bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Changes in MicroRNA-146a Expression Negatively Regulate the IL-1β-Induced Inflammatory Response in Human Lung Alveolar Epithelial Cells
Authors: Perry Mark M. , Moschos Sterghios A. , Williams Andrew E. , Shepherd Neil J. , Larner-Svensson Hanna M. , Lindsay Mark A.
Published: April 2014

Posttranscriptional regulation of gene expression by microRNAs (miRNAs) has been implicated in the regulation of chronic physiological and pathological responses. In this report, we demonstrate that changes in the expression of miRNAs can also regulate acute inflammatory responses in human lung alveolar epithelial cells. Thus, stimulation with IL-1β results in a rapid time- and concentration-dependent increase in miRNA-146a and, to a lesser extent, miRNA-146b expression, although these increases were only observed at high IL-1β concentration. Examination of miRNA function by overexpression and inhibition showed that increased miRNA-146a expression negatively regulated the release of the proinflammatory chemokines IL-8 and RANTES. Subsequent examination of the mechanism demonstrated that the action of miRNA-146a was mediated at the translational level and not through the down-regulation of proteins involved in the IL-1β signaling pathway or chemokine transcription or secretion. Overall, these studies indicate that rapid increase in miRNA-146a expression provides a novel mechanism for the negative regulation of severe inflammation during the innate immune response.
Depletion of β-Arrestin-2 Promotes Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer
Authors: Raghuwanshi Sandeep K. , Nasser Mohd W. , Chen Xiaoxin , Strieter Robert M. , Richardson Ricardo M.
Published: April 2014

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that β-arrestin-2 (βarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of βarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in βarr2-deficient mice (βarr2−/−) and control littermates (βarr2+/+) were used. βarr2−/− mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to βarr2+/+ mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR+ chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-κB activity was also enhanced in βarr2−/− mice, whereas hypoxia-inducible factor-1α expression was decreased. Inhibition of CXCR2 or NF-κB reduced tumor growth in both βarr2−/− and βarr2+/+ mice. NF-κB inhibition also decreased ELR+ chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that βarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-κB.
Programmed Death (PD)-1:PD-Ligand 1/PD-Ligand 2 Pathway Inhibits T Cell Effector Functions during Human Tuberculosis
Authors: Jurado Javier O. , Alvarez Ivana B. , Pasquinelli Virginia , Martínez Gustavo J. , Quiroga María F. , Abbate Eduardo , Musella Rosa M. , Chuluyan H. Eduardo , García Verónica E.
Published: April 2014

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-γ production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-γ production from these individuals. Moreover, M. tuberculosis-induced IFN-γ participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-γ-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-γ responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.
Retrogenic Modeling of Experimental Allergic Encephalomyelitis Associates T Cell Frequency but Not TCR Functional Affinity with Pathogenicity
Authors: Alli Rajshekhar , Nguyen Phuong , Geiger Terrence L.
Published: April 2014

The properties of a self-specific T cell’s TCR that determine its pathogenicity are not well understood. We developed TCR retroviral transgenic, or retrogenic, models of myelin oligodendroglial glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) to compare the pathologic potential of five H-2 Ab/MOG35–55-specific TCRs. The TCRs were cloned and retrovirally transduced into either TCRαβ-deficient hybridoma cells or Rag1−/− bone marrow progenitor cells. Comparison of the hybridomas, identical except for TCR sequence, revealed distinct responsiveness, or functionally determined affinity, for cognate Ag. Retrogenic mice were produced by transfer of transduced progenitor cells into Rag1−/− recipients. T cells were detected within 4 wk. Engraftment levels varied considerably among the different TCRs and showed separate variability among individual mice. T cells were predominantly naive and virtually exclusively CD4+ and CD25−. Relative responses of the retrogenic T cells to Ag paralleled those of the hybridoma cells. Induction of EAE through active immunization led to rapid and severe disease in all mice expressing MOG-specific TCR. The mice additionally developed spontaneous disease, the incidence of which varied with the individual receptors. Interestingly, spontaneous disease frequency and intensity could not be correlated with the functional affinity of the respective TCR. Instead, it was associated with engraftment level, even when measured weeks before the onset of disease symptoms. Our results demonstrate the feasibility of using retrogenic modeling to compare TCRs in the EAE system. They further suggest that affinity is not a primary determinant in spontaneous EAE development in mice expressing monotypic TCRs and that autoreactive T cell frequency is of greater significance.
Defective MHC Class II Presentation by Dendritic Cells Limits CD4 T Cell Help for Antitumor CD8 T Cell Responses
Authors: Gerner Michael Y. , Casey Kerry A. , Mescher Matthew F.
Published: April 2014

Cancer immunosurveillance failure is largely attributed to insufficient activation signals and dominant inhibitory stimuli for tumor Ag (TAg)-specific CD8 T cells. CD4 T cells have been shown to license dendritic cells (DC), thereby having the potential for converting CD8 T cell responses from tolerance to activation. To understand the potential cooperation of TAg-specific CD4 and CD8 T cells, we have characterized the responses of naive TCR transgenic CD8 and CD4 T cells to poorly immunogenic murine tumors. We found that whereas CD8 T cells sensed TAg and were tolerized, the CD4 T cells remained ignorant throughout tumor growth and did not provide help. This disparity in responses was due to normal TAg MHC class I cross-presentation by immature CD8α+ DC in the draining lymph node, but poor MHC class II presentation on all DC subsets due to selective inhibition by the tumor microenvironment. Thus, these results reveal a novel mechanism of cancer immunosubversion, in which inhibition of MHC-II TAg presentation on DC prevents CD4 T cell priming, thereby blocking any potential for licensing CD8α+ DC and helping tolerized CD8 T cells.
Alloreactive CD8 T Cell Tolerance Requires Recipient B Cells, Dendritic Cells, and MHC Class II
Authors: Fehr Thomas , Haspot Fabienne , Mollov Joshua , Chittenden Meredith , Hogan Timothy , Sykes Megan
Published: April 2014

Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.
Cutting Edge: Inflammasome Activation by Alum and Alum’s Adjuvant Effect Are Mediated by NLRP3
Authors: Li Hanfen , Willingham Stephen B. , Ting Jenny P.-Y. , Re Fabio
Published: April 2014

Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1β and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1β, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.
B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones
Authors: Takagi Rie , Higashi Takehiro , Hashimoto Kumiko , Nakano Kazuhisa , Mizuno Yosuke , Okazaki Yasushi , Matsushita Sho
Published: April 2014

Th 17 cells represent a novel subset of CD4+ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.
Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis Differently Modulate Human Dendritic Cell Functions Resulting in Divergent Prevalence of Th17-Polarized Responses
Authors: Fedele Giorgio , Nasso Maria , Spensieri Fabiana , Palazzo Raffaella , Frasca Loredana , Watanabe Mineo , Ausiello Clara M.
Published: April 2014

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1β production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1β, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IκBα and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.
cis-Urocanic Acid Initiates Gene Transcription in Primary Human Keratinocytes
Authors: Kaneko Kazuyo , Smetana-Just Ulrike , Matsui Mary , Young Antony R. , John Susan , Norval Mary , Walker Susan L.
Published: April 2014

It is well established that solar UV radiation (UVR) suppresses cutaneous cell-mediated immunity in humans. trans-Urocanic acid (trans-UCA) is a major UVR-absorbing skin molecule that undergoes a photoisomerization to its cis-isomer following UVR exposure. Animal studies have demonstrated that cis-UCA plays a role in UVR-induced immune suppression, but the molecular mechanisms of action of cis-UCA are not fully understood. In this study, we examined changes in gene expression and synthesis of cytokines and PGE2 following UCA treatment of primary human keratinocytes. A limited microarray analysis of keratinocytes from two donors indicated that ∼400 genes were induced by solar-simulated radiation (SSR), 16 of which were also up-regulated by cis-UCA. In contrast, trans-UCA had little or no effect on gene expression. The genes up-regulated by both cis-UCA and SSR were associated with apoptosis, cell growth arrest, cytokines, and oxidative stress. Further studies using primary keratinocytes from four new donors showed that PG-endoperoxide synthase-2 was dramatically induced by cis-UCA, resulting in an enhanced secretion of PGE2 into the cell culture supernatant. cis-UCA also increased cytokine protein production such as that of TNF-α, IL-6, and IL-8 in a dose-dependent manner. SSR had the same effect as cis-UCA, but trans-UCA had no effect. In addition, activation of NF-κB and lipid peroxidation were induced by cis-UCA and SSR, but not trans-UCA, suggesting possible upstream events of the gene expression changes. The data suggest that the induction of immune suppression by cis-UCA may involve the initiation of gene transcription of immunomodulatory mediators in primary human keratinocytes.
A Function for IL-7R for CD4+CD25+Foxp3+ T Regulatory Cells
Authors: Bayer Allison L. , Lee Joon Youb , de la Barrera Anabel , Surh Charles D. , Malek Thomas R.
Published: April 2014

The IL-2/IL-2R interaction is important for development and peripheral homeostasis of T regulatory (Treg) cells. IL-2- and IL-2R-deficient mice are not completely devoid of Foxp3+ cells, but rather lack population of mature CD4+CD25+Foxp3high Treg cells and contain few immature CD4+CD25−Foxp3low T cells. Interestingly, common γ chain (γc) knockout mice have been shown to have a near complete absence of Foxp3+ Treg cells, including the immature CD25−Foxp3low subset. Therefore, other γc-cytokine(s) must be critically important during thymic development of CD4+CD25+Foxp3+ Treg cells apart from the IL-2. The present study was undertaken to determine whether the γc-cytokines IL-7 or IL-15 normally contribute to expression of Foxp3 and Treg cell production. These studies revealed that mice double deficient in IL-2Rβ and IL-7Rα contained a striking lack in the CD4+Foxp3+ population and the Treg cell defect recapitulated the γc knockout mice. In the absence of IL-7R signaling, IL-15/IL-15R interaction is dispensable for the production of CD4+CD25+Foxp3+ Treg cells, indicating that normal thymic Treg cell production likely depends on signaling through both IL-2 and IL-7 receptors. Selective thymic reconstitution of IL-2Rβ in mice double deficient in IL-2Rβ and IL-7Rα established that IL-2Rβ is dominant and sufficient to restore production of Treg cells. Furthermore, the survival of peripheral CD4+Foxp3low cells in IL-2Rβ−/− mice appears to depend upon IL-7R signaling. Collectively, these data indicate that IL-7R signaling contributes to Treg cell development and peripheral homeostasis.
KDEL-Retained Antigen in B Lymphocytes Induces a Proinflammatory Response: A Possible Role for Endoplasmic Reticulum Stress in Adaptive T Cell Immunity
Authors: Wheeler Matthew C. , Rizzi Marta , Sasik Roman , Almanza Gonzalo , Hardiman Gary , Zanetti Maurizio
Published: April 2014

Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-γ and TNF-α, in responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial pathogens and the pathogenesis of certain autoimmune diseases.
Systematic Comparison of Gene Expression between Murine Memory and Naive B Cells Demonstrates That Memory B Cells Have Unique Signaling Capabilities
Authors: Tomayko Mary M. , Anderson Shannon M. , Brayton Catherine E. , Sadanand Saheli , Steinel Natalie C. , Behrens Timothy W. , Shlomchik Mark J.
Published: April 2014

Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Ag-specific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.
Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF
Authors: Xu Weifeng , Santini Paul A. , Matthews Allysia J. , Chiu April , Plebani Alessandro , He Bing , Chen Kang , Cerutti Andrea
Published: April 2014

Class switch DNA recombination (CSR) from IgM to IgG and IgA is crucial for antiviral immunity. Follicular B cells undergo CSR upon engagement of CD40 by CD40 ligand on CD4+ T cells. This T cell-dependent pathway requires 5–7 days, which is too much of a delay to block quickly replicating pathogens. To compensate for this limitation, extrafollicular B cells rapidly undergo CSR through a T cell-independent pathway that involves innate Ag receptors of the TLR family. We found that a subset of upper respiratory mucosa B cells expressed TLR3 and responded to viral dsRNA, a cognate TLR3 ligand. In the presence of dsRNA, mucosal B cells activated NF-κB, a transcription factor critical for CSR. Activation of NF-κB required TRIF (Toll/IL-1R domain-containing protein inducing IFN-β), a canonical TLR3 adapter protein, and caused germline transcription of downstream CH genes as well as expression of AID (activation-induced cytidine deaminase), a DNA-editing enzyme essential for CSR. Subsequent IgG and IgA production was enhanced by BAFF (B cell-activating factor of the TNF family), an innate mediator released by TLR3-expressing mucosal dendritic cells. Indeed, these innate immune cells triggered IgG and IgA responses upon exposure to dsRNA. By showing active TLR3 signaling and ongoing CSR in upper respiratory mucosa B cells from patients with CD40 signaling defects, our findings indicate that viral dsRNA may initiate frontline IgG and IgA responses through an innate TLR3-dependent pathway involving BAFF.
CD4 Help Regulates Expression of Crucial Genes Involved in CD8 T Cell Memory and Sensitivity to Regulatory Elements
Authors: Rapetti Laetitia , Meunier Sylvain , Pontoux Christiane , Tanchot Corinne
Published: April 2014

The role of CD4 help during CD8 memory differentiation has been clearly demonstrated in different experimental models. However, the mechanisms involved to mediate CD4 help and the extent of its effects remain largely unknown. Using gene analysis at a single cell level, which allows the study of gene expression in terms of frequency, intensity and coxpression, we show that unhelped CD8 T cells harbor severe defects in the expression of crucial genes involved in proliferation, survival, and cytotoxic functions, the three main characteristics of CD8 memory differentiation described so far. Importantly, during secondary response, unhelped CD8 T cells exhibit blockade in all cytotoxic pathways (perforin, Fas ligand, IFN-γ), demonstrating the highly ubiquitous effect of CD4 help. Secondly, resting unhelped CD8 T cells extinguish the majority of their stimulated genes, showing that CD4 help favors the persistence of gene expression. Indeed, during secondary response, unhelped CD8 T cells exhibit a profile very similar to naive T cells, demonstrating that no instructive program has been imprinted in these cells. Finally unhelped CD8 T cells exhibit a higher sensitivity to immunoregulatory genes during secondary immune response. Therefore, these results characterize the multiple effects of CD4 help on CD8 memory differentiation and provide important insights for the understanding of protective memory responses.
Development of Proteoglycan-Induced Arthritis Is Independent of IL-17
Authors: Doodes Paul D. , Cao Yanxia , Hamel Keith M. , Wang Yumei , Farkas Balint , Iwakura Yoichiro , Finnegan Alison
Published: April 2014

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-γ are secreted in CIA and EAE, disease is exacerbated in IFN-γ- or IFN-γ receptor-deficient mice due to the ability of IFN-γ to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-γ. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17−/−) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17−/− mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17−/− mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17−/− mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-κB ligand) expression. There were only subtle differences between WT and IL-17−/− mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.
Dendritic Cells Require the NF-κB2 Pathway for Cross-Presentation of Soluble Antigens
Authors: Lind Evan F. , Ahonen Cory L. , Wasiuk Anna , Kosaka Yoko , Becher Burkhard , Bennett Kathy A. , Noelle Randolph J.
Published: April 2014

NF-κB-inducing kinase (NIK) is responsible for activation of the non-canonical p100 processing pathway of NF-κB activation. This kinase has been shown to be critical for activation of this pathway after signaling through several TNF family members including CD40. The functional importance of this pathway in CD40 and TLR-induced dendritic cell (DC) differentiation was studied in vivo in the alymphoplasia (Aly) mouse. The Aly mouse expresses a mutant NIK molecule that prohibits the induction of the non-canonical pathway. We show that while MHC class II presentation and in vivo migration of Aly DCs is intact, these cells are unable to cross-prime CD8+ T cells to exogenous Ag. Gene expression array analysis of DCs matured in vivo indicates multiple defects in Ag processing pathways after maturation and provide a global view of the genes that are regulated by the NF-κB2 pathway in DCs. These experiments indicate a possible role for NIK in mediating cross-priming of soluble Ag. In addition, our findings explain the profound immune unresponsiveness of the Aly mouse.
Comprehensive Analysis of HLA-DR- and HLA-DP4-Restricted CD4+ T Cell Response Specific for the Tumor-Shared Antigen Survivin in Healthy Donors and Cancer Patients
Authors: Wang Xiao-Fei , Kerzerho Jerome , Adotevi Olivier , Nuyttens Hélène , Badoual Cecile , Munier Gaetan , Oudard Stéphane , Tu Shuiping , Tartour Eric , Maillère Bernard
Published: April 2014

Because of the wide distribution of the survivin Ag in a variety of tumors, we have investigated the survivin-specific CD4+ T cell response in healthy donors and cancer patients. Screening of the entire sequence of survivin for HLA class II binding led to the identification of seven HLA-DR promiscuous peptides, including four HLA-DP4 peptides. All of the peptides were able to prime in vitro CD4+ T cells of eight different healthy donors. The peptide-specific T cell lines were stimulated by dendritic cells loaded with the recombinant protein or with the lysates of tumor cells. The high frequency of responders (i.e., immunoprevalence) was provided by a wide reactivity of multiple peptides. Six peptides were T cell stimulating in at least half of the donors and were close to CD8+ T cell epitopes. HLA-DR molecules were more frequently involved in T cell stimulation than were HLA-DP4 molecules, and hence immunoprevalence relies mainly on HLA-DR promiscuity in the survivin Ag. In two cancer patients a spontaneous CD4+ T cell response specific for one of these peptides was also observed. Based on these observations, the tumor-shared survivin does not appear to be the target of immune tolerance in healthy donors and cancer patients and is a relevant candidate for cancer vaccine.
Human TCR α/β+ CD4−CD8− Double-Negative T Cells in Patients with Autoimmune Lymphoproliferative Syndrome Express Restricted Vβ TCR Diversity and Are Clonally Related to CD8+ T Cells
Authors: Bristeau-Leprince Anne , Mateo Véronique , Lim Annick , Magerus-Chatinet Aude , Solary Eric , Fischer Alain , Rieux-Laucat Frédéric , Gougeon Marie-Lise
Published: April 2014

The peripheral expansion of α/β+-CD4−CD8− double negative (DN) T cells in patients with autoimmune lymphoproliferative syndrome (ALPS) is a consistent feature of this disease, and part of the diagnostic criteria of ALPS. The origin of these cells remains undetermined. They could derive from mature T cells that have lost coreceptor expression, or represent a special minor cell lineage. To investigate relationship of DN and single positive (SP) T cells in ALPS, we used Immunoscope technology to analyze the TCRVβ repertoire diversity of sorted DN and SP T cells, and we performed CDR3 sequence analyses of matching clonotypes. We show that DN T cells express all the Vβ gene families that are used by their SP counterparts, though they dominantly use some Vβ genes. Analysis of CDR3 length distribution revealed a diverse polyclonal TCR repertoire for sorted CD4+ T cells, whereas both DN and CD8+ T cells showed a skewed TCR repertoire with oligoclonal expansions throughout most of the Vβ families. CDR3 sequencing of matching clonotypes revealed a significant sharing of CDR3 sequences from selected Vβ-Jβ transcripts between DN and CD8+ T cells. Altogether, these data strongly argue for a CD8 origin of DN T cells in ALPS.
Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG
Authors: Liu Xindong , Ye Lilin , Bai Yu , Mojidi Habi , Simister Neil E. , Zhu Xiaoping
Published: April 2014

Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-γ. However, in this study we found that IFN-γ down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-γ resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-γ activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-γ, whereas expression of JAK1 or STAT-1 protein restored the IFN-γ inhibitory effect on luciferase activity. The repressive effect of IFN-γ on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-γ activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-γ stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-γ.
CD73 Participates in Cellular Multiresistance Program and Protects against TRAIL-Induced Apoptosis
Authors: Mikhailov Andrey , Sokolovskaya Alice , Yegutkin Gennady G. , Amdahl Hanne , West Anne , Yagita Hideo , Lahesmaa Riitta , Thompson Linda F. , Jalkanen Sirpa , Blokhin Dmitry , Eriksson John E.
Published: April 2014

The molecular mechanisms underlying the multiresistant phenotype of leukemic and other cancer cells are incompletely understood. We used expression arrays to reveal differences in the gene expression profiles of an apoptosis-resistant T cell leukemia clone (A4) and normally apoptosis-sensitive parental Jurkat cells. CD73 (ecto-5′-nucleotidase) was the most up-regulated gene in the resistant A4 cell clone. A4 cells displayed CD73 surface expression and significant ecto-5′-nucleotidase activity. The role of CD73 was confirmed by transfection of wild-type CD73 into native Jurkat cells, which led to specific resistance against TRAIL-induced apoptosis, but not other types of apoptosis. The protective role of CD73 was further confirmed by small interfering RNA-mediated down-regulation of CD73, restoring TRAIL sensitivity. CD73-mediated resistance was independent of enzymatic activity of CD73, but was reliant on the anchoring of the protein to the membrane via GPI. We suggest that the inhibition of TRAIL signaling works through interaction of CD73 with death receptor 5, as CD73 and death receptor 5 could be coimmunoprecipitated and were shown to be colocalized in the plasma membrane by confocal microscopy. We propose that CD73 is a component of multiresistance machinery, the transcription of which is activated under selective pressure of the immune system.
Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function
Authors: Cao Shang , Carlesso Gianluca , Osipovich Anna B. , Llanes Joan , Lin Qing , Hoek Kristen L. , Khan Wasif N. , Ruley H. Earl
Published: April 2014

Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells in the bone marrow and from CD4−CD8− double-negative to CD4+CD8+ double-positive T cells in the thymus. In addition, mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation.
Innate Responses to Systemic Infection by Intracellular Bacteria Trigger Recruitment of Ly-6Chigh Monocytes to the Brain
Authors: Drevets Douglas A. , Schawang Jennifer E. , Dillon Marilyn J. , Lerner Megan R. , Bronze Michael S. , Brackett Daniel J.
Published: April 2014

Blood borne Listeria monocytogenes enter the CNS via migration of parasitized Ly-6Chigh monocytes, but the signals that trigger this migration are not known. To understand more completely events leading to monocyte recruitment, experiments presented here combined microarray analysis of gene expression in the brains of experimentally infected mice with measurements of bacterial CFU and serum cytokines following i.v. infection with L. monocytogenes. At 24 and 48 h postinfection, the brain was sterile but there were significant changes in transcriptional activity related to serum proinflammatory cytokines. Real-time PCR confirmed mRNA up-regulation of genes related to IFN-γ, IL-1, and TNF-α, although IFN-γ itself was not up-regulated in the brain. Infection with Δacta, but not Δhly mutants, increased serum concentrations of IFN-γ, IL-6, and to a lesser extent TNF-α. The brain was not infected but there was widespread mRNA up-regulation in it and an influx of Ly-6Chigh monocytes in Δacta-infected mice. Moreover, ΔactA-infected IFN-γ−/− mice had no brain influx of Ly-6Chigh monocytes despite normal monocyte trafficking from bone marrow to blood and spleen. Additionally, IFN-γ−/− mice showed diminished mRNA expression for monocyte-attracting chemokines, and significantly less CXCL9 and CXCL10 protein in the brain compared with normal mice. These data demonstrate that monocyte recruitment to the brain is independent of bacterial invasion of the CNS and is triggered by proinflammatory cytokines, in particular IFN-γ, produced by the innate immune response to intracellular infection in peripheral organs.
Differential CMV-Specific CD8+ Effector T Cell Responses in the Lung Allograft Predominate over the Blood during Human Primary Infection
Authors: Pipeling Matthew R. , West Erin E. , Osborne Christine M. , Whitlock Amanda B. , Dropulic Lesia K. , Willett Matthew H. , Forman Michael , Valsamakis Alexandra , Orens Jonathan B. , Moller David R. , Lechtzin Noah , Migueles Stephen A. , Connors Mark , McDyer John F.
Published: April 2014

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient− (D+R−) individuals. In 15 D+R− LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA− during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA− phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.
Nonmucosal Alphavirus Vaccination Stimulates a Mucosal Inductive Environment in the Peripheral Draining Lymph Node
Authors: Thompson Joseph M. , Nicholson Michael G. , Whitmore Alan C. , Zamora Melodie , West Ande , Iwasaki Akiko , Staats Herman F. , Johnston Robert E.
Published: April 2014

The strongest mucosal immune responses are induced following mucosal Ag delivery and processing in the mucosal lymphoid tissues, and much is known regarding the immunological parameters which regulate immune induction via this pathway. Recently, experimental systems have been identified in which mucosal immune responses are induced following nonmucosal Ag delivery. One such system, footpad delivery of Venezuelan equine encephalitis virus replicon particles (VRP), led to the local production of IgA Abs directed against both expressed and codelivered Ags at multiple mucosal surfaces in mice. In contrast to the mucosal delivery pathway, little is known regarding the lymphoid structures and immunological components that are responsible for mucosal immune induction following nonmucosal delivery. In this study, we have used footpad delivery of VRP to probe the constituents of this alternative pathway for mucosal immune induction. Following nonmucosal VRP delivery, J chain-containing, polymeric IgA Abs were detected in the peripheral draining lymph node (DLN), at a time before IgA detection at mucosal surfaces. Further analysis of the VRP DLN revealed up-regulated α4β7 integrin expression on DLN B cells, expression of mucosal addressin cell adhesion molecule 1 on the DLN high endothelia venules, and production of IL-6 and CC chemokines, all characteristics of mucosal lymphoid tissues. Taken together, these results implicate the peripheral DLN as an integral component of an alternative pathway for mucosal immune induction. A further understanding of the critical immunological and viral components of this pathway may significantly improve both our knowledge of viral-induced immunity and the efficacy of viral-based vaccines.
MyD88 Regulation of Fusarium Keratitis Is Dependent on TLR4 and IL-1R1 but Not TLR2
Authors: Tarabishy Ahmad B. , Aldabagh Bishr , Sun Yan , Imamura Yoshifumi , Mukherjee Pranab K. , Lass Jonathan H. , Ghannoum Mahmoud A. , Pearlman Eric
Published: April 2014

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88−/− mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2−/−, TLR4−/−, and TLR2/4−/− mice were similar to those of C57BL/6 mice; however, TLR4−/− and TLR2/4−/− mice had impaired antifungal responses. The phenotype of infected IL-1R1−/− mice was similar to that of MyD88−/− mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1−/− mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.
Surfactant Protein A Binds to HIV and Inhibits Direct Infection of CD4+ Cells, but Enhances Dendritic Cell-Mediated Viral Transfer
Authors: Gaiha Gaurav D. , Dong Tao , Palaniyar Nades , Mitchell Daniel A. , Reid Kenneth B. M. , Clark Howard W.
Published: April 2014

The identification of surfactant protein A (SP-A) as an important innate immune factor of the lungs, amniotic fluid, and the vaginal tract suggests that it could play an important role during various stages of HIV disease progression and transmission. Therefore, we examined whether SP-A could bind to HIV and also had any effect on viral infectivity. Our data demonstrate that SP-A binds to HIV in a calcium-dependent manner that is inhibitable by mannose and EDTA. Affinity capture of the HIV viral lysate reveals that SP-A targets the envelope glycoprotein of HIV (gp120), which was confirmed by ELISA using recombinant gp120. Digestion of gp120 with endoglycosidase H abrogates the binding of SP-A, indicating that the high mannose structures on gp120 are the target of the collectin. Infectivity studies reveal that SP-A inhibits the infection of CD4+ T cells by two strains of HIV (BaL, IIIB) by >80%. Competition assays with CD4 and mAbs F105 and b12 suggest that SP-A inhibits infectivity by occlusion of the CD4-binding site. Studies with dendritic cells (DCs) demonstrate that SP-A enhances the binding of gp120 to DCs, the uptake of viral particles, and the transfer of virus from DCs to CD4+ T cells by >5-fold at a pH representative of the vaginal tract. Collectively, these results suggest that SP-A acts as a dual modulator of HIV infection by protecting CD4+ T cells from direct infection but enhancing the transfer of infection to CD4+ T cells mediated by DCs.
Terminal Deoxynucleotidyl Transferase Establishes and Broadens Antiviral CD8+ T Cell Immunodominance Hierarchies
Authors: Haeryfar S. M. Mansour , Hickman Heather D. , Irvine Kari R. , Tscharke David C. , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

The action of TdT on mouse TCR genes accounts for ∼90% of T cell repertoire diversity. We report that in TdT−/− mice, total TCD8+ responses to influenza and vaccinia viruses are reduced by ∼30% relative to wild-type mice. We find that TCD8+ responses to three subdominant influenza virus determinants are reduced to background values in TdT−/− mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in TCD8+ responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT−/− mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral TCD8+ responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.
Potent Anti-Inflammatory and Neuroprotective Effects of TGF-β1 Are Mediated through the Inhibition of ERK and p47phox-Ser345 Phosphorylation and Translocation in Microglia
Authors: Qian Li , Wei Sung-Jen , Zhang Dan , Hu Xiaoming , Xu Zongli , Wilson Belinda , El-Benna Jamel , Hong Jau-Shyong , Flood Patrick M.
Published: April 2014

TGF-β1 is one of the most potent endogenous immune modulators of inflammation. The molecular mechanism of its anti-inflammatory effect on the activation of the transcription factor NF-κB has been well-studied; however, the potential effects of TGF-β1 on other proinflammatory signaling pathways is less clear. In this study, using the well-established LPS and the 1-methyl-4-phenylpyridinium-mediated models of Parkinson’s disease, we demonstrate that TGF-β1 exerts significant neuroprotection in both models via its anti-inflammatory properties. The neuroprotective effects of TGF-β1 are mainly attributed to its ability to inhibit the production of reactive oxygen species from microglia during their activation or reactivation. Moreover, we demonstrate that TGF-β1 inhibited LPS-induced NADPH oxidase (PHOX) subunit p47phox translocation from the cytosol to the membrane in microglia within 10 min. Mechanistic studies show that TGF-β1 fails to protect dopaminergic neurons in cultures from PHOX knockout mice, and significantly reduced LPS-induced translocation of the PHOX cytosolic subunit p47phox to the cell membrane. In addition, LPS-induced ERK phosphorylation and subsequent Ser345 phosphorylation on p47phox were significantly inhibited by TGF-β1 pretreatment. Taken together, our results show that TGF-β1 exerted potent anti-inflammatory and neuroprotective properties, either through the prevention of the direct activation of microglia by LPS, or indirectly through the inhibition of reactive microgliosis elicited by 1-methyl-4-phenylpyridinium. The molecular mechanisms of TGF-β1-mediated anti-inflammatory properties is through the inhibition of PHOX activity by preventing the ERK-dependent phosphorylation of Ser345 on p47phox in microglia to reduce oxidase activities induced by LPS.