bluuebunny/crossref_metadata_2025 huggingface dataset:
Nerve Growth Factor Promotes TLR4 Signaling-Induced Maturation of Human Dendritic Cells In Vitro through Inducible p75NTR 1
Authors: Jiang Yingming , Chen Guoyou , Zhang Yi , Lu Lin , Liu Shuxun , Cao Xuetao
Published: April 2014

Nerve growth factor (NGF) has been shown to play important roles in the differentiation, function, and survival of immune cells, contributing to immune responses and pathogenesis of autoimmune diseases. Dendritic cells (DCs) are a potent initiator for immune and inflammatory responses upon recognition of pathogens via Toll-like receptors (TLR). However, expression of NGF and its receptors on human monocyte-derived DCs (MoDCs) and the role of NGF in the response of DCs to TLR ligands remain to be investigated. In the present study, we demonstrate that there were weak expressions of NGF and no expression of NGF receptors p140TrkA and p75NTR on human immature MoDCs, however, the expression of NGF and p75NTR on MoDCs could be significantly up-regulated by LPS in a dose- and time-dependent manner. NGF could markedly promote LPS-induced expression of HLA-DR, CD40, CD80, CD83, CD86, CCR7, secretion of IL-12p40 and proinflammatory cytokines IL-1, IL-6, TNF-α, and the T cell-stimulating capacity of MoDCs, indicating that NGF can promote LPS-induced DC maturation. The promoting effect of NGF on LPS-induced MoDCs maturation could be completely abolished by pretreatment of MoDCs with p75NTR antagonist, suggesting that LPS-induced p75NTR mediates the effect. Furthermore, increased activation of the p38MAPK and NF-κB pathways has been shown to be responsible for the NGF-promoted DC maturation. Therefore, NGF facilitates TLR4 signaling-induced maturation of human DCs through LPS-up-regulated p75NTR via activation of p38 MAPK and NF-κB pathways, providing another mechanism for the involvement of NGF in the immune responses and pathogenesis of autoimmune diseases.
HVEM Signaling in Monocytes Is Mediated by Intracellular Calcium Mobilization
Authors: Heo Sook-Kyoung , Yoon Min-A , Lee Sang-Chul , Ju Seong-A , Choi Jang-Hyun , Suh Pann-Ghill , Kwon Byoung S. , Kim Byung-Sam
Published: April 2014

Herpes virus entry mediator (HVEM) is a member of the TNF receptor (TNFR) superfamily and is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Interaction of HVEM with its ligand, LIGHT, costimulates T cells and increases the bactericidal activity of monocytes and neutrophils. The interaction recruits cytoplasmic TNFR-associated factor adaptor proteins to the intracellular domain of HVEM. This leads to NFκB activation as a result of IκBα degradation and/or JNK/AP-1 activation, and ultimately results in the expression of genes required for cell survival, cytokine production, or cell proliferation. In this study, we show that treatment of human monocytes with recombinant human LIGHT (rhLIGHT) induces rapid elevation of intracellular calcium concentration ([Ca2+]i) in a HVEM-specific manner in parallel with TNF-α production, and enhances the bactericidal activities of monocytes. Immunoprecipitation and Western blotting analyses revealed phosphorylation of phospholipase Cγ1 (PLCγ1) but not PLCγ2. rhLIGHT-induced Ca2+response was completely abolished by silencing PLCγ1, or preincubating monocytes with PLC inhibitors, antagonists of the inositol-1,4,5-triphosphate receptor, or [Ca2+]i chelators. Furthermore, these PLC/Ca2+ inhibitors also blocked rhLIGHT-mediated IκBα degradation, generation of reactive oxygen species, TNF-α production and the bactericidal activities of monocytes. Our results indicate that Ca2+is a downstream mediator of the LIGHT/HVEM interaction in monocytes.
Complement Dependent Amplification of the Innate Response to a Cognate Microbial Ligand by the Long Pentraxin PTX3
Authors: Cotena Alessia , Maina Virginia , Sironi Marina , Bottazzi Barbara , Jeannin Pascale , Vecchi Annunciata , Corvaia Nathalie , Daha Mohamed R. , Mantovani Alberto , Garlanda Cecilia
Published: April 2014

The long pentraxin PTX3 is a fluid-phase pattern recognition receptor, which plays a nonredundant role in resistance against selected pathogens. PTX3 has properties similar to Abs; its production is induced by pathogen recognition, it recognizes microbial moieties, activates complement, and facilitates cellular recognition by phagocytes. The mechanisms responsible for the effector function of PTX3 in vivo have not been elucidated. OmpA, a major outer membrane protein of Gram-negative Enterobacteriaceae, is a microbial moiety recognized by PTX3. In the air pouch model, KpOmpA induces an inflammatory response, which is amplified by coadministration of PTX3 in terms of leukocyte recruitment and proinflammatory cytokine production. PTX3 did not affect the inflammatory response to LPS, a microbial moiety not recognized by PTX3. As PTX3 binds to C1q and modulates the activation of the complement cascade, we assessed the involvement of complement in the amplification of the response elicited by KpOmpA and PTX3. Experiments performed using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor 1 indicate that PTX3 amplifies the inflammatory response to KpOmpA through complement activation. The results reported here demonstrate that PTX3 activates a complement-dependent humoral amplification loop of the innate response to a microbial ligand.
Hsp72 Induces Inflammation and Regulates Cytokine Production in Airway Epithelium through a TLR4- and NF-κB-Dependent Mechanism
Authors: Chase Margaret A. , Wheeler Derek S. , Lierl Kristin M. , Hughes Valerie S. , Wong Hector R. , Page Kristen
Published: April 2014

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-κB inhibitor parthenolide. Hsp72 induced activation of NF-κB, as evidenced by NF-κB trans-activation and by p65 RelA and p50 NF-κB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-α, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-κB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-κB-dependent mechanism.
Decline in the Frequencies of Borrelia burgdorferi OspA161–175-Specific T Cells after Antibiotic Therapy in HLA-DRB1*0401-Positive Patients with Antibiotic-Responsive or Antibiotic-Refractory Lyme Arthritis
Authors: Kannian Priya , Drouin Elise E. , Glickstein Lisa , Kwok William W. , Nepom Gerald T. , Steere Allen C.
Published: April 2014

Synovitis in patients with antibiotic-refractory Lyme arthritis persists for months to several years after antibiotic therapy. This course, which may result from infection-induced autoimmunity, is associated with T cell recognition of Borrelia burgdorferi outer surface protein A (OspA161–175) and with HLA-DR molecules that bind this epitope, including the DRB1*0401 molecule. In this study, we used tetramer reagents to determine the frequencies of OspA161–175-specific T cells in samples of PBMC and synovial fluid mononuclear cells (SFMC) from 13 DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory arthritis. Initially, three of the six patients (50%) with antibiotic-responsive arthritis and four of the seven patients (57%) with antibiotic-refractory arthritis had frequencies of OspA161–175-specific CD4+ T cells in peripheral blood above the cutoff value of 4 per 105 cells. Among the five patients with concomitant PBMC and SFMC, four (80%) had OspA tetramer-positive cells at both sites, but the mean frequency of such cells was 16 times higher in SFMC, reaching levels as high as 1,177 per 105 cells. In the two patients in each patient group in whom serial samples were available, the frequencies of OspA161–175-specific T cells declined to low or undetectable levels during or soon after antibiotic therapy, months before the resolution of synovitis in the two patients with antibiotic-refractory arthritis. Thus, the majority of patients with Lyme arthritis initially have increased frequencies of OspA161–175-specific T cells. However, the marked decline in the frequency of such cells with antibiotic therapy suggests that persistent synovitis in the refractory group is not perpetuated by these cells.
VH4 Gene Segments Dominate the Intrathecal Humoral Immune Response in Multiple Sclerosis
Authors: Owens Gregory P. , Winges Kimberly M. , Ritchie Alanna M. , Edwards Sydni , Burgoon Mark P. , Lehnhoff Laura , Nielsen Kirsten , Corboy John , Gilden Donald H. , Bennett Jeffrey L.
Published: April 2014

A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138+ plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138+ cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138+ cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138+ cells with functionally rearranged VH4 gene segments as an overriding feature of MS CeSF repertoires. VH4 dominance was attributed to the preferential selection of specific VH4 genes, particularly gene segment VH4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in VH4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis.
Demethylation of CD40LG on the Inactive X in T Cells from Women with Lupus
Authors: Lu Qianjin , Wu Ailing , Tesmer Laura , Ray Donna , Yousif Neda , Richardson Bruce
Published: April 2014

Why systemic lupus erythematosus primarily affects women is unknown. Recent evidence indicates that human lupus is an epigenetic disease characterized by impaired T cell DNA methylation. Women have two X chromosomes; one is inactivated by mechanisms including DNA methylation. We hypothesized that demethylation of sequences on the inactive X may cause gene overexpression uniquely in women, predisposing them to lupus. We therefore compared expression and methylation of CD40LG, a B cell costimulatory molecule encoded on the X chromosome, in experimentally demethylated T cells from men and women and in men and women with lupus. Controls included TNFSF7, a methylation-sensitive autosomal B cell costimulatory molecule known to be demethylated and overexpressed in lupus. Bisulfite sequencing revealed that CD40LG is unmethylated in men, while women have one methylated and one unmethylated gene. 5-Azacytidine, a DNA methyltransferase inhibitor, demethylated CD40LG and doubled its expression on CD4+ T cells from women but not men, while increasing TNFSF7 expression equally between sexes. Similar studies demonstrated that CD40LG demethylates in CD4+ T cells from women with lupus, and that women but not men with lupus overexpress CD40LG on CD4+ T cells, while both overexpress TNFSF7. These studies demonstrate that regulatory sequences on the inactive X chromosome demethylate in T cells from women with lupus, contributing to CD40LG overexpression uniquely in women. Demethylation of CD40LG and perhaps other genes on the inactive X may contribute to the striking female predilection of this disease.
The Rheumatoid Arthritis Shared Epitope Triggers Innate Immune Signaling via Cell Surface Calreticulin
Authors: Ling Song , Pi Xiujun , Holoshitz Joseph
Published: April 2014

The shared epitope (SE), carried by the vast majority of rheumatoid arthritis patients, is a 5-aa sequence motif in the third allelic hypervariable region of the HLA-DRβ chain. We have recently demonstrated that the SE acts as an allele-specific ligand that triggers NO-mediated pro-oxidative signaling in opposite cells. The identity of the cell surface molecule that interacts with the SE is unknown. Using affinity chromatography purification, cell-binding assays, surface plasmon resonance, and time-resolved fluorescence resonance energy transfer techniques, we have identified cell surface calreticulin (CRT) as the SE-binding molecule. SE-triggered signaling could be blocked by anti-CRT Abs or Abs against CD91 and by CRT-specific antisense or small-interfering RNA oligonucleotides. Embryonic fibroblasts from crt−/− or CD91-deficient mice failed to transduce SE-triggered signals. Exogenously added soluble CRT attached to the cell surface and restored SE-triggered signaling responsiveness in crt−/− cells. These data indicate that cell surface CRT, a known innate immunity receptor, which has been previously proposed as a culprit in autoimmunity, plays a critical role in SE-triggered signal transduction.
Cutting Edge: Expression of TNFR2 Defines a Maximally Suppressive Subset of Mouse CD4+CD25+FoxP3+ T Regulatory Cells: Applicability to Tumor-Infiltrating T Regulatory Cells
Authors: Chen Xin , Subleski Jeffrey J. , Kopf Heather , Howard O. M. Zack , Männel Daniela N. , Oppenheim Joost J.
Published: April 2014

TNFR2 is predominantly expressed by a subset of human and mouse CD4+CD25+FoxP3+ T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2+ Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30–40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2− Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4+CD25−TNFR2+ cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2+ Tregs was considerably more potent than that of reportedly highly suppressive CD103+ Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2+ Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.
The Critical Role of IL-15 in the Antitumor Effects Mediated by the Combination Therapy Imatinib and IL-2
Authors: Mignot Grégoire , Ullrich Evelyn , Bonmort Mathieu , Ménard Cédric , Apetoh Lionel , Taieb Julien , Bosisio Daniela , Sozzani Silvano , Ferrantini Maria , Schmitz Jürg , Mack Matthias , Ryffel Bernard , Bulfone-Paus Silvia , Zitvogel Laurence , Chaput Nathalie
Published: April 2014

The synergistic antitumor effects of the combination therapy imatinib mesylate (IM) and IL-2 depended upon NK1.1- expressing cells and were associated with the accumulation of CD11cintB220+NK1.1+ IFN-producing killer dendritic cells (IKDC) into tumor beds. In this study, we show that the antitumor efficacy of the combination therapy was compromised in IL-15 and IFN-type 1R loss-of-function mice. IL-15Rα was required for the proliferation of IKDC during IM plus IL-2 therapy. Trans-presentation of IL-15/IL-15Rα activated IKDC to express CCR2 and to respond to type 1 IFN by producing CCL2. Moreover, the antitumor effects of the combination therapy correlated with a CCL2-dependent recruitment of IKDC, but not B220− NK cells, into tumor beds. Altogether, the IL-15-driven peripheral expansion and the CCL-2-dependent intratumoral chemoattraction of IKDC are two critical parameters dictating the antitumor efficacy of IM plus IL-2 in mice.
Contribution of TCR-β Locus and HLA to the Shape of the Mature Human Vβ Repertoire
Authors: Melenhorst J. Joseph , Lay Matthew D. H. , Price David A. , Adams Sharon D. , Zeilah Josette , Sosa Edgardo , Hensel Nancy F. , Follmann Dean , Douek Daniel C. , Davenport Miles P. , Barrett A. John
Published: April 2014

T cells that survive thymic selection express a diverse array of unique heterodimeric αβ TCRs that mediate peptide-MHC Ag recognition. The proportion of the total T cell repertoire that expresses a particular Vβ protein may be determined by a variety of factors: 1) germline preference for use of particular Vβ genes; 2) allelic effects on the expression of different Vβ genes; and 3) HLA effects on the expression of different Vβ genes (acting via thymic selection and/or peripheral mechanisms). In this study, we show that Vβ usage by human CD4+ and CD8+ T cells in neonatal and adult donors is highly correlated between unrelated individuals, suggesting that a large proportion of the observed pattern of Vβ expression is determined by factors intrinsic to the TCR-β locus. The presence of identical TCR alleles (within an individual) leads to a significantly better correlation between CD4+ and CD8+ T cells with respect to Vβ expression; these effects are, however, relatively minor. The sharing of HLA alleles between individuals also leads to an increased correlation between their Vβ expression patterns, although this did not reach statistical significance. We therefore conclude that the correlation in Vβ expression patterns between CD4+ and CD8+ T cells can be explained predominantly by germline TCR-β locus factors and not TCR-β allelic or HLA effects.
Long-Term Functionality of TCR-Transduced T Cells In Vivo
Authors: Coccoris Miriam , Swart Erwin , de Witte Moniek A. , van Heijst Jeroen W. J. , Haanen John B. A. G. , Schepers Koen , Schumacher Ton N. M.
Published: April 2014

To broaden the applicability of adoptive T cell therapy to cancer types for which tumor-specific T cells cannot routinely be isolated, an effort has been made to develop the transfer of tumor-specific TCR genes into autologous T cells as a novel immunotherapeutic approach. Although such TCR-modified T cells have been shown to react to Ag encounter and can be used to break tolerance to defined self-Ags, the persistence and capacity for renewed expansion of TCR-modified T cells has not been analyzed. To establish whether TCR-transduced T cells can provide recipients with long-term Ag-specific immune protection, we analyzed long-term function of TCR transduced T cells in mouse model systems. We demonstrate that polyclonal populations of T cells transduced with a class I restricted OVA-specific TCR are able to persist in vivo and respond upon re-encounter of cognate Ag as assessed by both proliferation and cytolytic capacity. These experiments indicate that TCR gene transfer can be used to generate long-term Ag-specific T cell responses and provide a useful model system to assess the factors that can promote high-level persistence of TCR-modified T cells.
Naive and Innate Memory Phenotype CD4+ T Cells Have Different Requirements for Active Itk for Their Development
Authors: Hu Jianfang , August Avery
Published: April 2014

The Tec family kinase Itk regulates the development of conventional and innate CD8+ T cells. However, little is known about the role of Itk in the development of CD4+ T cell lineages, although the role of Itk in the T cell activation and function is well defined. We show in this study that Itk null mice have increased percentage of CD62LlowCD44high memory phenotype CD4+ T cells compared with wild-type mice. These cells arise directly in the thymus, express high levels of transcripts for the T-bet and IFN-γ and are able to produce IFN-γ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with CD62LhighCD44low naive phenotype, but has no effect on the number of memory phenotype CD4+ T cells, indicating that the development of memory phenotype CD4+ T cells is Itk-independent. We further show that the development of the naive phenotype CD4+ T cells is dependent on active Itk signals and can be rescued by expression of Itk specifically in T cells. Our data also show that Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or Listeria monocytogenes stimulation. These results indicate that CD62LhighCD44low “naive” CD4+ and CD62LlowCD44high “innate memory phenotype” CD4+ T cells may be independent populations that differ in their requirement for Itk signals for development. Our data also suggest that CD4+CD62LlowCD44high memory phenotype T cells have innate immune function.
Long-Term Cardiac Allograft Survival across an MHC Mismatch after “Pruning” of Alloreactive CD4 T Cells
Authors: Hu Min , Watson Debbie , Zhang Geoff Y. , Graf Nicole , Wang Yuan M. , Sartor Mary , Howden Brian , Fletcher Jeffrey , Alexander Stephen I.
Published: April 2014

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4+ T cells from an MLR or “pruning” delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4+ T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4+ T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4+ T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4+ T cells. TCR β-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4+ T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.
Administration of PLP139–151 Primes T Cells Distinct from Those Spontaneously Responsive In Vitro to This Antigen
Authors: Penitente Romina , Nicolò Chiara , Van den Elzen Peter , Di Sante Gabriele , Agrati Chiara , Aloisi Francesca , Sercarz Eli E. , Ria Francesco
Published: April 2014

We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139–151 by Vβ-Jβ spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vβ10-Jβ1.1, but naive mice do not; in contrast, TCR CDR3-β rearrangements of Vβ18-Jβ1.2 and Vβ19-Jβ1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP139–151 presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vβ10-Jβ1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vβ10-Jβ1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vβ18-Jβ1.2 and Vβ19-Jβ1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP139–151 primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This “immunized” repertoire substitutes for the naive one and becomes dominant at the time of disease onset.
Cigarette Smoke Exposure Impairs Dendritic Cell Maturation and T Cell Proliferation in Thoracic Lymph Nodes of Mice
Authors: Robbins Clinton S. , Franco Francesca , Mouded Majd , Cernadas Manuela , Shapiro Steven D.
Published: April 2014

Respiratory tract dendritic cells (DCs) are juxtaposed to directly sample inhaled environmental particles. Processing and presentation of these airborne Ags could result in either the development of immunity or tolerance. The purpose of this study was to determine the consequences of cigarette smoke exposure on DC function in mice. We demonstrate that while cigarette smoke exposure decreased the number of DCs in the lungs, Ag-induced DC migration to the regional thoracic lymph nodes was unaffected. However, cigarette smoking suppressed DC maturation within the lymph nodes as demonstrated by reduced cell surface expression of MHC class II and the costimulatory molecules CD80 and CD86. Consequently, DCs from cigarette smoke-exposed animals had a diminished capacity to induce IL-2 production by T cells that was associated with diminished Ag-specific T cell proliferation in vivo. Smoke-induced defects in DC function leading to impaired CD4+ T cell function could inhibit tumor surveillance and predispose patients with chronic obstructive pulmonary disease to infections and exacerbations.
Regulatory T Cell Expression of Herpesvirus Entry Mediator Suppresses the Function of B and T Lymphocyte Attenuator-Positive Effector T Cells
Authors: Tao Ran , Wang Liqing , Murphy Kenneth M. , Fraser Christopher C. , Hancock Wayne W.
Published: April 2014

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA−/− CD4+CD25+ Tregs had normal suppressive activity, whereas BTLA−/− Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM−/− Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM−/− Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.
Mannan Chain Length Controls Lipoglycans Signaling via and Binding to TLR2
Authors: Nigou Jérôme , Vasselon Thierry , Ray Aurélie , Constant Patricia , Gilleron Martine , Besra Gurdyal S. , Sutcliffe Iain , Tiraby Gérard , Puzo Germain
Published: April 2014

TLR2 is a pattern-recognition receptor that is activated by a large variety of conserved microbial components, including lipoproteins, lipoteichoic acids, and peptidoglycan. Lipoglycans are TLR2 agonists found in some genera of the phylogenetic order Actinomycetales, including Mycobacterium. They are built from a mannosyl-phosphatidyl-myo-inositol anchor attached to a (α1→6)-linked d-mannopyranosyl chain whose units can be substituted by d-mannopyranosyl and/or d-arabinofuranosyl units. At this time, little is known about the molecular bases underlying their ability to induce signaling via this receptor. We have recently shown that the anchor must be at least triacylated, including a diacylglyceryl moiety, whereas the contribution of the glycosidic moiety is not yet clearly defined. We show herein that lipoglycan activity is directly determined by mannan chain length. Indeed, activity increases with the number of units constituting the (α1→6)-mannopyranosyl backbone but is also critically dependent on the substitution type of the 2-hydroxyl of these units. We thus provide evidence for the definition of a new pattern that includes the nonlipidic moiety of the molecules, most probably as a result of the (α1→6)-mannopyranosyl backbone being a highly conserved structural feature among lipoglycans. Moreover, we demonstrate that lipoglycans can bind cell surface-expressed TLR2 and that their ability to induce signaling might be, at least in part, dictated by their avidity for the receptor. Finally, our data suggest that lipoglycans and lipoproteins have a common binding site. The present results are thus discussed in the light of the recently published crystal structure of a TLR1-TLR2-lipopeptide complex.
Common Genetic Determinants of Uveitis Shared with Other Autoimmune Disorders
Authors: Mattapallil Mary J. , Sahin Azize , Silver Phyllis B. , Sun Shu-Hui , Chan Chi-Chao , Remmers Elaine F. , Hejtmancik J. Fielding , Caspi Rachel R.
Published: April 2014

Uveitis is a complex multifactorial autoimmune disease of the eye characterized by inflammation of the uvea and retina, degeneration of the retina, and blindness in genetically predisposed patients. Using the rat model of experimental autoimmune uveitis (EAU), we previously identified three quantitative trait loci (QTL) associated with EAU on rat chromosomes 4, 12, and 10 (Eau1, Eau2, and Eau3). The primary goal of the current study is to delineate additional non-MHC chromosomal regions that control susceptibility to EAU, and to identify any QTLs that overlap with the QTLs of other autoimmune diseases. Using a set of informative microsatellite markers and F2 generations of resistant and susceptible MHC class II-matched rat strains (F344 and LEW), we have identified several new significant or suggestive QTLs on rat chromosomes 2, 3, 7, 10, and 19 that control susceptibility to EAU. A protective allele was identified in the susceptible LEW strain in the Eau5 locus at D7Wox18, and epistatic interactions between QTLs were found to influence the severity of disease. The newly identified regions (Eau4 through Eau9) colocalize with the genetic determinants of other autoimmune disease models, and to disease-regulating syntenic regions identified in autoimmune patients on human chromosomes 4q21-31, 5q31-33, 16q22-24, 17p11-q12, 20q11-13, and 22q12-13. Our results suggest that uveitis shares some of the pathogenic mechanisms associated with other autoimmune diseases, and lends support to the “common gene, common pathway” hypothesis for autoimmune disorders.
Ly49H+ NK Cells Migrate to and Protect Splenic White Pulp Stroma from Murine Cytomegalovirus Infection
Authors: Bekiaris Vasileios , Timoshenko Olga , Hou Tie Zheng , Toellner Kai , Shakib Saba , Gaspal Fabrina , McConnell Fiona M. , Parnell Sonia M. , Withers David , Buckley Chris D. , Sweet Clive , Yokoyama Wayne M. , Anderson Graham , Lane Peter J. L.
Published: April 2014

In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients.
Munc13-4 Regulates Granule Secretion in Human Neutrophils
Authors: Pivot-Pajot Christophe , Varoqueaux Frederique , de Saint Basile Geneviève , Bourgoin Sylvain G.
Published: April 2014

The neutrophil plays a central role in the innate host immune defense. Regulated exocytosis of its granules and release of antimicrobial and cytotoxic substances are key events to limit the spread of pathogens. However, the molecular mechanisms that control exocytosis of neutrophil granules are ill-defined. Recently, it was shown that Munc13-4 is essential for the priming of granules in several hematopoietic cells. In this study, we show that Munc13-4 is expressed in human neutrophils, and that its expression is increased during granulocytic differentiation of HL-60 and PLB-985 cells. Cell fractionation analysis reveals that Munc13-4 is mainly cytosolic and is recruited rapidly to membranes following stimulation with fMLF (N-formyl-methionyl-leucyl-phenylalanine). Moreover, a pool of Munc13-4 associated with mobilizable secondary and tertiary granules is relocalized to the plasma membrane after stimulation with fMLF. The fMLF-induced translocation of Munc13-4 is strictly dependent on calcium in neutrophils. C2 domains of Munc13-4 are essential for binding to phospholipid vesicles in a Ca2+-independent manner. Finally, down-regulation of Munc13-4 using small interfering RNA decreases exocytosis of tertiary granules in PLB-985 cells, whereas overexpression of Munc13-4 enhances secretion of MMP-9 (matrix metalloproteinase-9) from tertiary granules. Our findings suggest a role for Munc13-4 as a component of the secretory machinery in neutrophils.
IL-6-Dependent Mucosal Protection Prevents Establishment of a Microbial Niche for Attaching/Effacing Lesion-Forming Enteric Bacterial Pathogens
Authors: Dann Sara M. , Spehlmann Martina E. , Hammond Dustin C. , Iimura Mitsutoshi , Hase Koji , Choi Lillian J. , Hanson Elaine , Eckmann Lars
Published: April 2014

Enteric infections with attaching/effacing lesion-inducing bacterial pathogens are a worldwide health problem. A murine infection model with one such pathogen, Citrobacter rodentium, was used to elucidate the importance of the pleiotropic immune regulator, IL-6, in the pathogenesis of infection. IL-6 was strongly induced in colonic epithelial cells and macrophages upon C. rodentium infection and was required for effective host defense, because mice lacking IL-6 failed to control bacterial numbers 2–3 wk after infection and exhibited increased mortality. IL-6 was not needed for mounting effective T and B cell responses to the pathogens, nor was it important for induction of IFN-γ or TNF-α, cytokines involved in host defense against the bacteria, or the antibacterial effector, NO. Instead, IL-6 played a key role in mucosal protection, since its absence was associated with marked infection-induced apoptosis in the colonic epithelium and subsequent ulcerations. Cell culture studies confirmed that IL-6 protected colon epithelial cells directly against inducible apoptosis, which was accompanied by increased expression of an array of genes encoding antiapoptotic proteins, including Bcl-xL, Mcl-1, cIAP-2, and Bcl-3. Ulcerations appeared to be pathogenetically important, because bacteria localized preferentially to those regions, and chemically induced colonic ulcerations promoted bacterial colonization. Furthermore, blood components likely present in ulcer exudates, particularly alanine, asparagine, and glycine, promoted bacterial growth. Thus, IL-6 is an important regulator of host defense against C. rodentium by protecting the mucosa against ulcerations which can act as a microbial niche for the bacteria.
CEACAM1 Dynamics during Neisseria gonorrhoeae Suppression of CD4+ T Lymphocyte Activation
Authors: Lee Hannah S. W. , Ostrowski Mario A. , Gray-Owen Scott D.
Published: April 2014

Neisseria gonorrhoeae colony opacity-associated (Opa) proteins bind to human carcinoembryonic antigen cellular adhesion molecules (CEACAM) found on host cells including T lymphocytes. Opa binding to CEACAM1 suppresses the activation of CD4+ T cells in response to a variety of stimuli. In this study, we use primary human CD4+ T cells isolated from peripheral blood to define the molecular events occurring subsequent to Opa-CEACAM1 binding. We establish that, in contrast to other cell types, T cells do not engulf N. gonorrhoeae upon CEACAM1 binding. Instead, the bacteria recruit CEACAM1 from intracellular stores and maintain it on the T cell surface. Upon TCR ligation, the co-engaged CEACAM1 becomes phosphorylated on tyrosine residues within the ITIMs apparent in the cytoplasmic domain. This allows the recruitment and subsequent activation of the src homology domain 2-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3ζ-chain and ZAP-70 kinase in response to TCR engagement. Combined, this dynamic response allows the bacteria to effectively harness the coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step.
Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages
Authors: Cornish E. Jean , Hurtgen Brady J. , McInnerney Kate , Burritt Nancy L. , Taylor Ross M. , Jarvis James N. , Wang Shirley Y. , Burritt James B.
Published: April 2014

The fungal pathogen Aspergillus fumigatus is responsible for increasing numbers of fatal infections in immune-compromised humans. Alveolar macrophages (AM) are important in the innate defense against aspergillosis, but little is known about their molecular responses to fungal conidia in vivo. We examined transcriptional changes and superoxide release by AM from C57BL/6 and gp91phox−/− mice in response to conidia. Following introduction of conidia into the lung, microarray analysis of AM showed the transcripts most strongly up-regulated in vivo to encode chemokines and additional genes that play a critical role in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM to fungal conidia. Of the 73 AM genes showing ≥2-fold changes, 8 were also increased in gp91phox−/− mice by conidia and in C57BL/6 mice by polystyrene beads, suggesting a common innate response to particulate matter. Ingenuity analysis of the microarray data from C57BL/6 mice revealed immune cell signaling and gene expression as primary mechanisms of this response. Despite the well-established importance of phagocyte NADPH oxidase in resisting aspergillosis, we found no evidence of this mechanism in AM following introduction of conidia into the mouse lung using transcriptional, luminometry, or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91phox−/− mice inhibit conidial germination equally in vitro. Our results indicate that early transcription in mouse AM exposed to conidia in vivo targets neutrophil recruitment, and that NADPH oxidase-independent mechanisms in AM contribute to inhibition of conidial germination.
Lactoferrin Acts as an Alarmin to Promote the Recruitment and Activation of APCs and Antigen-Specific Immune Responses
Authors: de la Rosa Gonzalo , Yang De , Tewary Poonam , Varadhachary Atul , Oppenheim Joost J.
Published: April 2014

Lactoferrin is an 80-kDa iron-binding protein present at high concentrations in milk and in the granules of neutrophils. It possesses multiple activities, including antibacterial, antiviral, antifungal, and even antitumor effects. Most of its antimicrobial effects are due to direct interaction with pathogens, but a few reports show that it has direct interactions with cells of the immune system. In this study, we show the ability of recombinant human lactoferrin (talactoferrin alfa (TLF)) to chemoattract monocytes. What is more, addition of TLF to human peripheral blood or monocyte-derived dendritic cell cultures resulted in cell maturation, as evidenced by up-regulated expression of CD80, CD83, and CD86, production of proinflammatory cytokines, and increased capacity to stimulate the proliferation of allogeneic lymphocytes. When injected into the mouse peritoneal cavity, lactoferrin also caused a marked recruitment of neutrophils and macrophages. Immunization of mice with OVA in the presence of TLF promoted Th1-polarized Ag-specific immune responses. These results suggest that lactoferrin contributes to the activation of both the innate and adaptive immune responses by promoting the recruitment of leukocytes and activation of dendritic cells.
Host and Donor Immune Responses Contribute to Antiviral Effects of Amotosalen-Treated Donor Lymphocytes following Early Posttransplant Cytomegalovirus Infection
Authors: Hossain Mohammad S. , Roback John D. , Wang Fengrong , Waller Edmund K.
Published: April 2014

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6→CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 × 106 untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 × 106 treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8+ T cells, amotosalen-treated CD8+ T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8+ T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4−CD8− (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-γ and TNF-α in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.
Agonists of Proteinase-Activated Receptor-2 Enhance IFN-γ-Inducible Effects on Human Monocytes: Role in Influenza A Infection
Authors: Feld Micha , Shpacovitch Victoria M. , Ehrhardt Christina , Kerkhoff Claus , Hollenberg Morley D. , Vergnolle Nathalie , Ludwig Stephan , Steinhoff Martin
Published: April 2014

Proteinase-activated receptor-2 (PAR2) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR2 agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR2 agonist to enhance IFN-γ-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IκBα after costimulation of cells with PAR2 agonist and IFN-γ. Remarkably, coapplication of PAR2 agonist and IFN-γ also enhances the effects of IFN-γ on IFN-γ-inducible protein 10 kDa release, and CD64 and αVβ3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR2 activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR2 agonists to enhance IFN-γ-induced protective effects on human monocytes.
Blockade of Virus Infection by Human CD4+ T Cells via a Cytokine Relay Network
Authors: Davis Ann M. , Hagan Kristan A. , Matthews Loderick A. , Bajwa Gagan , Gill Michelle A. , Gale Michael , Farrar J. David
Published: April 2014

CD4+ T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8+ T cell functions, we sought to determine whether human CD4+ T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-γ and TNF-α, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-γ and TNF-α acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-γ and TNF-α also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-γ and TNF-α secreted by human Th1 cells acted indirectly through the IFN-α/β receptor. These results highlight the importance of CD4+ T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.