bluuebunny/crossref_metadata_2025 huggingface dataset:
Blimp-1 Induced by IL-4 Plays a Critical Role in Suppressing IL-2 Production in Activated CD4 T Cells
Authors: Wang Lu , van Panhuys Nicholas , Hu-Li Jane , Kim Sohee , Le Gros Graham , Min Booki
Published: April 2014

Although an inhibitory function of IL-4 in CD4 T cell IL-2 production has long been recognized, a mechanism mediating the inhibition remains unclear. In this study we demonstrate that IL-4 displays a potent suppressive function in IL-2 production of activated CD4 T cells through STAT6. IL-4-induced IL-2 suppression required IL-2 because IL-2 neutralization restored the production of IL-2 even in the presence of IL-4. In vivo, enhanced IL-2 production was found in nematode-infected IL-4- or STAT6-deficient animals, whereas immunization in the presence of IL-4 substantially diminished IL-2 production by Ag-specific CD4 T cells. IL-2 mRNA expression was reduced when T cells were stimulated in the presence of IL-4, whereas IL-2 mRNA decay was unaltered, suggesting that IL-4 mediates the suppression at a transcriptional level. Blimp-1 induced by IL-4 stimulation in activated CD4 T cells was found to be necessary to mediate the IL-2 inhibition as IL-4-mediated IL-2 suppression was less pronounced in activated CD4 T cells deficient in Blimp-1. Taken together, our results demonstrate a potential link with IL-4, Blimp-1, and IL-2 production, suggesting that Blimp-1 may play an important role in controlling IL-2 production in activated T cells and in adaptive T cell immunity.
CD4 T Cell-Mediated Rejection of Cardiac Allografts in B Cell-Deficient Mice
Authors: Nozaki Taiji , Rosenblum Joshua M. , Ishii Daisuke , Tanabe Kazunari , Fairchild Robert L.
Published: April 2014

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.μMT−/− recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.μMT−/− CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.μMT−/− spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.μMT−/− mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.μMT−/− CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.μMT−/− mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.
Maintenance of Thymic Epithelial Phenotype Requires Extrinsic Signals in Mouse and Zebrafish
Authors: Soza-Ried Cristian , Bleul Conrad C. , Schorpp Michael , Boehm Thomas
Published: April 2014

Thymopoiesis strictly depends on proper differentiation of the thymic epithelial anlage. Differentiation of thymic epithelial cells (TECs) is controlled by the Foxn1 transcription factor. The in vivo signals initiating and maintaining Foxn1 expression in the future thymus anlage are unknown. In the mouse, bone morphogenetic protein (BMP) signaling is required for the maintenance of Foxn1 expression in TECs, as shown here by lineage tracing using a Foxn1-driven Cre transgene. Loss of Foxn1 expression after BMP inhibition reverts TECs to a basal state of pharyngeal epithelium unable to support T cell development; it does not divert them into a parathyroid fate. In zebrafish larvae, BMP inhibition likewise causes loss of foxn1 expression in the thymic anlage and subsequent impairment of thymopoiesis. These results indicate an evolutionarily conserved role of BMP signaling in the maintenance of Foxn1 expression.
Priming of a Novel Subset of CD28+ Rapidly Expanding High-Avidity Effector Memory CTL by Post Maturation Electroporation-CD40L Dendritic Cells Is IL-12 Dependent
Authors: DeBenedette Mark A. , Calderhead David M. , Ketteringham Helen , Gamble Alicia H. , Horvatinovich Joe M. , Tcherepanova Irina Y. , Nicolette Charles A. , Healey Don G.
Published: April 2014

Dendritic cell (DC)-based immunotherapeutics must induce robust CTL capable of killing tumor or virally infected cells in vivo. In this study, we show that RNA electroporated post maturation and coelectroporated with CD40L mRNA (post maturation electroporation (PME)-CD40L DC) generate high-avidity CTL in vitro that lyse naturally processed and presented tumor Ag. Unlike cytokine mixture-matured DC which induce predominantly nonproliferative effector memory CD45RA+ CTL, PME-CD40L DC prime a novel subset of Ag-specific CTL that can be expanded to large numbers upon sequential DC stimulation in vitro. We have defined these cells as rapidly expanding high-avidity (REHA) CTL based on: 1) the maintenance of CD28 expression, 2) production of high levels of IFN-γ and IL-2 in response to Ag, and 3) the demonstration of high-avidity TCR that exhibit strong cytolytic activity toward limiting amounts of native Ag. We demonstrate that induction of REHA CTL is dependent at least in part on the production of IL-12. Interestingly, neutralization of IL-12 did not effect cytolytic activity of REHA CTL when Ag is not limiting, but did result in lower TCR avidity of Ag-reactive CTL. These results suggest that PME-CD40L DC are uniquely capable of delivering the complex array of signals needed to generate stable CD28+ REHA CTL, which if generated in vivo may have significant clinical benefit for the treatment of infectious disease and cancer.
Human Memory B Lymphocyte Subsets Fulfill Distinct Roles in the Anti-Polysaccharide and Anti-Protein Immune Response
Authors: Moens Leen , Wuyts Margareta , Meyts Isabelle , De Boeck Kris , Bossuyt Xavier
Published: April 2014

There is controversy on the role of IgM memory and switched memory B lymphocytes in the Ab response to T cell-independent and T cell-dependent Ags. We transplanted SCID/SCID mice with human B lymphocyte subsets and immunized them with heat-inactivated Streptococcus pneumoniae or with a pneumococcal vaccine. Inactivated S. pneumoniae and soluble pneumococcal capsular polysaccharides elicited an IgM anti-polysaccharide and anti-protein Ab response from IgM memory B lymphocytes and an IgG anti-polysaccharide and anti-protein response from switched memory B lymphocytes. In addition to the IgM Ab response, IgM memory B cells elicited an IgG anti-polysaccharide and anti-protein Ab response after immunization with inactivated S. pneumoniae or soluble pneumococcal capsular polysaccharides. In conclusion, our findings provide evidence for a versatile role of IgM memory B cells in T-independent and T-dependent immune responses.
Activation of Human Peripheral IgM+ B Cells Is Transiently Inhibited by BCR-Independent Aggregation of FcγRIIB
Authors: Fournier Emilie M. , Sibéril Sophie , Costes Anne , Varin Audrey , Fridman Wolf-Herman , Teillaud Jean-Luc , Sautès-Fridman Catherine
Published: April 2014

Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG+ B cells and plasma cells by binding to FcγRIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of FcγRIIB on human peripheral IgM+ B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human FcγRIIB (IIA1.6-FcγRIIB), we also show that the unique aggregation of human FcγRIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by FcγRIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-FcγRIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated FcγRIIB (FcγRIIBY292G), suggesting that ITIM phosphorylation is necessary for FcγRIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of FcγRIIB on human peripheral IgM+ B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that FcγRIIB could negatively regulate IgM+ B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM+ B cells.
Cyclooxygenase Inhibition during Allergic Sensitization Increases STAT6-Independent Primary and Memory Th2 Responses
Authors: Zhou Weisong , Newcomb Dawn C. , Moore Martin L. , Goleniewska Kasia , O'Neal Jamye F. , Peebles R. Stokes
Published: April 2014

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.
XCL1 Enhances Regulatory Activities of CD4+CD25highCD127low/− T Cells in Human Allergic Asthma
Authors: Nguyen Khoa D. , Fohner Alison , Booker Jerome D. , Dong Chen , Krensky Alan M. , Nadeau Kari C.
Published: April 2014

Chemokine-mediated recruitment of regulatory cell subsets to the airway during inflammation and enhancement of their activities are potential strategies for therapeutic development in allergic asthma (AA). In this study, we aim to explore the role of XCL1, a chemokine associated with immune suppression and allergy, on CD4+CD25highCD127low/− regulatory T cell (Treg) function in AA. Flow cytometry and PCR analysis showed a reduction in XCL1 and XCR1 expression in AA Treg compared with healthy control and nonallergic asthmatic counterparts. This reduction in XCL1 expression was associated with the suboptimal regulatory function of Treg in AA. Interestingly, incubation with recombinant human XCL1 significantly increased Treg-mediated suppression and cytotoxicity by up-regulating expression of XCL1 and chief effector molecules of Treg function. Altogether, these results suggest an association between dysregulated XCL1 expression and reduced Treg activities in AA, as well as a potential role of XCL1 in reversing defective Treg function in the disease.
The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation
Authors: Chen Wei , Liang Xueqing , Peterson Amanda J. , Munn David H. , Blazar Bruce R.
Published: April 2014

Human plasmacytoid dendritic cells (PDCs) can drive naive, allogeneic CD4+CD25− T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the intracellular mechanism or mechanisms underlying PDC-induced Treg generation are unknown. In this study, we show that human PDCs express high levels of IDO, an intracellular enzyme that catabolizes tryptophan degradation. Triggering of TLR 9 with CpG oligodeoxynucleotides activates PDCs to up-regulate surface expression of B7 ligands and HLA-DR Ag, but also significantly increases the expression of IDO and results in the generation of inducible Tregs from CD4+CD25− T cells with potent suppressor cell function. Blocking IDO activity with the pharmacologic inhibitor 1-methyl-d-tryptophan significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine, the immediate downstream metabolite of tryptophan, bypasses the 1-methyl-d-tryptophan effect and restores PDC-driven Treg generation. Our results demonstrate that the IDO pathway is essential for PDC-driven Treg generation from CD4+CD25− T cells and implicate the generation of kynurenine pathway metabolites as the critical mediator of this process.
Complexes of Two Cohorts of CLIP Peptides and HLA-DQ2 of the Autoimmune DR3-DQ2 Haplotype Are Poor Substrates for HLA-DM
Authors: Fallang Lars-Egil , Roh Sujin , Holm Anders , Bergseng Elin , Yoon Taejin , Fleckenstein Burkhard , Bandyopadhyay Arunima , Mellins Elizabeth D. , Sollid Ludvig M.
Published: April 2014

Atypical invariant chain (Ii) CLIP fragments (CLIP2) have been found in association with HLA-DQ2 (DQ2) purified from cell lysates. We mapped the binding register of CLIP2 (Ii 96–104) to DQ2 and found proline at the P1 position, in contrast to the canonical CLIP1 (Ii 83–101) register with methionine at P1. CLIP1/2 peptides are the predominant peptide species, even for DQ2 from HLA-DM (DM)-expressing cells. We hypothesized that DQ2-CLIP1/2 might be poor substrates for DM. We measured DM-mediated exchange of CLIP and other peptides for high-affinity indicator peptides and found it is inefficient for DQ2. DM-DQ-binding and DM chaperone effects on conformation and levels of DQ are also reduced for DQ2, compared with DQ1. We suggest that the unusual interaction of DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2.
NKG2D Is Critical for NK Cell Activation in Host Defense against Pseudomonas aeruginosa Respiratory Infection
Authors: Wesselkamper Scott C. , Eppert Bryan L. , Motz Gregory T. , Lau Gee W. , Hassett Daniel J. , Borchers Michael T.
Published: April 2014

Pseudomonas aeruginosa is a major cause of nosocomial respiratory infections. The eradication of P. aeruginosa from the lung involves the orchestrated actions of the pulmonary epithelium and both resident and recruited immune cells. The NKG2D receptor is constitutively expressed on the surface of circulating and tissue-resident NK cells (and other cytotoxic lymphocytes), and is capable of controlling NK cell activation and production of cytokines, such as IFN-γ via interactions with ligands expressed on the surface of stressed cells. Previously, we demonstrated that NKG2D mediates pulmonary clearance of P. aeruginosa. In the present study, we investigated the cellular and molecular mechanisms of NKG2D-mediated clearance of P. aeruginosa using a novel transgenic mouse model of doxycycline-inducible conditional expression of NKG2D ligands (retinoic acid early transcript 1, α) in pulmonary epithelial cells. NKG2D ligand expression in this model increased pulmonary clearance, cellular phagocytosis, and survival following P. aeruginosa respiratory infection. Additionally, NK cell sensitivity to ex vivo LPS stimulation was greater in lung cells isolated from naive transgenic mice administered doxycycline. We also showed that NK cells are the primary source of lymphocyte-derived IFN-γ in response to P. aeruginosa respiratory infection. Significantly, we demonstrated that NKG2D is critical to the nonredundant IFN-γ production by pulmonary NK cells following acute P. aeruginosa infection. These results represent the principal report of NKG2D-mediated activation of lung NK cells following respiratory infection with an opportunistic pathogen and further establish the importance of NKG2D in the host response against P. aeruginosa respiratory infection.
The Efficacy of T Cell-Mediated Immune Responses Is Reduced by the Envelope Protein of the Chimeric HIV-1/SIV-KB9 Virus In Vivo
Authors: Stevceva Liljana , Yoon Victor , Carville Angela , Pacheco Beatriz , Santosuosso Michael , Korioth-Schmitz Birgit , Mansfield Keith , Poznansky Mark C.
Published: April 2014

Gp120 is a critical component of the envelope of HIV-1. Its role in viral entry is well described. In view of its position on the viral envelope, gp120 is a part of the retrovirus that immune cells encounter first and has the potential to influence antiretroviral immune responses. We propose that high levels of gp120 are present in tissues and may contribute to the failure of the immune system to fully control and ultimately clear the virus. Herein, we show for the first time that lymphoid tissues from acutely HIV-1/SIV (SHIV)-KB9-infected macaques contain deposits of gp120 at concentrations that are high enough to induce suppressive effects on T cells, thus negatively regulating the antiviral CTL response and contributing to virus survival and persistence. We also demonstrate that SHIV-KB9 gp120 influences functional T cell responses during SHIV infection in a manner that suppresses degranulation and cytokine secretion by CTLs. Finally, we show that regulatory T cells accumulate in lymphoid tissues during acute infection and that they respond to gp120 by producing TGFβ, a known suppressant of cytotoxic T cell activity. These findings have significant implications for our understanding of the contribution of non-entry-related functions of HIV-1 gp120 to the pathogenesis of HIV/AIDS.
Dynamics of Human Respiratory Virus-Specific CD8+ T Cell Responses in Blood and Airways during Episodes of Common Cold
Authors: Heidema Jojanneke , Rossen John W. A. , Lukens Michaël V. , Ketel Marianne S. , Scheltens Eva , Kranendonk Mariette E. G. , van Maren Wendy W. C. , van Loon Anton M. , Otten Henny G. , Kimpen Jan L. L. , van Bleek Grada M.
Published: April 2014

We determined the dynamics of CD8+ T cells specific for influenza virus and respiratory syncytial virus in blood and tracheostoma aspirates of children during the course of respiratory infections. We showed that during localized respiratory infections the ratio of activated effector CD8+ T cells to resting memory/naive CD8+ T cells in peripheral blood increased significantly. Furthermore, the number of effector/memory T cells specific for respiratory viruses declined in blood and increased in the airways, suggesting that these T cells redistributed from blood to airways. T cells specific for the infecting virus were present in the airways for longer periods at increased levels than nonspecifically recruited bystander T cells. After clearance of the infection, the ratio of resting memory and naive CD8+ T cells normalized in peripheral blood and also memory T cell numbers specific for unrelated viruses that declined during the infection due to bystander recruitment were restored. Taken together, these results showed a significant systemic T cell response during relatively mild secondary infections and extensive dynamics of virus-specific and nonspecific Ag-experienced T cells.
A Phosphatidylserine Species Inhibits a Range of TLR- but Not IL-1β-Induced Inflammatory Responses by Disruption of Membrane Microdomains
Authors: Parker Lisa C. , Prestwich Elizabeth C. , Ward Jon R. , Smythe Elizabeth , Berry Anthony , Triantafilou Martha , Triantafilou Kathy , Sabroe Ian
Published: April 2014

TLRs detect conserved molecular patterns that are unique to microbes, enabling tailored responses to invading pathogens and modulating a multitude of immunopathological conditions. We investigated the ability of a naturally occurring stearoyl-arachidonoyl form of phosphatidylserine (SAPS) to inhibit the proinflammatory effects of TLR agonists in models of inflammation investigating the interaction of leukocytes with epithelial and endothelial cells. The responses to LPS of both epithelial and endothelial cells were highly amplified in the presence of PBMCs. Coincubation with SAPS markedly inhibited activation of cocultures by LPS, principally through inhibition of the TLR4 signaling pathway in PBMCs; however, this was not through downmodulation of TLR4 or coreceptor expression, nor was IL-1β-induced cytokine release affected. SAPS also impaired Pam3CSK4 (TLR2/1), Gardiquimod (TLR7/8), and Streptococcus pneumoniae-induced cytokine release, but had only modest effects on poly(I:C) (TLR3)-induced responses. Fluorescence resonance energy transfer analysis of molecular associations revealed that SAPS disrupted the association of both TLR4 and TLR2 with their respective membrane partners that are required for signaling. Thus, our data reinforce the existence and importance of cooperative networks of TLRs, tissue cells, and leukocytes in mediating innate immunity, and identify a novel disrupter of membrane microdomains, revealing the dependence of TLR signaling on localization within these domains.
Prostaglandin E2 Signals Monocyte/Macrophage Survival to Peroxynitrite via Protein Kinase A Converging in Bad Phosphorylation with the Protein Kinase Cα-Dependent Pathway Driven by 5-Hydroxyeicosatetraenoic Acid
Authors: Tommasini Ilaria , Cerioni Liana , Palomba Letizia , Cantoni Orazio
Published: April 2014

Monocytes/macrophages committed to death by peroxynitrite nevertheless survive with a signaling response promoting Bad phosphorylation, as well as its cytosolic localization, via upstream activation of cytosolic phospholipase A2, 5-lipoxygenase, and protein kinase Cα. We now report evidence for an alternative mechanism converging in Bad phosphorylation when the expression/activity of the above enzymes are suppressed. Under these conditions, also associated with peroxynitrite-dependent severe inhibition of Akt, an additional Bad kinase, Bad dephosphorylation promoted its accumulation in the mitochondria and a prompt lethal response. PGE2 prevented toxicity via EP2 receptor-mediated protein kinase A-dependent Bad phosphorylation. This notion was established in U937 cells by the following criteria: 1) there was a strong correlation between survival and cAMP accumulation, both in the absence and presence of phosphodiesterase inhibitors; 2) direct activation of adenylyl cyclase afforded cytoprotection; and 3) PGE2 promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP2 receptor agonists, and prevented by EP2 receptor antagonists or protein kinase A inhibitors. Finally, selected experiments performed in human monocytes/macrophages and in rat peritoneal macrophages indicated that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage to both exogenous and endogenous peroxynitrite. The notion that two different pathways mediated by downstream products of arachidonic acid metabolism converge in Bad phosphorylation emphasizes the relevance of this strategy for the regulation of macrophage survival to peroxynitrite at the inflammatory sites.
The Vitamin D3/Hox-A10 Pathway Supports MafB Function during the Monocyte Differentiation of Human CD34+ Hemopoietic Progenitors
Authors: Gemelli Claudia , Orlandi Claudia , Marani Tommaso Zanocco , Martello Andrea , Vignudelli Tatiana , Ferrari Francesco , Montanari Monica , Parenti Sandra , Testa Anna , Grande Alexis , Ferrari Sergio
Published: April 2014

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.
Macrophage Migration Inhibitory Factor Plays a Role in the Regulation of Microfold (M) Cell-Mediated Transport in the Gut
Authors: Man Angela L. , Lodi Federica , Bertelli Eugenio , Regoli Mari , Pin Carmen , Mulholland Francis , Satoskar Abhay R. , Taussig Michael J. , Nicoletti Claudio
Published: April 2014

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer’s patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c+ cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer’s patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF−/− mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.
Neutrophil-mediated Activation of Epithelial Protease-Activated Receptors-1 and -2 Regulates Barrier Function and Transepithelial Migration
Authors: Chin Alex C. , Lee Winston Y. , Nusrat Asma , Vergnolle Nathalie , Parkos Charles A.
Published: April 2014

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn’s disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.
IL-23 Inhibits Osteoclastogenesis Indirectly through Lymphocytes and Is Required for the Maintenance of Bone Mass in Mice
Authors: Quinn Julian M. W. , Sims Natalie A. , Saleh Hasnawati , Mirosa Danijela , Thompson Keith , Bouralexis Stelios , Walker Emma C. , Martin T. John , Gillespie Matthew T.
Published: April 2014

IL-23 stimulates the differentiation and function of the Th17 subset of CD4+ T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4+ T lymphocyte-dependent manner. When sufficiently enriched, γδ T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19−/−) had ∼30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19−/− mice also had reduced BV/TV. IL-23p19−/− mice had no detectable osteoclast defect in trabecular bone but IL-23p19−/− had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.
Tolerance without Clonal Expansion: Self-Antigen-Expressing B Cells Program Self-Reactive T Cells for Future Deletion
Authors: Frommer Friederike , Heinen Tobias J. A. J. , Wunderlich F. Thomas , Yogev Nir , Buch Thorsten , Roers Axel , Bettelli Estelle , Müller Werner , Anderton Stephen M. , Waisman Ari
Published: April 2014

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.
B Cells from Patients with Graves’ Disease Aberrantly Express the IGF-1 Receptor: Implications for Disease Pathogenesis
Authors: Douglas Raymond S. , Naik Vibharavi , Hwang Catherine J. , Afifiyan Nikoo F. , Gianoukakis Andrew G. , Sand Daniel , Kamat Shweta , Smith Terry J.
Published: April 2014

Graves’ disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R+ memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R+ phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R+ B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.
Periodontitis Associates with a Type 1 IFN Signature in Peripheral Blood Neutrophils
Authors: Wright Helen J. , Matthews John B. , Chapple Iain L. C. , Ling-Mountford Nic , Cooper Paul R.
Published: April 2014

Peripheral blood neutrophils from periodontitis patients exhibit a hyperreactive and hyperactive phenotype (collectively termed hyperresponsivity) in terms of production of reactive oxygen species (ROS). The molecular basis for this phenomenon, however, has yet to be determined. Our objectives were to identify genes differentially expressed in hyperresponsive peripheral blood neutrophils from chronic periodontitis patients relative to periodontally healthy controls and use these data to identify potential contributory pathways to the hyperresponsive neutrophil phenotype. Using microarray technology we demonstrated differential expression of 163 genes (149 increased, 14 decreased) representing a range of ontological classes. There was increased expression of a significant number of IFN-stimulated genes (ISG). RT-PCR analysis of ISG transcripts in individual and pooled samples further corroborated these data, and indicated that levels decreased to near those of controls following successful therapy. Significantly enhanced FcγR-stimulated ROS production was subsequently achieved by priming control neutrophils with IFN-α/-β/-γ, but not LPS, and gene expression analysis indicated that exposure to the type I IFN (in particular IFN-α) better replicated the mRNA profile observed in vivo. Further studies demonstrated that plasma levels of IFN-α were significantly higher in samples from patients relative to unaffected controls. Following successful periodontitis treatment, plasma IFN-α levels, neutrophil ISG expression, and FcγR-stimulated neutrophil ROS output of patients, all decreased to levels comparable with those of controls. In conclusion, although chronic periodontitis is a complex disease, raised IFN-α may be one determinant of the distinct molecular phenotype and hyperresponsivity exhibited by patients’ peripheral blood neutrophils.
Cutting Edge: Local Recall Responses by Memory T Cells Newly Recruited to Peripheral Nonlymphoid Tissues
Authors: Wakim Linda M. , Gebhardt Thomas , Heath William R. , Carbone Francis R.
Published: April 2014

Infection results in the formation of a circulating effector memory T cell population able to enter peripheral tissues either in the steady state or in response to localized infection. As a consequence, recall is thought to result from a phased response first involving those T cells already at the site of infection followed by the infiltration of memory cells from the wider circulation. We have recently reported that tissue-resident T cells can undergo stimulation and proliferation in response to local infection. In this study, we examine the proliferation of memory T cells newly recruited from the circulation. Our results show that although recruitment of circulating memory cells is nonspecific in nature, there is preferential proliferation of specific T cells within infected tissues. Thus, expansion represents a means of local Ag-specific enrichment of T cells recruited from a circulating memory pool of mixed specificities.
Autoreactive B Cells Discriminate CpG-Rich and CpG-Poor DNA and This Response Is Modulated by IFN-α
Authors: Uccellini Melissa B. , Busconi Liliana , Green Nathaniel M. , Busto Patricia , Christensen Sean R. , Shlomchik Mark J. , Marshak-Rothstein Ann , Viglianti Gregory A.
Published: April 2014

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-α lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-α can contribute to the pathogenesis of systemic lupus erythematosus.
SIT and TRIM Determine T Cell Fate in the Thymus
Authors: Koelsch Uwe , Schraven Burkhart , Simeoni Luca
Published: April 2014

Thymic selection is a tightly regulated developmental process essential for establishing central tolerance. The intensity of TCR-mediated signaling is a key factor for determining cell fate in the thymus. It is widely accepted that low-intensity signals result in positive selection, whereas high-intensity signals induce negative selection. Transmembrane adaptor proteins have been demonstrated to be important regulators of T cell activation. However, little is known about their role during T cell development. Herein, we show that SIT (SHP2 Src homology domain containing tyrosine phosphatase 2-interacting transmembrane adaptor protein) and TRIM (TCR-interacting molecule), two structurally related transmembrane adaptors, cooperatively regulate TCR signaling potential, thereby influencing the outcome of thymic selection. Indeed, loss of both SIT and TRIM resulted in the up-regulation of CD5, CD69, and TCRβ, strong MAPK activation, and, consequently, enhanced positive selection. Moreover, by crossing SIT/TRIM double-deficient mice onto transgenic mice bearing TCRs with different avidity/affinity, we found profound alterations in T cell development. Indeed, in female HY TCR transgenic mice, positive selection was completely converted into negative selection resulting in small thymi devoided of double-positive thymocytes. More strikingly, in a nonselecting background, SIT/TRIM double-deficient single-positive T cells developed, were functional, and populated the periphery. In summary, we demonstrated that SIT and TRIM regulate cell fate of developing thymocytes, thus identifying them as essential regulators of central tolerance.
Identification of CD25+ γδ T Cells As Fetal Thymus-Derived Naturally Occurring IL-17 Producers
Authors: Shibata Kensuke , Yamada Hisakata , Nakamura Risa , Sun Xun , Itsumi Momoe , Yoshikai Yasunobu
Published: April 2014

We previously reported that resident γδ T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing γδ T cells did not produce IFN-γ or IL-4, similar to Th17 cells. IL-17-producing γδ T cells specifically express CD25 but not CD122, whereas CD122+ γδ T cells produced IFN-γ. IL-17-producing γδ T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-γ-producing CD122+ γδ T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing γδ T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing γδ T cells. The number of thymic IL-17-producing γδ T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vγ6+Vδ1+ γδ T cells. The number of IL-17-producing γδ T cells was decreased in fetal thymus of Vδ1-deficient mice, whereas Vγ5+ fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vγ6+Vδ1+ T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.
IL-23 Promotes Maintenance but Not Commitment to the Th17 Lineage
Authors: Stritesky Gretta L. , Yeh Norman , Kaplan Mark H.
Published: April 2014

IL-23 plays a critical role establishing inflammatory immunity and enhancing IL-17 production in vivo. However, an understanding of how it performs those functions has been elusive. In this report, using an IL-17-capture technique, we demonstrate that IL-23 maintains the IL-17-secreting phenotype of purified IL-17+ cells without affecting cell expansion or survival. IL-23 maintains the Th17 phenotype over multiple rounds of in vitro stimulation most efficiently in conjunction with IL-1β. However, in contrast to Th1 and Th2 cells, the Th17 phenotype is not stable and when long-term IL-23-stimulated Th17 cultures are exposed to Th1- or Th2-inducing cytokines, the Th17 genetic program is repressed and cells that previously secreted IL-17 assume the cytokine secreting profile of other Th subsets. Thus, while IL-23 can maintain the Th17 phenotype, it does not promote commitment to an IL-17-secreting lineage.
Rho-ROCK-Dependent Ezrin-Radixin-Moesin Phosphorylation Regulates Fas-Mediated Apoptosis in Jurkat Cells
Authors: Hébert Marylise , Potin Sophie , Sebbagh Michaël , Bertoglio Jacques , Bréard Jacqueline , Hamelin Jocelyne
Published: April 2014

Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.
An IL-1 Cytokine Member, IL-33, Induces Human Basophil Activation via Its ST2 Receptor
Authors: Suzukawa Maho , Iikura Motoyasu , Koketsu Rikiya , Nagase Hiroyuki , Tamura Chise , Komiya Akiko , Nakae Susumu , Matsushima Kouji , Ohta Ken , Yamamoto Kazuhiko , Yamaguchi Masao
Published: April 2014

Basophils are thought to play pivotal roles in allergic inflammation through rapid release of chemical mediators in addition to sustained production of Th2 cytokines, including IL-4. A newly identified cytokine, IL-33, has been recognized as one of the key cytokines enhancing Th2-balanced immune regulation through its receptor, ST2. The present study was conducted to elucidate whether IL-33 acts directly on, and affects the functions of, human basophils. Real-time PCR analysis showed that basophils express transcripts for ST2. The expression levels were significantly higher compared with eosinophils and neutrophils, and treatment with IL-33 significantly up-regulated basophil ST2 mRNA expression. Expressions of IL-4 and IL-13 mRNA were also up-regulated by IL-33, and there was also enhanced secretion of IL-4 protein. IL-33 increased the surface levels of basophil CD11b expression and enhanced basophil adhesiveness. Although IL-33 failed to directly induce degranulation or attract basophils, it exerted priming effects on basophils. It enhanced degranulation in response to IgE-crosslinking stimulus and also enhanced basophil migration toward eotaxin without changing surface CCR3. Also, IL-33 synergistically enhanced IL-4 production and CD11b expression by IL-3-stimulated basophils. Neutralization using Ab specific for ST2 significantly diminished the enhancing effects of IL-33 on both basophil CD11b expression and migration toward eotaxin, indicating that IL-33 signals via ST2 expressed on basophils. This study revealed that IL-33 potently regulates migration and activation of human basophils. IL-33 may be a key cytokine in the pathogenesis of Th2-dominant inflammation by acting not only on lymphocytes but also on effector cells such as basophils.