response to Ag re-exposure and the ability to self-renew. However, memory lymphocytes in different lymphocyte lineages are functionally and phenotypically diverse. It is not known whether discrete populations of T and B cells use similar transcriptional programs during differentiation into the memory state. We used cross-species genomic analysis to examine the pattern of genes up-regulated during the differentiation of naive lymphocytes into memory cells in multiple populations of human CD4, CD8, and B cell lymphocytes as well as two mouse models of memory development. We identified and validated a signature of genes that was up-regulated in memory cells compared with naive cells in both human and mouse CD8 memory differentiation, suggesting marked evolutionary conservation of this transcriptional program. Surprisingly, this conserved CD8 differentiation signature was also up-regulated during memory differentiation in CD4 and B cell lineages. To validate the biologic significance of this signature, we showed that alterations in this signature of genes could distinguish between functional and exhausted CD8 T cells from a mouse model of chronic viral infection. Finally, we generated genome-wide microarray data from tetramer-sorted human T cells and showed profound differences in this differentiation signature between T cells specific for HIV and those specific for influenza. Thus, our data suggest that in addition to lineage-specific differentiation programs, T and B lymphocytes use a common transcriptional program during memory development that is disrupted in chronic viral infection.
Modulation of T Cell Activation by Stomatin-Like Protein 2
Authors: Kirchhof Mark G. , Chau Luan A. , Lemke Caitlin D. , Vardhana Santosh , Darlington Peter J. , Márquez Maria E. , Taylor Roy , Rizkalla Kamilia , Blanca Isaac , Dustin Michael L. , Madrenas Joaquín
Published: April 2014

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.
Identification of Residues in the Cμ4 Domain of Polymeric IgM Essential for Interaction withPlasmodium falciparumErythrocyte Membrane Protein 1 (PfEMP1)
Authors: Ghumra Ashfaq , Semblat Jean-Philippe , McIntosh Richard S. , Raza Ahmed , Rasmussen Ingunn B. , Braathen Ranveig , Johansen Finn-Eirik , Sandlie Inger , Mongini Patricia K. , Rowe J. Alexandra , Pleass Richard J.
Published: April 2014

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.
Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus
Authors: Achour Ikbel , Cavelier Patricia , Tichit Magali , Bouchier Christiane , Lafaye Pierre , Rougeon François
Published: April 2014

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as VHH and CHH, respectively, and which differ from conventional VH and CH counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both VHH and VH genes followed by a unique DH-JH cluster and C region genes, which include both CHH and CH genes. Although this general gene organization greatly resembles that of other typical mammalian Vn-Dn-Jn-Cn translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM+ stage, similar to the case for conventional IgG.
Genetic Analysis of SH2D4A, a Novel Adapter Protein Related to T Cell-Specific Adapter and Adapter Protein in Lymphocytes of Unknown Function, Reveals a Redundant Function in T Cells
Authors: Lapinski Philip E. , Oliver Jennifer A. , Kamen Lynn A. , Hughes Elizabeth D. , Saunders Thomas L. , King Philip D.
Published: April 2014

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.
Lipoproteins of Listeria monocytogenes Are Critical for Virulence and TLR2-Mediated Immune Activation
Authors: Machata Silke , Tchatalbachev Svetlin , Mohamed Walid , Jänsch Lothar , Hain Torsten , Chakraborty Trinad
Published: April 2014

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-κB activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-κB, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2−/− mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the Δlgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.
Type I IFNs Enhance Susceptibility toChlamydia muridarumLung Infection by Enhancing Apoptosis of Local Macrophages
Authors: Qiu Hongyu , Fan Yijun , Joyee Antony George , Wang Shuhe , Han Xiaobing , Bai Hong , Jiao Lei , Van Rooijen Nico , Yang Xi
Published: April 2014

Type I IFNs (IFNIs) have pleiotropic functions in regulating host innate and adaptive immune responses to pathogens. To elucidate the role of IFNIs in host resistance to chlamydial infection in vivo, we compared IFN-α/β receptor knockout (IFNAR−/−) and wild-type control mice in susceptibility to Chlamydia trachomatis mouse pneumonitis (Chlamydia muridarum) lung infection. We found that the IFNAR−/− mice were significantly more resistant to C. muridarum infection showing less bacterial burden and bodyweight loss, and milder pathological changes. However, IFN-γ response, which is believed to be critical in host defense against chlamydial infection, was similar between the wild-type and IFNAR−/− mice. More importantly, TUNEL analysis showed less macrophage apoptosis in IFNAR−/− mice, which was consistent with lower expressions of IFNI-induced apoptotic factors, TRAIL, Daxx, and PKR. Furthermore, depletion of lung macrophages with dichloromethylene diphosphonate-liposome significantly increased the susceptibility of the IFNAR−/− mice to C. muridarum, confirming the importance of macrophages. Overall, the data indicate that IFNIs play a promoting role in C. muridarum lung infection, largely through increase of local macrophage apoptosis.
CD8+ T Lymphocytes Control Murine Cytomegalovirus Replication in the Central Nervous System of Newborn Animals
Authors: Bantug Glenn R. B. , Cekinovic Djurdijca , Bradford Russell , Koontz Thad , Jonjic Stipan , Britt William J.
Published: April 2014

Human CMV infection of the neonatal CNS results in long-term neurologic sequelae. To define the pathogenesis of fetal human CMV CNS infections, we investigated mechanisms of virus clearance from the CNS of neonatal BALB/c mice infected with murine CMV (MCMV). Virus titers peaked in the CNS between postnatal days 10–14 and infectious virus was undetectable by postnatal day 21. Congruent with virus clearance was the recruitment of CD8+ T cells into the CNS. Depletion of CD8+ T cells resulted in death by postnatal day 15 in MCMV-infected animals and increased viral loads in the liver, spleen, and the CNS, suggesting an important role for these cells in the control of MCMV replication in the newborn brain. Examination of brain mononuclear cells revealed that CD8+ T cell infiltrates expressed high levels of CD69, CD44, and CD49d. IE1168-specific CD8+ T cells accumulated in the CNS and produced IFN-γ and TNF-α but not IL-2 following peptide stimulation. Moreover, adoptive transfer of brain mononuclear cells resulted in decreased virus burden in immunodepleted MCMV-infected syngeneic mice. Depletion of the CD8+ cell population following transfer eliminated control of virus replication. In summary, these results show that functionally mature virus-specific CD8+ T cells are recruited to the CNS in mice infected with MCMV as neonates.
Blimp-1 Attenuates Th1 Differentiation by Repression of ifng, tbx21, and bcl6 Gene Expression
Authors: Cimmino Luisa , Martins Gislaine A. , Liao Jerry , Magnusdottir Erna , Grunig Gabriele , Perez Rocio K. , Calame Kathryn L.
Published: April 2014

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-γ production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-γ, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.
Human Leukocyte Antigen Class I Genotypes in Relation to Heterosexual HIV Type 1 Transmission within Discordant Couples
Authors: Tang Jianming , Shao Wenshuo , Yoo Yun Joo , Brill Ilene , Mulenga Joseph , Allen Susan , Hunter Eric , Kaslow Richard A.
Published: April 2014

Differences in immune control of HIV-1 infection are often attributable to the highly variable HLA class I molecules that present viral epitopes to CTL. In our immunogenetic analyses of 429 HIV-1 discordant Zambian couples (infected index partners paired with cohabiting seronegative partners), several HLA class I variants in index partners were associated with contrasting rates and incidence of HIV-1 transmission within a 12-year study period. In particular, A*3601 on the A*36-Cw*04-B*53 haplotype was the most unfavorable marker of HIV-1 transmission by index partners, while Cw*1801 (primarily on the A*30-Cw*18-B*57 haplotype) was the most favorable, irrespective of the direction of transmission (male to female or female to male) and other commonly recognized cofactors of infection, including age and GUI. The same HLA markers were further associated with contrasting viral load levels in index partners, but they had no clear impact on HIV-1 acquisition by the seronegative partners. Thus, HLA class I gene products not only mediate HIV-1 pathogenesis and evolution but also influence heterosexual HIV-1 transmission.
Attenuated Inflammatory Responses in Hemochromatosis Reveal a Role for Iron in the Regulation of Macrophage Cytokine Translation
Authors: Wang Lijian , Johnson Erin E. , Shi Hai Ning , Walker W. Allan , Wessling-Resnick Marianne , Cherayil Bobby J.
Published: April 2014

Disturbances of iron homeostasis are associated with altered susceptibility to infectious disease, but the underlying molecular mechanisms are poorly understood. To study this phenomenon, we examined innate immunity to oral Salmonella infection in Hfe knockout (Hfe−/−) mice, a model of the human inherited disorder of iron metabolism type I hemochromatosis. Salmonella- and LPS-induced inflammatory responses were attenuated in the mutant animals, with less severe enterocolitis observed in vivo and reduced macrophage TNF-α and IL-6 secretion measured in vitro. The macrophage iron exporter ferroportin (FPN) was up-regulated in the Hfe−/− mice, and correspondingly, intramacrophage iron levels were lowered. Consistent with the functional importance of these changes, the abnormal cytokine production of the mutant macrophages could be reproduced in wild-type cells by iron chelation, and in a macrophage cell line by overexpression of FPN. The results of analyzing specific steps in the biosynthesis of TNF-α and IL-6, including intracellular concentrations, posttranslational stability and transcript levels, were consistent with reduced translation of cytokine mRNAs in Hfe−/− macrophages. Polyribosome profile analysis confirmed that elevated macrophage FPN expression and low intracellular iron impaired the translation of specific inflammatory cytokine transcripts. Our results provide molecular insight into immune function in type I hemochromatosis and other disorders of iron homeostasis, and reveal a novel role for iron in the regulation of the inflammatory response.