bluuebunny/crossref_metadata_2025 huggingface dataset:
Annexin 1 Negatively Regulates IL-6 Expression via Effects on p38 MAPK and MAPK Phosphatase-1
Authors: Yang Yuan H. , Toh Myew-Ling , Clyne Colin D. , Leech Michelle , Aeberli Daniel , Xue Jin , Dacumos April , Sharma Laveena , Morand Eric F.
Published: April 2014

Annexin 1 (Anx-1) is a mediator of the anti-inflammatory actions of glucocorticoids, but the mechanism of its anti-inflammatory effects is not known. We investigated the role of Anx-1 in the regulation of the proinflammatory cytokine, IL-6. Lung fibroblast cell lines derived from Anx-1−/− and wild-type (WT) mice were treated with dexamethasone and/or IL-1. IL-6 mRNA and protein were measured using real-time PCR and ELISA, and MAPK pathway activation was studied. Compared with WT cells, unstimulated Anx-1−/− cells exhibited dramatically increased basal IL-6 mRNA and protein expression. In concert with this result, Anx-1 deficiency was associated with increased basal phosphorylated p38, JNK, and ERK1/2 MAPKs. IL-1-inducible phosphorylated p38 was also increased in Anx-1−/− cells. The increase in IL-6 release in Anx-1−/− cells was inhibited by inhibition of p38 MAPK. Anx-1−/− cells were less sensitive to dexamethasone inhibition of IL-6 mRNA expression than WT cells, although inhibition by dexamethasone of IL-6 protein was similar. MAPK phosphatase-1 (MKP-1), a glucocorticoid-induced negative regulator of MAPK activation, was up-regulated by dexamethasone in WT cells, but this effect of dexamethasone was significantly impaired in Anx-1−/− cells. Treatment of Anx-1−/− cells with Anx-1 N-terminal peptide restored MKP-1 expression and inhibited p38 MAPK activity. These data demonstrate that Anx-1 is an endogenous inhibitory regulator of MAPK activation and IL-6 expression, and that Anx-1 is required for glucocorticoid up-regulation of MKP-1. Therapeutic manipulation of Anx-1 could provide glucocorticoid-mimicking effects in inflammatory disease.
The Role of TLR2 In Vivo following Challenge withStaphylococcus aureusand Prototypic Ligands
Authors: Mullaly Sarah C. , Kubes Paul
Published: April 2014

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2−/− or C5aR−/− mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2−/− mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.
Antigen-Specific Central Memory CD4+ T Lymphocytes Produce Multiple Cytokines and Proliferate In Vivo in Humans
Authors: Stubbe Muriel , Vanderheyde Nathalie , Goldman Michel , Marchant Arnaud
Published: April 2014

The function of Ag-specific central (TCM) and effector (TEM) memory CD4+ T lymphocytes remains poorly characterized in vivo in humans. Using CD154 as a marker of Ag-specific CD4+ T cells, we studied the differentiation of memory subsets following anti-hepatitis B immunization. Hepatitis B surface Ag (HBs)-specific memory CD4+ T cells were heterogeneous and included TCM (CCR7+CD27+) and TEM (CCR7−CD27+/−). HBs-specific TCM and TEM shared the capacity to produce multiple cytokines, including IL-2 and IFN-γ. Several years postimmunization, ∼10% of HBs-specific memory CD4+ T cells were in cycle (Ki67+) and the proliferating cells were CCR7+. These results suggest that the model of functional specialization of TCM and TEM cannot be applied to protein vaccine Ags and support the concept that TCM are capable of self-renewal and contribute to maintain the pool of memory cells.
CD8-Mediated Type 1 Antitumor Responses Selectively Modulate Endogenous Differentiated and Nondifferentiated T Cell Localization, Activation, and Function in Progressive Breast Cancer
Authors: Dobrzanski Mark J. , Reome Joyce B. , Hylind James C. , Rewers-Felkins Kathleen A.
Published: April 2014

CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-γ, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Using a murine TCR transgenic T cell/breast tumor model, we show that adoptively transferred Ag-specific Tc1 cells are more effective in delaying mammary tumor growth and progression than that of functionally distinct Tc2 cells. Donor Tc1 cells administered 7 days posttumor challenge localized and persisted at sites of primary tumor growth with antitumor responses that were dependent, in part, on effector cell-derived IFN-γ. Tc1-mediated responses markedly enhanced the appearance and local accumulation of highly differentiated (CD44high) CD4 and CD8 endogenous tumor-infiltrating T cells when compared with that of untreated tumor-bearing mice. Conversely, Tc1 cell transfer markedly delayed the appearance of corresponding nondifferentiated (CD44low) endogenous T cells. Such cells were acutely activated as defined by coexpression of surface markers associated with TCR engagement (CD69) and early T cell activation (CD25). Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IFN-γ-inducible protein-10 in vivo. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD4 and CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment at various stages of tumor progression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.
Antibodies against the Activated Coagulation Factor X (FXa) in the Antiphospholipid Syndrome That Interfere with the FXa Inactivation by Antithrombin
Authors: Yang Yao-Hsu , Hwang Kwan-Ki , FitzGerald John , Grossman Jennifer M. , Taylor Mihaela , Hahn Bevra H. , Chen Pojen P.
Published: April 2014

Antiphospholipid Ab have been shown to promote thrombosis and fetal loss in the antiphospholipid syndrome (APS). Previously, we found IgG anti-thrombin Ab in some APS patients that could interfere with inactivation of thrombin by antithrombin (AT). Considering that activated coagulation factor X (FXa) is homologous to thrombin in the catalytic domains and is also regulated primarily by AT, we hypothesized that some thrombin-reactive Ab may bind to FXa and interfere with AT inactivation of FXa. To test these hypotheses, we studied reactivity of eight patient-derived monoclonal IgG antiphospholipid Ab with FXa and the presence of IgG anti-FXa Ab in APS patients and investigated the effects of FXa-reactive mAb on AT inactivation of FXa. The results revealed that six of six thrombin-reactive IgG mAb bound to FXa and that the levels of plasma IgG anti-FXa Ab in 38 APS patients were significantly higher than those in 30 normal controls (p < 0.001). When the mean plus 3 SDs of the 30 normal controls was used as the cutoff, 5 of 38 APS patients (13.2%) had IgG anti-FXa Ab. Importantly, three of six FXa-reactive mAb significantly inhibited AT inactivation of FXa. Combined, these results indicate that anti-FXa Ab may contribute to thrombosis by interfering with the anticoagulant function of AT on FXa in some APS patients.
Role for Nephritogenic T Cells in Lupus Glomerulonephritis: Progression to Renal Failure Is Accompanied by T Cell Activation and Expansion in Regional Lymph Nodes
Authors: Bagavant Harini , Deshmukh Umesh S. , Wang Hongyang , Ly Timothy , Fu Shu Man
Published: April 2014

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c+ dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vβ families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.
Cutting Edge: Programed Death (PD) Ligand-1/PD-1 Interaction Is Required for CD8+ T Cell Tolerance to Tissue Antigens
Authors: Martin-Orozco Natalia , Wang Yi-Hong , Yagita Hideo , Dong Chen
Published: April 2014

Constitutive presentation of tissue Ags by dendritic cells results in tolerance of autoreactive CD8+ T cells; however, the underlying molecular mechanisms are not well understood. In this study we show that programmed death (PD)-1, an inhibitory receptor of the CD28 family, is required for tolerance induction of autoreactive CD8+ T cells. An antagonistic Ab against PD-1 provoked destructive autoimmune diabetes in RIP-mOVA mice expressing chicken OVA in the pancreatic islet cells, which received naive OVA-specific CD8+ OT-I cells. This effect was mediated by the PD ligand (PD-L) PD-L1 but not by PD-L2. An increased number of effector OT-I cells recovered from the pancreatic lymph nodes of anti-PD-L1-treated mice showed down-regulation of PD-1. Furthermore, the blockade of PD-1/PD-L1 interaction during the priming phase did not significantly affect OT-I cell division but enhanced its granzyme B, IFN-γ, and IL-2 production. Thus, during the presentation of tissue Ags to CD8+ T cells, PD-1/PD-L1 interaction crucially controls the effector differentiation of autoreactive T cells to maintain self-tolerance.
Lunatic Fringe Controls T Cell Differentiation through Modulating Notch Signaling
Authors: Tsukumo Shin-ichi , Hirose Kayo , Maekawa Yoichi , Kishihara Kenji , Yasutomo Koji
Published: April 2014

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4−CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4−CD8− (double-negative (DN)) and mature CD4+CD8− (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25− stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.
T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection
Authors: Stiles Linda N. , Hardison Jenny L. , Schaumburg Chris S. , Whitman Lucia M. , Lane Thomas E.
Published: April 2014

The chemokine CXCL10 is expressed within the CNS in response to intracerebral infection with mouse hepatitis virus (MHV). Blocking CXCL10 signaling results in increased mortality accompanied by reduced T cell infiltration and increased viral titers within the brain suggesting that CXCL10 functions in host defense by attracting T cells into the CNS. The present study was undertaken to extend our understanding of the functional role of CXCL10 in response to MHV infection given that CXCL10 signaling has been implicated in coordinating both effector T cell generation and trafficking. We show that MHV infection of CXCL10+/+ or CXCL10−/− mice results in comparable levels of T cell activation and similar numbers of virus-specific CD4+ and CD8+ T cells. Subsequent analysis revealed no differences in T cell proliferation, IFN-γ secretion by virus-specific T cells, or CD8+ T cell cytolytic activity. Analysis of chemokine receptor expression on CD4+ and CD8+ T cells obtained from MHV-immunized CXCL10+/+ and CXCL10−/− mice revealed comparable levels of CXCR3 and CCR5, which are capable of responding to ligands CXCL10 and CCL5, respectively. Adoptive transfer of splenocytes acquired from MHV-immunized CXCL10−/− mice into MHV-infected RAG1−/− mice resulted in T cell infiltration into the CNS, reduced viral burden, and demyelination comparable to RAG1−/− recipients of immune CXCL10+/+ splenocytes. Collectively, these data imply that CXCL10 functions primarily as a T cell chemoattractant and does not significantly influence T cell effector response following MHV infection.
Identification of Cellular Intermediates and Molecular Pathways Induced by IL-21 in Human B Cells
Authors: Konforte Danijela , Paige Christopher J.
Published: April 2014

The complex process of B cell development is controlled by multiple factors from the surrounding microenvironment including cytokines. IL-21 is a recently identified type I cytokine, mainly produced by activated CD4+ T cells. It has been shown to promote differentiation of human primary B cells into Ig-secreting plasma cells. The objective of our study was to describe cellular intermediates that exist during IL-21-induced transition from an activated B cell to an Ig-secreting cell and to identify molecular mechanisms involved in this process. Novel Epstein-Barr Virus-positive human B cell lines with phenotypes characteristic of Ag-activated IgG+ B cell blasts were used as a model system to study IL-21 effects in vitro. We show that IL-21 increased both proliferation and survival of B cell lines during the first 3 days of in vitro culture. This process was associated with CD38low/intCD23intHLA-DRhighCD19highCD20int cell surface phenotype. Continued culture with IL-21 resulted in accumulation of cells in G0/G1 stage of the cell cycle and increased apoptosis. This coincided with differentiation into small, CD38highCD23low/−HLA-DRintCD19intCD20low late plasmablasts/early plasma cells that expressed lower levels of c-Myc protein, and secreted greater amounts of Ig than the control cells. Partial inhibition of IL-21-induced JAK/STAT signaling by the low-dose pharmacological agent, JAK inhibitor I, did not prevent the initial increase in proliferation. However, decrease in c-Myc protein expression and subsequent differentiation to late plasmablasts/early plasma cells were strongly inhibited. Our study is the first to show the link between IL-21-induced JAK/STAT signaling, c-Myc regulation, and differentiation of human B cells.
Innate Cytokine Responses in Porcine Macrophage Populations: Evidence for Differential Recognition of Double-Stranded RNA
Authors: Loving Crystal L. , Brockmeier Susan L. , Ma Wenjun , Richt Juergen A. , Sacco Randy E.
Published: April 2014

Pulmonary airways are vulnerable to infection because of exposure to Ag during respiration. The innate, antiviral response must be activated rapidly after pathogen recognition, and alveolar macrophages (AMφ) play a role in this response. TLR3 and protein kinase R (PKR) recognize dsRNA, a replication intermediate of RNA viruses, and initiate transcription of IFN-αβ. In this study, synthetic dsRNA poly(I:C) was used to investigate innate responses of porcine AMφ compared with responses of peritoneal macrophages (PMφ). Poly(I:C) triggered IFN-αβ in AMφ and PMφ, but levels in AMφ were higher. In contrast, mRNA levels of IFN-stimulated genes, Mx and PKR, were greater in PMφ than AMφ. Low levels of Mx and PKR transcription in AMφ were not due to deficient type I IFN receptor signaling, as exogenous IFN-α induced nuclear translocation of phosphorylated STAT1. To investigate the differential mechanism by which IFN-αβ transcription is activated in AMφ and PMφ, 2-aminopurine (2-AP) was used to block dsRNA-mediated activation of PKR. IFN-αβ, Mx, and PKR mRNA levels in AMφ after poly(I:C) treatment were unaffected by 2-AP; conversely, transcription of IFN-αβ, Mx, or PKR remained at baseline levels in PMφ. Phosphorylated PKR was detected in PMφ, but not AMφ, after poly(I:C) treatment. In addition to IFN-αβ gene induction, mRNA levels of TNF-α and RANTES were higher in AMφ than PMφ after poly(I:C) stimulation. In summary, differential dsRNA-induced cytokine expression patterns between AMφ and PMφ provide evidence that dsRNA recognition and subsequent signaling is likely mediated via TLR3 in AMφ and PKR in PMφ.
Regulated Expression of FcγR in Human Dendritic Cells Controls Cross-Presentation of Antigen-Antibody Complexes
Authors: Liu Yi , Gao Xiaoni , Masuda Emi , Redecha Patricia B. , Blank Marissa C. , Pricop Luminita
Published: April 2014

Receptors for IgG (FcγR) expressed in dendritic cells (DCs) influence the initiation of Ab-mediated immunity. Dynamic variations in FcγR expression allow DCs to adjust their capacity to capture Ab-opsonized Ag. The current paradigm predicts a progressive decline in FcγR-mediated phagocytic function upon DC maturation. Surprisingly, we find that expression of the phagocytic receptor FcγRIIa is preserved in immature and mature DCs at comparable levels with macrophages. Moreover, phagocytosis of antigenic peptides directed to FcγRIIa on DCs leads to dramatic increases in Ag cross-presentation and T cell activation. In immature DCs, high expression of inhibitory FcγRIIb correlates with decreased uptake and cross-presentation of Ab-Ag complexes. In contrast, engagement of FcγRIIb is not associated with changes in cross-presentation in mature DCs. We provide evidence that FcγRIIb expression is patently reduced in mature DCs, an effect that is modulated by treatment with cytokines. The regulated expression of activating and inhibitory FcγRs in DCs emerges as a critical checkpoint in the process of Ag uptake and cross-presentation
Interactions between T Cells Responding to Concurrent Mycobacterial and Influenza Infections
Authors: Co Dominic O. , Hogan Laura H. , Karman Jozsef , Heninger Erika , Vang Shoua , Wells Krisna , Kawaoka Yoshihiro , Sandor Matyas
Published: April 2014

CD4+ T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-γ secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-γ and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.
Structurally Distinct Membrane Nanotubes between Human Macrophages Support Long-Distance Vesicular Traffic or Surfing of Bacteria
Authors: Önfelt Björn , Nedvetzki Shlomo , Benninger Richard K. P. , Purbhoo Marco A. , Sowinski Stefanie , Hume Alistair N. , Seabra Miguel C. , Neil Mark A. A. , French Paul M. W. , Davis Daniel M.
Published: April 2014

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > ∼0.7 μm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at ∼1 μm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.
Prenatal Stress Enhances Susceptibility of Murine Adult Offspring toward Airway Inflammation
Authors: Pincus-Knackstedt Maike K. , Joachim Ricarda A. , Blois Sandra M. , Douglas Alison J. , Orsal Arif S. , Klapp Burghard F. , Wahn Ulrich , Hamelmann Eckard , Arck Petra C.
Published: April 2014

Allergic asthma is one of the most prevalent and continuously increasing diseases in developed countries. Its clinical features include airway hyperresponsiveness and inflammation upon allergen contact. Furthermore, an emerging area of research subsumed as fetal programming evaluates the impact of environmental insults in utero on the incidence of diseases in later life. The aim of this study was to identify whether prenatal exposure to stress, which constitutes a severe environmental insult, perpetuates airway inflammation in later life. Our experiments were performed in mice and revealed that prenatally stressed adult offspring indeed show an increased vulnerability toward airway hyperresponsiveness and inflammation. Furthermore, we provide persuasive insights on dysregulated pathways of the cellular and humoral immune response upon Ag challenge in prenatally stressed adult offspring, reflected by a Th2 greater Th1 adaptive immune response and increased CCR3 and IgE levels in vivo. Additionally, APCs derived from prenatally stressed offspring trigger clonal expansion of Th2 cells in vitro. We also deliver experimental evidence for a reduced corticotrophin-releasing hormone expression in the paraventricular nucleus of adult offspring in response to prenatal stress. Furthermore, behavioral analyses indicate an increase in anxiety in these mice. In conclusion, our data will facilitate future research aiming to identify the individual impact, hierarchy, and redundancy of multiple key protagonists in airway inflammation in an interdisciplinary context. This will foster the substantiation of disease-prevention strategies, such as asthma, during the prenatal period.
Phagocytosis Induces Lysosome Remodeling and Regulated Presentation of Particulate Antigens by Activated Dendritic Cells
Authors: Nayak Jayakar V. , Hokey David A. , Larregina Adriana , He Yukai , Salter Russell D. , Watkins Simon C. , Falo Louis D.
Published: April 2014

Immunization with particulate Ag effectively induces antitumor and antiviral T cell-mediated immunity. Immature dendritic cells (DCs) efficiently internalize, process, and present a variety of particulate Ags; however, previously published data suggest that both the uptake of soluble Ag through micropinocytosis, and phagocytosis of particulates are significantly curtailed in activated DC populations. In this study, we demonstrate that although macropinocytosis of soluble Ag is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags. Live cell imaging of activated DCs reveals that phagocytosis of particulates can result in cytoskeletal remodeling and perinuclear lysosome cluster disruption in a time-dependent manner. Interestingly, our results suggest that in activated DC populations, presentation of phagocytosed particulate Ags is dependent on the nature of the activation signal. These results provide direct evidence of functional heterogeneity in DC populations and contribute to the development of particle-based immunization strategies.
Treatment of Experimental Autoimmune Prostatitis in Nonobese Diabetic Mice by the Vitamin D Receptor Agonist Elocalcitol
Authors: Penna Giuseppe , Amuchastegui Susana , Cossetti Chiara , Aquilano Francesca , Mariani Roberto , Sanvito Francesca , Doglioni Claudio , Adorini Luciano
Published: April 2014

On the basis of on the marked inhibitory activity of the vitamin D receptor agonist Elocalcitol on basal and growth factor-induced proliferation of human prostate cells and on its potent anti-inflammatory properties, we have tested its capacity to treat experimental autoimmune prostatitis (EAP) induced by injection of prostate homogenate-CFA in nonobese diabetic (NOD) mice. Administration of Elocalcitol, at normocalcemic doses, for 2 wk in already established EAP significantly inhibits the intraprostatic cell infiltrate, leading to a profound reduction in the number of CD4+ and CD8+ T cells, B cells, macrophages, dendritic cells, and I-Ag7-positive cells. Immunohistological analysis demonstrates reduced cell proliferation and increased apoptosis of resident and infiltrating cells. Significantly decreased production of the proinflammatory cytokines IFN-γ and IL-17 is observed in prostate-draining lymph node T cells from Elocalcitol-treated NOD mice stimulated by TCR ligation. In addition, Elocalcitol treatment reduces IFN-γ production by prostate-infiltrating CD4+ T cells and draining lymph node T cells specific for an immunodominant peptide naturally processed from prostate steroid-binding protein, a prostate-specific autoantigen. Finally, CD4+ splenic T cells from Elocalcitol-treated NOD mice show decreased ability, upon adoptive transfer into NOD.SCID recipients, to induce autoimmune prostatitis, paralleled by a reduced capacity to produce IFN-γ in response to prostate steroid-binding protein. The results indicate that Elocalcitol is able to interfere with key pathogenic events in already established EAP in the NOD mouse. These data show a novel indication for vitamin D receptor agonists and indicate that treatment with Elocalcitol may inhibit the intraprostatic inflammatory response in chronic prostatitis/chronic pelvic pain syndrome patients.
Defective Chemokine-Directed Lymphocyte Migration and Development in the Absence of Rho Guanosine Diphosphate-Dissociation Inhibitors α and β
Authors: Ishizaki Hiroyoshi , Togawa Atsushi , Tanaka-Okamoto Miki , Hori Keiko , Nishimura Miyuki , Hamaguchi Akiko , Imai Toshio , Takai Yoshimi , Miyoshi Jun
Published: April 2014

Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) α and Rho GDIβ (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDIα and Rho GDIβ genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDIα and Rho GDIβ thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.
Decidual NK Cells Alter In Vitro First Trimester Extravillous Cytotrophoblast Migration: A Role for IFN-γ
Authors: Hu Yuxiang , Dutz Jan P. , MacCalman Colin D. , Yong Paul , Tan Rusung , von Dadelszen Peter
Published: April 2014

Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-γ. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi.
CD5-CK2 Binding/Activation-Deficient Mice Are Resistant to Experimental Autoimmune Encephalomyelitis: Protection Is Associated with Diminished Populations of IL-17-Expressing T Cells in the Central Nervous System
Authors: Axtell Robert C. , Xu Liang , Barnum Scott R. , Raman Chander
Published: April 2014

Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-γ and IL-17 and, to a lesser extent, cells that express IFN-γ or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-γ+/IL-17+ infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.
Functional Requirements for the Lysosomal Thiol Reductase GILT in MHC Class II-Restricted Antigen Processing
Authors: Hastings K. Taraszka , Lackman Rebecca L. , Cresswell Peter
Published: April 2014

Ag processing and presentation via MHC class II is essential for activation of CD4+ T lymphocytes. γ-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys46 and Cys49, present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys46 or Cys49 abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys200 or Cys211, previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.
Cross-Species Dependence of Ly49 Recognition on the Supertype Defining B-Pocket of a Class I MHC Molecule
Authors: Lavender Kerry J. , Kane Kevin P.
Published: April 2014

Ly49 recognition of MHC class I (MHC I) can be allele specific. However, the site of interaction on MHC I consists of highly conserved solvent-exposed amino acids, leaving it unclear how allele specificity occurs. In examining the specificity of mouse and rat Ly49, we noticed that MHC I ligands for mouse Ly49G and W, and the rat Ly49i2, typically share the HLA-B7 supertype, defined by a B-pocket that prefers a proline at position 2 in bound peptides. Through mutagenesis, we show that the supertype-defining B-pocket of RT1-A1c controls its allele-specific recognition by the syngeneic rat Ly49i2 inhibitory receptor and xenogeneic mouse inhibitory Ly49G and activating Ly49W receptors. Single amino acid substitutions in the B-pocket that did not prevent peptide binding disrupted Ly49 recognition. In contrast, single mutations in other regions of the peptide-binding groove had no effect. We provide a model whereby the B-pocket dictates the conformation of conserved residues at the Ly49 interaction site below, defining Ly49 allele specificity for MHC I. Therefore, at least some Ly49 may recognize supertypes, detectable even across species, and are sensitive to polymorphisms in the supertype-defining B-pocket. This would ensure that expression of specific MHC I supertypes capable of Ag presentation to T cells is sensed by NK cells, and if lacking, targets a cell for elimination, suggesting a supertype-mediated link between innate and adaptive immunity.
Th2 Cell-Selective Enhancement of HumanIL13Transcription byIL13-1112C>T, a Polymorphism Associated with Allergic Inflammation
Authors: Cameron Lisa , Webster Robin B. , Strempel Jannine M. , Kiesler Patricia , Kabesch Michael , Ramachandran Harikrishnan , Yu Lizhi , Stern Debra A. , Graves Penelope E. , Lohman I. Carla , Wright Anne L. , Halonen Marilyn , Klimecki Walter T. , Vercelli Donata
Published: April 2014

IL-13 is a central mediator of allergic inflammation. The single nucleotide polymorphism IL13-1112C>T (rs1800925) is associated with allergic phenotypes in ethnically distinct populations, but the underlying mechanism(s) remain unknown. Using in vivo, in vitro, and in silico analysis, we show that the IL13-1112T allele enhanced IL13 promoter activity in primary human and murine CD4+ Th2 lymphocytes. Increased expression of IL13-1112T in Th2 cells was associated with the creation of a Yin-Yang 1 binding site that overlapped a STAT motif involved in negative regulation of IL13 expression and attenuated STAT6-mediated transcriptional repression. Because IL-13 secretion was increased in IL13-1112TT homozygotes, we propose that increased expression of IL13-1112T in vivo may underlie its association with susceptibility to allergic inflammation. Interestingly, IL13-1112T had opposite transcriptional effects in nonpolarized CD4+ T cells, paralleled by distinct patterns of DNA-protein interactions at the IL13 promoter. Our findings suggest the nuclear milieu dictates the functional outcome of genetic variation.
Human α- and β-Defensins Block Multiple Steps in Herpes Simplex Virus Infection
Authors: Hazrati Ehsan , Galen Benjamin , Lu Wuyuan , Wang Wei , Ouyang Yan , Keller Marla J. , Lehrer Robert I. , Herold Betsy C.
Published: April 2014

This study examined the ability of nine human defensins (HD) to protect against herpes simplex virus infection. Noncytotoxic concentrations of all six α-defensins (HNP1–4, HD5, and HD6) and human β-defensin (hBD) 3 inhibited HSV infection. Two other β-defensins, hBD1 and 2, lacked this protective activity. Synchronized assays revealed that HNP-4, HD6, and hBD3 acted primarily by preventing binding and entry, whereas HNP1–3 and HD5 also inhibited postentry events. Even when added several hours after entry, substantial reduction in viral gene expression ensued. Human cervical epithelial cells incubated with HNP-1 or HD5 accumulated the peptides intracellularly. Surface plasmon resonance studies revealed that HNPs 1, 2, 3, and HD5 bound HSV glycoprotein B (gB) with high affinity, but showed minimal binding to heparan sulfate, the receptor for attachment. In contrast, HNP-4 and HD6 bound heparan sulfate, but not gB. HBD3 bound both gB and heparan sulfate, but hBD1 and hBD2 bound neither. Admixture of HD5 with hydroxyethylcellulose significantly protected mice from a viral challenge lethal to controls receiving an inactive peptide or hydroxyethylcellulose alone. These findings demonstrate that HDs act at multiple steps in the HSV life cycle and support the development of defensins or defensin-like peptides as microbicides.
Interleukin-23 Restores Immunity to Mycobacterium tuberculosis Infection in IL-12p40-Deficient Mice and Is Not Required for the Development of IL-17-Secreting T Cell Responses
Authors: Wozniak Teresa M. , Ryan Anthony A. , Britton Warwick J.
Published: April 2014

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-γ and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rβ1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40−/− mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Codelivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-γ-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-γ-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-γ production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.
Human Effector CD8+ T Lymphocytes Express TLR3 as a Functional Coreceptor
Authors: Tabiasco Julie , Devêvre Estelle , Rufer Nathalie , Salaun Bruno , Cerottini Jean-Charles , Speiser Daniel , Romero Pedro
Published: April 2014

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-γ secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-γ production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.
Deficiency of P-Selectin or P-Selectin Glycoprotein Ligand-1 Leads to Accelerated Development of Glomerulonephritis and Increased Expression of CC Chemokine Ligand 2 in Lupus-Prone Mice
Authors: He Xiaodong , Schoeb Trenton R. , Panoskaltsis-Mortari Angela , Zinn Kurt R. , Kesterson Robert A. , Zhang Junxuan , Samuel Sharon , Hicks M. John , Hickey Michael J. , Bullard Daniel C.
Published: April 2014

The selectins and their ligands mediate leukocyte rolling on endothelial cells, the initial step in the emigration cascade leading to leukocyte infiltration of tissue. These adhesion molecules have been shown to be key promoters of acute leukocyte emigration events; however, their roles in the development of long-term inflammatory responses, including those that occur during chronic inflammatory diseases such as systemic lupus erythematosus, are unclear. To assess participation of P-selectin in such disorders, we studied the progression of systemic lupus erythematosus-like disease in P-selectin-deficient and control MRL/MpJ-Faslpr (Faslpr) mice. Surprisingly, we found that P-selectin deficiency resulted in significantly earlier mortality, characterized by a more rapid development of glomerulonephritis and dermatitis. Expression of CCL2 (MCP-1) was increased in the kidneys of P-selectin mutant mice and in supernatants of LPS-stimulated primary renal endothelial cell cultures from these mice. A closely similar phenotype, including elevated renal expression of CCL2, was also observed in Faslpr mice deficient in the major P-selectin ligand, P-selectin glycoprotein ligand-1. These results indicate that P-selectin and P-selectin glycoprotein ligand-1 are not required for leukocyte infiltration and the development of autoimmune disease in Faslpr mice, but rather expression of these adhesion molecules is important for modulating the progression of glomerulonephritis, possibly through down-regulation of endothelial CCL2 expression.
Biomechanical Signals Suppress Proinflammatory Responses in Cartilage: Early Events in Experimental Antigen-Induced Arthritis
Authors: Ferretti Mario , Gassner Robert , Wang Zheng , Perera Priyangi , Deschner James , Sowa Gwendolyn , Salter Robert B. , Agarwal Sudha
Published: April 2014

Although biomechanical signals generated during joint mobilization are vital in maintaining integrity of inflamed cartilage, the molecular mechanisms of their actions are little understood. In an experimental model of arthritis, we demonstrate that biomechanical signals are potent anti-inflammatory signals that repress transcriptional activation of proinflammatory genes and augment expression of anti-inflammatory cytokine IL-10 to profoundly attenuate localized joint inflammation.