bluuebunny/crossref_metadata_2025 huggingface dataset:
PECAM-1 Ligation Negatively Regulates TLR4 Signaling in Macrophages
Authors: Rui Yuxiang , Liu Xingguang , Li Nan , Jiang Yingming , Chen Guoyou , Cao Xuetao , Wang Jianli
Published: April 2014

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-α, IL-6, and IFN-β production by inhibiting JNK, NF-κB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.
CCL2 Inhibits the Apoptosis Program Induced by Growth Factor Deprivation, Rescuing Functional T Cells
Authors: Diaz-Guerra Eva , Vernal Rolando , del Prete M. Julieta , Silva Augusto , Garcia-Sanz Jose A.
Published: April 2014

The precise mechanisms involved in the switch between the clonal expansion and contraction phases of a CD8+ T cell response remain to be fully elucidated. One of the mechanisms implicated in the contraction phase is cytokine deprivation, which triggers apoptosis in these cells. CCR2 chemokine receptor is up-regulated following IL-2 deprivation, and its ligand CCL2 plays an essential role preventing apoptosis induced by IL-2 withdrawal not only in CTLL2 cells, but also in mouse Ag-activated primary CD8+ T cells because it rescued functional CD8+ T cells from deprivation induced apoptosis, promoting proliferation in response to subsequent addition of IL-2 or to secondary antigenic challenges. Thus, up-regulation of the CCR2 upon growth factor withdrawal together with the protective effects of CCL2, represent a double-edged survival strategy, protecting cells from apoptosis and enabling them to migrate toward sites where Ag and/or growth factors are available.
Pivotal Roles of CD4+ Effector T cells in Mediating Agonistic Anti-GITR mAb-Induced-Immune Activation and Tumor Immunity in CT26 Tumors
Authors: Zhou Pengfei , L’italien Lawrence , Hodges Douglas , Schebye Xiao Min
Published: April 2014

Glucocorticoid-induced TNF receptor family related protein (GITR) is a member of the TNFR superfamily. Previous studies have shown that in vivo administration of a GITR agonistic Ab (DTA-1) is able to overcome tolerance and induce tumor rejection in several murine syngeneic tumor models. However, little is known about the in vivo targets and the mechanisms of how this tolerance is overcome in a tumor-bearing host, nor is much known about how the immune network is regulated to achieve this antitumor response. In this study, we demonstrate that the in vivo ligation of GITR on CD4+ effector T cells renders them refractory to suppression by regulatory T (Treg) cells in the CT26 tumor-bearing mouse. GITR engagement on Treg cells does not appear to directly abrogate their suppressive function; rather, it increases the expansion of Treg cells and promotes IL-10 production, a cytokine important for their suppressive function. Moreover, CD4+ effector T cells play a crucial role in mediating DTA-1-induced immune activation and expansion of CD8+, NK, and B cells in the tumor-draining lymph nodes. This includes increased CD69 expression on all of these subsets. In addition, NK and tumor-specific CD8+ T cells are generated that are cytolytic, which show increased intracellular IFN-γ production and CD107a mobilization, the latter a hallmark of cytolytic activities that lead to tumor killing.
Activation of Immature Hepatic NK Cells As Immunotherapy for Liver Metastatic Disease
Authors: Bahjat Keith S. , Prell Rodney A. , Allen Heather E. , Liu Weiqun , Lemmens Edward E. , Leong Meredith L. , Portnoy Daniel A. , Dubensky Thomas W. , Brockstedt Dirk G. , Giedlin Martin A.
Published: April 2014

NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.
Functional Outcome of B Cell Activation by Chromatin Immune Complex Engagement of the B Cell Receptor and TLR9
Authors: Busconi Liliana , Bauer Jason W. , Tumang Joseph R. , Laws Amy , Perkins-Mesires Kristin , Tabor Abigail S. , Lau Christina , Corley Ronald B. , Rothstein Thomas L. , Lund Frances E. , Behrens Timothy W. , Marshak-Rothstein Ann
Published: April 2014

We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-κB activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.
Sensitive Gene Expression Profiling of Human T Cell Subsets Reveals Parallel Post-Thymic Differentiation for CD4+ and CD8+ Lineages
Authors: Appay Victor , Bosio Andreas , Lokan Stefanie , Wiencek Yvonne , Biervert Christian , Küsters Daniel , Devevre Estelle , Speiser Daniel , Romero Pedro , Rufer Nathalie , Leyvraz Serge
Published: April 2014

The differentiation of CD4+ or CD8+ T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4+ or CD8+ T cell differentiation. Surprisingly, our data revealed that while CD4+ and CD8+ T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4+ and CD8+ T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.
Therapeutic Effect of Anthracene-Based Anticancer Agent Ethonafide in an Animal Model of Multiple Sclerosis
Authors: Piao Wen-Hua , Wong Rudy , Bai Xue-Feng , Huang Jianhua , Campagnolo Denise I. , Dorr Robert T. , Vollmer Timothy L. , Shi Fu-Dong
Published: April 2014

The side effects of cancer chemotherapeutic agents such as mitoxantrone (MIT) in multiple sclerosis (MS) patients justify the search for less toxic drugs. Ethonafide is an anthracene-based antineoplastic drug similar to MIT. With reference to MIT, we examined the effect of ethonafide on experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, an animal model of human MS. We demonstrated that ethonafide is effective in preventing development of EAE as well as in ameliorating the severity of EAE when disease is ongoing. In relatively higher dosages, the effects of ethonafide and MIT on EAE were identical, whereas in lower dosages, MIT seemed more effective. Therapeutic effects of ethonafide were associated with the initial reduction in cellular counts of CD3+, CD4+, CD8+, B220+, CD11b+, NK cells, and NKT cells, followed by recovery of these cells from the bone marrow. Interestingly, the recovered autoreactive T cells in ethonafide-treated animals have reduced capacity to expand and produce cytokines in response to myelin Ag stimulation. Furthermore, CD4+CD25+ regulatory T cells were relatively resistant to depletion and/or recovered faster than T effector cells. The ability of regulatory T cells to resist depletion and replenish quickly during cell ablation therapy may provide an opportunity to reprogram the immune system. Moreover, we provided evidences that ethonafide has less cardiac toxicity compared with MIT. The effectiveness and the low cardiotoxicity of ethonafide might make it a promising immunosuppressive agent for clinical use in treating MS patients.
Keratinocyte Growth Factor Induces Expansion of Murine Peripheral CD4+Foxp3+ Regulatory T Cells and Increases Their Thymic Output
Authors: Bruinsma Marieke , van Soest Peter L. , Leenen Pieter J. M. , Lambrecht Bart N. , Cupedo Tom , Löwenberg Bob , Cornelissen Jan J. , Braakman Eric
Published: April 2014

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4+Foxp3+ Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4+Foxp3+ Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4+Foxp3− T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4+ T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4+Foxp3+ Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.
Loss of IL-7 Receptor α on CD4+ T Cells Defines Terminally Differentiated B Cell-Helping Effector T Cells in a B Cell-Rich Lymphoid Tissue
Authors: Lim Hyung W. , Kim Chang H.
Published: April 2014

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Rα (CD127) and the common γ-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4+ T cells uniformly express CD127, the memory/effector (non-FOXP3+) CD4+ T cells are divided into CD127+ and CD127− cells. The CD127− T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127+ T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127− T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127+ memory T cells, CD127− T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127− T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.
Functional Role of P-Selectin Glycoprotein Ligand 1/P-Selectin Interaction in the Generation of Tolerogenic Dendritic Cells
Authors: Urzainqui Ana , Martínez del Hoyo Gloria , Lamana Amalia , de la Fuente Hortensia , Barreiro Olga , Olazabal Isabel M. , Martin Pilar , Wild Martin K. , Vestweber Dietmar , González-Amaro Roberto , Sánchez-Madrid Francisco
Published: April 2014

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-β genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4+CD25+Foxp3+ regulatory T cells, which expressed high levels of TGF-β1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4+CD25− T cells. Accordingly, we found that DCs from PSGL-1−/− mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.
Programmed Death 1 Ligand (PD-L) 1 and PD-L2 Limit Autoimmune Kidney Disease: Distinct Roles
Authors: Menke Julia , Lucas Julie A. , Zeller Geraldine C. , Keir Mary E. , Huang Xiao R. , Tsuboi Naotake , Mayadas Tanya N. , Lan Han Y. , Sharpe Arlene H. , Kelley Vicki R.
Published: April 2014

The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell- and macrophage (Mφ)-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1−/−), PD-L2 (PD-L2−/−), or both (PD-L1/L2−/−) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L−/− strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1−/− and PD-L2−/− mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68+ cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2−/− mice, but not PD-L1−/− mice. In contrast, we detected a rise in activated CD8+ T cells in PD-L1−/− mice, but not PD-L2−/− mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.
Continuous RANKL Inhibition in Osteoprotegerin Transgenic Mice and Rats Suppresses Bone Resorption without Impairing Lymphorganogenesis or Functional Immune Responses
Authors: Stolina Marina , Dwyer Denise , Ominsky Michael S. , Corbin Timothy , Van Gwyneth , Bolon Brad , Sarosi Ildiko , McCabe James , Zack Debra J. , Kostenuik Paul
Published: April 2014

Receptor activator of NF-κB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. The effects of RANKL are inhibited by a soluble decoy receptor called osteoprotegerin (OPG). Total ablation of RANKL in knockout mice leads to high bone mass, lymph node agenesis, and altered lymphocyte differentiation. In contrast, RANKL inhibition via OPG suppresses bone resorption but not inflammation in animal models of inflammatory bone loss. This suggests that the immune phenotype of RANKL knockout mice is related to total RANKL ablation. We hypothesized that prenatal RANKL inhibition via OPG overexpression would suppress bone resorption without influencing lymph node formation or subsequent immune responses. Transgenic rats were created, wherein soluble OPG was overexpressed by 100-fold vs wild type (WT) controls, by gestational day 11 (i.e., before lymph node formation). The structure of lymph nodes, spleen, and thymus of OPG-transgenic (OPG-Tg) animals were comparable to those of age-matched WT rats at gestational day 19 and in adulthood. The OPG-Tg neonates had elevated bone mass, confirming the prenatal inhibition of RANKL. Adult OPG-Tg rats and OPG-Tg mice exhibited no significant functional alterations relative to WT controls when subjected to immune challenges to test for altered innate and humoral responses (e.g., contact hypersensitivity to oxazolone, IgM response to Pneumovax, IgG response to keyhole limpet hemocyanin, or cytokine response to LPS). In summary, prenatal RANKL inhibition did not impair lymph node development, nor did continuous life-long RANKL inhibition cause obvious changes in innate or humoral immune responses in mice or rats.
Dendritic Cells Present Lytic Antigens and Maintain Function throughout Persistent γ-Herpesvirus Infection
Authors: Kupresanin Fiona , Chow Jonathan , Mount Adele , Smith Christopher M. , Stevenson Philip G. , Belz Gabrielle T.
Published: April 2014

The activation and maintenance of Ag-specific CD8+ T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8+ T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8+ T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8+ T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8+ T cells capable of limiting persistent viral infection.
Transgenic Expression of Bcl-xL or Bcl-2 by Murine B Cells Enhances the In Vivo Antipolysaccharide, but Not Antiprotein, Response to IntactStreptococcus pneumoniae
Authors: Chattopadhyay Gouri , Khan Abdul Q. , Sen Goutam , Colino Jesus , duBois Wendy , Rubtsov Anatoly , Torres Raul M. , Potter Michael , Snapper Clifford M.
Published: April 2014

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4+ T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-xL or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc−/− mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.
Role of CD30 in B/T Segregation in the Spleen
Authors: Bekiaris Vasileios , Withers David , Glanville Stephanie H. , McConnell Fiona M. , Parnell Sonia M. , Kim Mi-Yeon , Gaspal Fabrina M. C. , Jenkinson Eric , Sweet Clive , Anderson Graham , Lane Peter J. L.
Published: April 2014

In this report, we identify an important function for CD30 signals in the effective segregation of B and T lymphocytes in the murine spleen, additional to the recognized requirement for lymphotoxin signals. We show that CD30 signals are not required for transcription or protein expression of homeostatic chemokines, but CD30-deficient mice display impaired B/T segregation. This defect correlates with defective expression as detected by Abs of the transmembrane mucin-type protein podoplanin on T zone stroma, although expression at other sites is normal. Defective segregation is not intrinsic to CD30-deficient lymphocytes which segregate normally following transfer into RAG-deficient mice and significantly up-regulate the expression of both CCL21 and podoplanin on T zone stroma of RAG-deficient mice. During development, induction of expression of the CD30 ligand by lymphoid tissue inducer cells and podoplanin by T zone stroma are temporally linked, and the spatial association of these cells suggests that lymphoid tissue inducer cells are capable of providing the CD30 signals. Finally, we show that the appearance of podoplanin on T zone stroma in development is associated with B/T segregation of splenic white pulp areas. Our studies indicate that homeostatic chemokine expression by itself is not sufficient for B/T segregation and our data point to a significant role for podoplanin or molecules associated with podoplanin expressing stroma in the effective segregation of lymphocytes.
X-linked Inhibitor of Apoptosis Regulates T Cell Effector Function
Authors: Zehntner Simone P. , Bourbonnière Lyne , Moore Craig S. , Morris Stephen J. , Methot Danielle , St. Jean Martine , Lacasse Eric , Hebb Andrea L. O. , Robertson George S. , Durkin Jon , Gillard John W. , Owens Trevor
Published: April 2014

To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with experimental autoimmune encephalomyelitis, correlating with disease severity. Daily administration (10 mg/kg/day i.p.) of a 19-mer antisense oligonucleotide specific for XIAP (ASO-XIAP) abolished disease-associated XIAP mRNA and protein expression, and given from day of onset, alleviated experimental autoimmune encephalomyelitis and prevented relapses. Prophylactic treatment also reduced XIAP expression and prevented disease. Random or 5-base mismatched ASO was not inhibitory, and ASO-XIAP did not affect T cell priming. In ASO-XIAP-treated animals, infiltrating cells and inflammatory foci were dramatically reduced within the CNS. Flow cytometry showed an 88–93% reduction in T cells. The proportion of TUNEL+ apoptotic CD4+ T cells in the CNS was increased from <1.6 to 26% in ASO-XIAP-treated mice, and the proportion of Annexin V-positive CD4+ T cells in the CNS increased. Neurons and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function.
Role of STAT3 in CD4+CD25+FOXP3+ Regulatory Lymphocyte Generation: Implications in Graft-versus-Host Disease and Antitumor Immunity
Authors: Pallandre Jean-René , Brillard Emilie , Créhange Gilles , Radlovic Amandine , Remy-Martin Jean-Paul , Saas Philippe , Rohrlich Pierre-Simon , Pivot Xavier , Ling Xiang , Tiberghien Pierre , Borg Christophe
Published: April 2014

Immunological tolerance is maintained by specialized subsets of T cells including CD4+CD25+FOXP3+ regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4+CD25− T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4+CD25− T lymphocytes leads to STAT3 Tyr705 phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4+CD25− T cell conversion into Treg through costimulation with TCR/CD28 and TGF-β1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-γ. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4+CD25+ T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4+CD25+ T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4+ lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.
Peptide-Mediated Disruption of NFκB/NRF Interaction Inhibits IL-8 Gene Activation by IL-1 or Helicobacter pylori
Authors: Bartels Myriam , Schweda Aike Torben , Dreikhausen Ursula , Frank Ronald , Resch Klaus , Beil Winfried , Nourbakhsh Mahtab
Published: April 2014

Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NFκB repressing factor (NRF) and NFκB play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NFκB p65 in cells. We identified a synthetic peptide corresponding to aa 223–238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NFκB interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NFκB interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.
Tapasin Increases Efficiency of MHC I Assembly in the Endoplasmic Reticulum but Does Not Affect MHC I Stability at the Cell Surface
Authors: Everett Maya W. , Edidin Michael
Published: April 2014

Cell surface expression of MHC I molecules depends on the chaperone tapasin; how tapasin functions is not fully understood. We created three fluorescent tapasin constructs: wild-type tapasin, soluble tapasin, which does not interact with TAP, and N300 tapasin, which does not interact with MHC I. In contrast to earlier reports, all three constructs localize to the endoplasmic reticulum (ER), though soluble tapasin is more mobile than wild type and N300. Soluble tapasin does not increase MHC I surface levels to the same extent as wild type, which suggests that proximity to TAP is necessary for full tapasin function. N300 acts as a dominant-negative perhaps by blocking wild-type tapasin access to TAP. None of the constructs affects MHC I stability at the cell surface, although stability of ER resident MHC I is decreased in tapasin-negative cells. We propose that tapasin acts primarily to increase efficiency of assembly of MHC I within the ER.
Host Defense Peptide LL-37, in Synergy with Inflammatory Mediator IL-1β, Augments Immune Responses by Multiple Pathways
Authors: Yu Jie , Mookherjee Neeloffer , Wee Kathleen , Bowdish Dawn M. E. , Pistolic Jelena , Li Yuexin , Rehaume Linda , Hancock Robert E. W.
Published: April 2014

The human cathelicidin LL-37 is a cationic host defense peptide and serves as an important component of innate immunity. It has been demonstrated to be a multifunctional modulator of innate immune responses, although the mechanism(s) underlying this have not been well characterized. In this study, it was demonstrated that LL-37 synergistically enhanced the IL-1β-induced production of cytokines (IL-6, IL-10) and chemokines such as macrophage chemoattractant proteins (MCP-1, MCP-3) in human PBMC, indicating a role in enhancing certain innate immune responses. Similarly, LL-37 synergistically enhanced chemokine production in the presence of GM-CSF, but IFN-γ, IL-4, or IL-12 addition led to antagonism, indicating that the role of LL-37 in reinforcing specific immune responses is selective and restricted to particular endogenous immune mediators. The inhibition of G protein-coupled receptors and PI3K substantially suppressed the ability of IL-1β and LL-37 to synergistically enhance the production of chemokine MCP-3. Consistent with this, the combination of IL-1β and LL-37 enhanced the activation/phosphorylation of kinase Akt and the transcription factor CREB. The role of transcription factor NF-κB was revealed through the demonstration of enhanced phosphorylation of IκBα and the consequent nuclear translocation of NF-κB subunits p50 and p65, as well as the antagonistic effects of an inhibitor of IκBα phosphorylation. These results together indicate that the human host defense peptide LL-37 can work in synergy with the endogenous inflammatory mediator IL-1β to enhance the induction of specific inflammatory effectors by a complex mechanism involving multiple pathways, thus reinforcing certain innate immune responses.
Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo
Authors: Alaniz Robert C. , Deatherage Brooke L. , Lara Jimmie C. , Cookson Brad T.
Published: April 2014

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-α, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4+ T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4+ T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.
An IL-17F/A Heterodimer Protein Is Produced by Mouse Th17 Cells and Induces Airway Neutrophil Recruitment
Authors: Liang Spencer C. , Long Andrew J. , Bennett Frann , Whitters Matthew J. , Karim Riyez , Collins Mary , Goldman Samuel J. , Dunussi-Joannopoulos Kyriaki , Williams Cara M. M. , Wright Jill F. , Fouser Lynette A.
Published: April 2014

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4+ T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.
NF-κB-Inducing Kinase Regulates Cyclooxygenase 2 Gene Expression in Macrophages by Phosphorylation of PU.1
Authors: Azim Anser C. , Wang Xuerong , Park Gye Young , Sadikot Ruxana T. , Cao Hongmei , Mathew Biji , Atchison Michael , van Breemen Richard B. , Joo Myungsoo , Christman John W.
Published: April 2014

Selective expression of cyclooxygenase 2 (COX-2) by macrophages could have an important role in the pathobiology of inflammation. We reported a functional synergism between PU.1 and other transcription factors that contributes to COX-2 gene expression in macrophages. PU.1 resides in the nuclear compartment and is activated by phosphorylation to bind to cognate DNA elements containing a 5′-GGAA/T-3′ motif, but the involved kinase has not been discovered. We tested the hypothesis that NF-κB-inducing kinase (NIK) regulates COX-2 gene expression in macrophages through inducible phosphorylation of PU.1. Our initial experiments showed an in vitro protein-protein binding interaction between myc-NIK and GST-PU.1. Purified myc-NIK had a strong in vitro kinase activity for purified GST-PU.1, and this activity and production of COX-2 protein is blocked by treatment with a nonspecific kinase inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an ∼50% decrease in COX-2 protein production and decreased generation of PGD2, and this was correlated with decreased binding of activated PU.1 to the COX-2 promoter in response to treatment with endotoxin. These findings suggest a novel role for NIK in mediating COX-2 gene expression in endotoxin-treated macrophages by a mechanism that involves phosphorylation of PU.1.
Activation of Protein Kinase D1 in Mast Cells in Response to Innate, Adaptive, and Growth Factor Signals
Authors: Murphy Thomas R. , Legere Henry J. , Katz Howard R.
Published: April 2014

Little is known about the serine/threonine kinase protein kinase D (PKD)1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of this enzyme to mast cell activation induced by diverse agonists. Mouse bone marrow-derived mast cells (BMMC) contained both PKD1 mRNA and immunoreactive PKD1 protein. Activation of BMMC through TLR2, Kit, or FcεRI with Pam3CSK4 (palmitoyl-3-cysteine-serine-lysine-4), stem cell factor (SCF), and cross-linked IgE, respectively, induced activation of PKD1, as determined by immunochemical detection of autophosphorylation. Activation of PKD1 was inhibited by the combined PKD1 and protein kinase C (PKC) inhibitor Gö 6976 but not by broad-spectrum PKC inhibitors, including bisindolylmaleimide (Bim) I. Pam3CSK4 and SCF also induced phosphorylation of heat shock protein 27, a known substrate of PKD1, which was also inhibited by Gö 6976 but not Bim I in BMMC. This pattern also extended to activation-induced increases in mRNA encoding the chemokine CCL2 (MCP-1) and release of the protein. In contrast, both pharmacologic agents inhibited exocytosis of β-hexosaminidase induced by SCF or cross-linked IgE. Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. In contrast with certain other cell types, activation of PKD1 in BMMC is largely independent of PKC activation. Furthermore, our findings also indicate that PKD1 preferentially influences transcription-dependent production of CCL2, whereas PKC predominantly regulates the rapid exocytosis of preformed secretory granule mediators.
Functional Expression of H4 Histamine Receptor in Human Natural Killer Cells, Monocytes, and Dendritic Cells
Authors: Damaj Bassam B. , Becerra Cecilia Barrena , Esber Henry J. , Wen Ying , Maghazachi Azzam A.
Published: April 2014

We describe here the protein expression of H4 histamine receptor in cells of the innate immune system, which include NK cells, monocytes, and dendritic cells (DCs). Anti-H4R specifically stained permeabilized NK cells, THP-1 clone 15 monocytes, and DCs. This binding was inhibited by incubating anti-H4R Ab with its corresponding peptide. Histamine induced NK cells, THP-1 clone 15 cells, and DCs chemotaxis with high affinity. The ED50 chemotactic effect was 5 nM, 6.8 nM, and 2.7 nM for NK cells, THP-1 clone 15 cells, and DCs, respectively. Thioperamide, an H3R/H4R antagonist, inhibited histamine-induced chemotaxis in all these cells. However, histamine failed to induce the mobilization of [Ca2+]i in NK cells and THP-1 clone 15 cells, but it induced calcium fluxes in DCs. Using a new method of detecting NK cell-mediated cytolysis, it was observed that NK cells efficiently lysed K562 target cells and that histamine did not affect this NK cell activity. In summary, this is the first demonstration of the protein expression of H4 receptor in NK cells. Also, the results of the chemotactic effects of histamine on NK cells and THP-1 cells are novel. These results may shed some light on the colocalization of cells of innate immune arm at sites of inflammation. They are also important for developing drugs that target H4R for the treatment of various disorders, such as autoimmune and immunodeficient diseases.
Effective Proliferation of Human Regulatory T Cells Requires a Strong Costimulatory CD28 Signal That Cannot Be Substituted by IL-2
Authors: Hombach Andreas A. , Kofler David , Hombach Anja , Rappl Gunter , Abken Hinrich
Published: April 2014

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4+C25+ Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4+CD25− T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4+CD25− T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.
TWEAK/Fn14 Interactions Are Instrumental in the Pathogenesis of Nephritis in the Chronic Graft-versus-Host Model of Systemic Lupus erythematosus
Authors: Zhao Zeguo , Burkly Linda C. , Campbell Sean , Schwartz Noa , Molano Alberto , Choudhury Arpita , Eisenberg Robert A. , Michaelson Jennifer S. , Putterman Chaim
Published: April 2014

TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a prominent inducer of proinflammatory cytokines in vitro and in vivo. We previously found that kidney cells display the TWEAK receptor Fn14, and that TWEAK stimulation of mesangial cells and podocytes induces a potent proinflammatory response. Several of the cytokines up-regulated in the kidney in response to TWEAK are instrumental in Lupus nephritis; we therefore hypothesized that TWEAK/Fn14 interactions may be important in the cascade(s) leading to renal damage in systemic Lupus erythematosus. In this study, we analyzed the effects of Fn14 deficiency in the chronic graft-vs-host model of SLE, and the benefits of treatment with an anti-TWEAK mAb in this mouse model. We found that anti-nuclear Ab titers were no different between C57BL/6 Fn14 wild-type and deficient mice injected with alloreactive bm12 splenocytes. However, kidney disease was significantly less severe in Fn14 knockout mice. Furthermore, kidney IgG deposition, IL-6, MCP-1, RANTES, and IP-10, as well as macrophage infiltration, were significantly decreased in Fn14-deficient mice with induced lupus. Similarly, mice with induced Lupus treated with an anti-TWEAK neutralizing mAb had significantly diminished kidney expression of IL-6, MCP-1, IL-10, as well as proteinuria, but similar autoantibody titers, as compared with control-treated mice. We conclude that TWEAK is an important mediator of kidney damage that acts by promoting local inflammatory events, but without impacting adaptive immunity in this experimental LN model. Thus, TWEAK blockade may be a novel therapeutic approach to reduce renal damage in SLE.
COPs and POPs: Modulators of Inflammasome Activity
Authors: Stehlik Christian , Dorfleutner Andrea
Published: April 2014

Inflammasomes represent molecular platforms for the activation of inflammatory caspases and are essential for processing and secretion of the inflammatory cytokines IL-1β and IL-18. Multiple key proteins of inflammasomes contain caspase recruitment domains (CARDs) or pyrin domains (PYDs). Dissecting CARD- and PYD-mediated interactions substantially improved our understanding of the mechanisms by which these protein platforms are activated and emphasized their essential role during the inflammatory cytokine response. However, their precise regulation is still poorly understood. A family of small proteins that are composed of either a CARD or a PYD only emerged as important inflammasome regulators. These CARD-only proteins (COPs) and PYD-only proteins (POPs) function as endogenous dominant negative proteins that modulate the activity of inflammasomes in response to pathogen infection and tissue destruction. In this review we will summarize the most recent advances in the regulation of inflammasomes and highlight their importance for immunity and inflammatory disease.
TLR Ligands Differentially Modulate T Cell Responses to Acute and Chronic Antigen Presentation
Authors: Singh Nevil J. , Cox Maureen , Schwartz Ronald H.
Published: April 2014

The outcome of peripheral T cell activation is thought to be largely determined by the context in which the cognate Ag is initially presented. In this framework, microbial products that can activate APCs via TLRs are considered critical in converting an otherwise tolerogenic context to an immunogenic one. We examine this idea using a model system where naive T cells are stimulated in the periphery by a persistent self Ag. The addition of multiple TLR ligands to this context, acutely or chronically, failed to significantly alter the tolerogenic phenotype in the responding T cells. This contrasts with the ability of such adjuvants to improve T cell responses to soluble peptide immunizations. We reconcile this difference by revealing a hitherto poorly appreciated property of TLR ligands, which extends the duration of soluble Ag presentation in vivo by an additional two to three days. Finally, we could replace the requirement for TLR-mediated APC activation in soluble-Ag-induced T cell expansion and differentiation, by maintaining the Ag depot in vivo using repeated immunizations. These data suggest a novel process by which TLR ligands modulate T cell responses to acute Ags, without disrupting the induction of tolerance to persistent self Ags.