bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysozyme-Modified Probiotic Components Protect Rats against Polymicrobial Sepsis: Role of Macrophages and Cathelicidin-Related Innate Immunity
Authors: Bu Heng-Fu , Wang Xiao , Zhu Ya-Qin , Williams Roxanne Y. , Hsueh Wei , Zheng Xiaotian , Rozenfeld Ranna A. , Zuo Xiu-Li , Tan Xiao-Di
Published: April 2014

Severe sepsis is associated with dysfunction of the macrophage/monocyte, an important cellular effector of the innate immune system. Previous investigations suggested that probiotic components effectively enhance effector cell functions of the immune system in vivo. In this study, we produced bacteria-free, lysozyme-modified probiotic components (LzMPC) by treating the probiotic bacteria, Lactobacillus sp., with lysozyme. We showed that oral delivery of LzMPC effectively protected rats against lethality from polymicrobial sepsis induced by cecal ligation and puncture. We found that orally administrated LzMPC was engulfed by cells such as macrophages in the liver after crossing the intestinal barrier. Moreover, LzMPC-induced protection was associated with an increase in bacterial clearance in the liver. In vitro, LzMPC up-regulated the expression of cathelicidin-related antimicrobial peptide (CRAMP) in macrophages and enhanced bactericidal activity of these cells. Furthermore, we demonstrated that surgical stress or cecal ligation and puncture caused a decrease in CRAMP expression in the liver, whereas enteral administration of LzMPC restored CRAMP gene expression in these animals. Using a neutralizing Ab, we showed that protection against sepsis by LzMPC treatment required endogenous CRAMP. In addition, macrophages from LzMPC-treated rats had an enhanced capacity of cytokine production in response to LPS or LzMPC stimulation. Together, our data suggest that the protective effect of LzMPC in sepsis is related to an enhanced cathelicidin-related innate immunity in macrophages. Therefore, LzMPC, a novel probiotic product, is a potent immunomodulator for macrophages and may be beneficial for the treatment of sepsis.
SHIP1 Negatively Regulates Proliferation of Osteoclast Precursors via Akt-Dependent Alterations in D-Type Cyclins and p27
Authors: Zhou Ping , Kitaura Hideki , Teitelbaum Steven L. , Krystal Gerald , Ross F. Patrick , Takeshita Sunao
Published: April 2014

Osteoclasts arise from macrophage progenitors in bone marrow (BMMs) as a consequence of signaling events elicited by M-CSF and receptor activator of NF-κB ligand, acting on their unique receptors, via c-Fms and receptor activator of NF-κB. Both receptors activate the PI3K and MAPK pathways, which promote cell proliferation and survival. SHIP1 is essential for normal bone homeostasis, as mice lacking the protein exhibit osteoporosis resulting from increased numbers of hyper-resorptive osteoclasts. In this study, we show that BMMs from SHIP1 null mice respond to M-CSF, but not receptor activator of NF-κB ligand, by increasing Akt activation. In consequence, there are up-regulation of D-type cyclins, down-regulation of the cyclin-dependent kinase inhibitor p27, and, therefore, increased phosphorylation of the retinoblastoma protein and cell proliferation. Surprisingly, cell survival of wild-type and knockout BMMs is unaltered. Finally, osteoclastogenesis and periarticular bone erosions are markedly increased in SHIP1−/− mice with inflammatory arthritis, a condition characterized by increased M-CSF expression. The SHIP1/Akt pathway therefore suppresses bone loss in pathological states associated with an excess of the cytokine.
Aberrant Regulation of Synovial T Cell Activation by Soluble Costimulatory Molecules in Rheumatoid Arthritis
Authors: Wan Bing , Nie Hong , Liu Ailian , Feng Guozhang , He Dongyi , Xu Rong , Zhang Qi , Dong Chen , Zhang Jingwu Z.
Published: April 2014

T cell activation and function are critically regulated by positive and negative costimulatory molecules. Aberrant expression and function of costimulatory molecules have been associated with persistent activation of self-reactive T cells in autoimmune diseases such as rheumatoid arthritis (RA). In this study, initial analysis of costimulatory molecules led to the unexpected observation that, in addition to CD80, several negative regulators (e.g., CTLA-4, programmed death-1 (PD-1), and PD ligand-1) were overexpressed in synovial T cells and macrophages derived from RA patients as opposed to controls. The expression of CD80 and PD ligand-1 on monocytes could be induced in vitro by IFN-γ and TNF-α that were produced abundantly in RA-derived synovial fluid (SF). Furthermore, the soluble form of negative costimulatory molecules occurred at high concentrations in sera and SF of RA patients and correlated with titers of rheumatoid factor in RA patients. In particular, the levels of soluble PD-1 were found to correlate significantly with those of TNF-α in SF derived from RA patients. Detailed characterization of soluble PD-1 revealed that it corresponded to an alternative splice variant (PD-1Δex3) and could functionally block the regulatory effect of membrane-bound PD-1 on T cell activation. Our data indicate a novel pathogenic pathway in which overexpression of negative costimulatory molecules to restrict synovial inflammation in RA is overruled by the excessive production of soluble costimulatory molecules.
Adenovirus-Specific CD4+ T Cell Clones Recognizing Endogenous Antigen Inhibit Viral Replication In Vitro through Cognate Interaction
Authors: Heemskerk Bianca , van Vreeswijk Tamara , Veltrop-Duits Louise A. , Sombroek Claudia C. , Franken Kees , Verhoosel Renate M. , Hiemstra Pieter S. , van Leeuwen Daphne , Ressing Maaike E. , Toes René E. M. , van Tol Maarten J. D. , Schilham Marco W.
Published: April 2014

Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4+ T cells. CD4+ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4+ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4+ T cell clones when derived from endogenously produced hexon protein. Together, these results show that cross-reactive HAdV-specific CD4+ T cells can control replication of HAdV in vitro and provide a rationale for the use of HAdV-specific T cells in adoptive immunotherapy protocols for control of life-threatening HAdV-infections in immunocompromised patients.
Synergism between CpG-Containing Oligodeoxynucleotides and IL-2 Causes Dramatic Enhancement of Vaccine-Elicited CD8+ T Cell Responses
Authors: Kochenderfer James N. , Chien Christopher D. , Simpson Jessica L. , Gress Ronald E.
Published: April 2014

Novel anticancer vaccination regimens that can elicit large numbers of Ag-specific T cells are needed. When we administered therapeutic vaccines containing the MHC class I-presented self-peptide tyrosinase-related protein (TRP)-2180–188 and CpG-containing oligodeoxynucleotides (CpG ODN) to mice, growth of the TRP-2-expressing B16F1 melanoma was not inhibited compared with growth in mice that received control vaccinations. When we added systemic IL-2 to the TRP-2180–188 plus CpG ODN vaccines, growth of B16F1 was inhibited in a CD8-dependent, epitope-specific manner. Vaccines containing TRP-2180–188 without CpG ODN did not cause epitope-specific tumor growth inhibition when administered with IL-2. The antitumor efficacy of the different regimens correlated with their ability to elicit TRP-2180–188-specific CD8+ T cell responses. When we administered TRP-2180–188 plus CpG ODN-containing vaccines with systemic IL-2, 18.2% of CD8+ T cells were specific for TRP-2180–188. Identical TRP-2180–188 plus CpG ODN vaccines given without IL-2 elicited a TRP-2180–188-specific CD8+ T cell response of only 1.1% of CD8+ T cells. Vaccines containing TRP-2180–188 without CpG ODN elicited TRP-2180–188-specific responses of 2.8% of CD8+ T cells when administered with IL-2. There was up to a 221-fold increase in the absolute number of TRP-2180–188-specific CD8+ T cells when IL-2 was added to TRP-2180–188 plus CpG ODN-containing vaccines. Peptide plus CpG ODN vaccines administered with IL-2 generated epitope-specific CD8+ T cells by a mechanism that depended on endogenous IL-6. This is the first report of synergism between CpG ODN and IL-2. This synergism caused a striking increase in vaccine-elicited CD8+ T cells and led to epitope-specific antitumor immunity.
Differential Pattern Recognition Receptor Expression but Stereotyped Responsiveness in Rat Spleen Dendritic Cell Subsets
Authors: Hubert François-Xavier , Voisine Cécile , Louvet Cédric , Heslan Jean-Marie , Ouabed Asmahan , Heslan Michèle , Josien Régis
Published: April 2014

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4−, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4− DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4− and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4− DC to mature and produce high IL-12p40, low IL-10, and TNF-α. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-α and low IFN-α. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.
Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates Invariant NKT Cell-Mediated Cross-Talk with Myeloid Dendritic Cells
Authors: Montoya Carlos J. , Jie Hyun-Bae , Al-Harthi Lena , Mulder Candice , Patiño Pablo J. , Rugeles María T. , Krieg Arthur M. , Landay Alan L. , Wilson S. Brian
Published: April 2014

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-α. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.
Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils
Authors: MacGlashan Donald , Vilariño Natalia
Published: April 2014

Previous studies of secretion from basophils have demonstrated the phenomenon called nonspecific desensitization, the ability of one IgE-mediated stimulus to alter the cell’s response to other non-cross-reacting IgE-mediated stimuli, and a process that would modify phosphatidylinositol 3,4,5-phosphate levels was speculated to be responsible for nonspecific desensitization. The current studies examined the changes and characteristics of SHIP1 phosphorylation as a measure of SHIP1 participation in the reaction. Based on the earlier studies, two predictions were made that were not observed. First, the kinetics of SHIP1 phosphorylation were similar to reaction kinetics of other early signals and returned to resting levels while nonspecific desensitization remained. Second, in contrast to an expected exaggerated SHIP phosphorylation, cells in a state of nonspecific desensitization showed reduced SHIP phosphorylation (compared with cells not previously exposed to a non-cross-reacting Ag). Discordant with expectations concerning partial recovery from nonspecific desensitization, treatment of cells with DNP-lysine to dissociate bound DNP-HSA, either enhanced or had no effect on SHIP phosphorylation following a second Ag. These experiments also showed a form of desensitization that persisted despite dissociation of the desensitizing Ag. Recent studies and the results of these studies suggest that loss of early signaling components like syk kinase may account for some of the effects of nonspecific desensitization and result in a form of immunological memory of prior stimulation. Taken together, the various characteristics of SHIP phosphorylation were not consistent with expectations for a signaling element involved in nonspecific desensitization, but instead one which itself undergoes nonspecific desensitization.
CTLA-4 Overexpression Inhibits T Cell Responses through a CD28-B7-Dependent Mechanism
Authors: Engelhardt John J. , Sullivan Timothy J. , Allison James P.
Published: April 2014

CTLA-4 has been shown to be an important negative regulator of T cell activation. To better understand its inhibitory action, we constructed CTLA-4 transgenic mice that display constitutive cell surface expression of CTLA-4 on CD4 and CD8 T cells. In both in vivo and in vitro T cell responses, CTLA-4 overexpression inhibits T cell activation. This inhibition is dependent on B7 and CD28, suggesting that overexpressed CTLA-4 inhibits responses by competing with CD28 for B7 binding or by interfering with CD28 signaling. In addition, expression of the transgene decreases the number of CD25+Foxp3+ T cells in these mice, but does not affect their suppressive ability. Our data confirm the activity of CTLA-4 as a negative regulator of T cell activation and that its action may be by multiple mechanisms.
Epigenetic Remodeling of theIL-2andIFN-γ Loci in Memory CD8 T Cells Is Influenced by CD4 T Cells
Authors: Northrop John K. , Thomas Rajan M. , Wells Andrew D. , Shen Hao
Published: April 2014

Memory T cells (TM) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as TM undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 TM by comparing them to defective CD8 TM generated in the absence of CD4 T cells. Unhelped CD8 TM are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-γ. Our data show that this defect in IL-2 and IFN-γ production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-γ loci and substantial histone acetylation at the IFN-γ promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-γ locus. Thus, CD4 T cell help influences epigenetic modification during CD8 TM differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a “ready-to-respond” state in CD8 TM.
Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation
Authors: Watanabe Ryosuke , Harada Yohsuke , Takeda Kei , Takahashi Jun , Ohnuki Kazunobu , Ogawa Shuhei , Ohgai Daisuke , Kaibara Nanako , Koiwai Osamu , Tanabe Kazunari , Toma Hiroshi , Sugamura Kazuo , Abe Ryo
Published: April 2014

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.
Prostaglandin E2 Augments IL-10 Signaling and Function
Authors: Cheon HyeonJoo , Rho Young Hee , Choi Seong Jae , Lee Young Ho , Song Gwan Gyu , Sohn Jeongwon , Won Nam Hee , Ji Jong Dae
Published: April 2014

In inflamed joints of rheumatoid arthritis, PGE2 is highly expressed, and IL-10 and IL-6 are also abundant. PGE2 is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE2 on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE2 significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE2 suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE2-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE2-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE2 selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE2 may modulate immune responses by alteration of cytokine signaling in THP-1 cells.
Germinal Center Function in the Spleen during Simian HIV Infection in Rhesus Monkeys
Authors: Margolin David H. , Saunders Erika H. , Bronfin Benjamin , de Rosa Nicole , Axthelm Michael K. , Goloubeva Olga G. , Eapen Sara , Gelman Rebecca S. , Letvin Norman L.
Published: April 2014

Infection with HIV-1, SIV, or simian HIV is associated with abnormalities in the number, size, and structure of germinal centers (GCs). To determine whether these histopathologic abnormalities are associated with abnormalities in Ab development, we analyzed nucleotide sequences of Igs from splenic GCs of simian HIV-infected macaques. Virus-specific GCs were identified in frozen splenic tissue sections by inverse immunohistochemistry using rHIV-1 gp120 as a probe. B cells from envelope-specific GCs were isolated from these sections using laser capture microdissection. Their Igs were amplified from cDNA using nested PCR, then cloned and sequenced. Nucleotide sequences were recovered from nine multimember clonal lineages. Within each lineage, sequences had similar V-D-J or V-J junctions but differed by somatic mutations distributed throughout the variable domain. The clones were highly mutated, similar to that previously reported for HIV-1-specific human IgG Abs. The average clone had 37 mutations in the V region, for a frequency of 0.11 mutations/base. The mutational pattern was strikingly nonrandom, with somatic mutations occurring preferentially at RGYW/WRCY hotspots. Transition mutations were favored over transversions, with C→T and G→A replacements together accounting for almost one-third of all mutations. Analysis of replacement and silent mutations in the framework and CDRs suggests that the Igs were subjected to affinity selection. These data demonstrate that the process of Ab maturation is not seriously disrupted in GCs during the early stages of immunodeficiency virus infection, and that Env-specific Igs developing in GCs are subject to extensive somatic mutation and profound selection pressures.
Sumoylation of Daxx Regulates IFN-Induced Growth Suppression of B Lymphocytes and the Hormone Receptor-Mediated Transactivation
Authors: Muromoto Ryuta , Ishida Masato , Sugiyama Kenji , Sekine Yuichi , Oritani Kenji , Shimoda Kazuya , Matsuda Tadashi
Published: April 2014

Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.
Granzyme M Directly Cleaves Inhibitor of Caspase-Activated DNase (CAD) to Unleash CAD Leading to DNA Fragmentation
Authors: Lu Hongxia , Hou Qiang , Zhao Tongbiao , Zhang Honglian , Zhang Qixiang , Wu Lianfeng , Fan Zusen
Published: April 2014

Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis.
GM1 Binding-Deficient Exotoxin Is a Potent Noninflammatory Broad Spectrum Intradermal Immunoadjuvant
Authors: Zoeteweij J. Paul , Epperson Diane E. , Porter Jackie D. , Zhang Chen X. , Frolova Olga Y. , Constantinides Anita P. , Fuhrmann Steven R. , El-Amine Moustapha , Tian Jing-Hui , Ellingsworth Larry R. , Glenn Gregory M.
Published: April 2014

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.
Epithelial Cell IκB-Kinase β Has an Important Protective Role inClostridium difficileToxin A-Induced Mucosal Injury
Authors: Chae Sungwon , Eckmann Lars , Miyamoto Yukiko , Pothoulakis Charalabos , Karin Michael , Kagnoff Martin F.
Published: April 2014

Toxin A released by Clostridium difficile interacts with the single layer of intestinal epithelial cells that lines the host’s intestinal tract and leads to mucosal damage and inflammation that manifests clinically as antibiotic-associated diarrhea and pseudomembranous colitis. Activation of the transcription factor NF-κB in intestinal epithelial cells is important for regulating the expression of epithelial cell proinflammatory genes and cell survival. However, the role of NF-κB activation in the pathogenesis of C. difficile toxin A-induced colitis is unknown. To determine the functional importance in vivo of NF-κB activation in intestinal epithelium in the pathogenesis of C. difficile-induced colitis, we used mutant mice that do not activate the classical NF-κB signaling pathway in intestinal epithelial cells due to a conditional deficiency in those cells of the IκB-kinase β (IKKβ) subunit of IKK. C. difficile toxin A challenge of intestinal loops in intestinal epithelial cell IKKβ-deficient mice induced a rapid and significant increase in intestinal epithelial apoptosis compared with littermate controls. This was accompanied by a significant increase in acute mucosal inflammation, mucosal injury, luminal fluid secretion, and bacterial translocation. We conclude that activation of intestinal epithelial cell NF-κB by toxin A plays an important host mucosal protective role after C. difficile toxin A exposure that is mediated, at least in part, through promoting epithelial cell survival by abrogating epithelial cell apoptosis.
Streptococcal M Protein: A Multipotent and Powerful Inducer of Inflammation
Authors: Påhlman Lisa I. , Mörgelin Matthias , Eckert Jana , Johansson Linda , Russell Wayne , Riesbeck Kristian , Soehnlein Oliver , Lindbom Lennart , Norrby-Teglund Anna , Schumann Ralf R. , Björck Lars , Herwald Heiko
Published: April 2014

Severe infections with Streptococcus pyogenes, an important human pathogen, are associated with massive inflammatory reactions in the human host. Here we show that streptococcal M protein interacts with TLR2 on human peripheral blood monocytes. As a consequence, monocytes express the cytokines IL-6, IL-1β, and TNF-α. This response is significantly increased in the presence of neutrophil-derived heparin-binding protein (HBP), which costimulates monocytes by interacting with CD11/CD18. Analysis of tissue biopsies from patients with necrotizing fasciitis revealed recruitment of neutrophils and monocytes to the infectious site, combined with the release of HBP. The results show that M protein, in synergy with HBP, evokes an inflammatory response that may contribute to the profound pathophysiological consequences seen in severe streptococcal infections.
NK Cell Responses toPlasmodiumInfection and Control of Intrahepatic Parasite Development
Authors: Roland Jacques , Soulard Valérie , Sellier Christèle , Drapier Anne-Marie , Di Santo James P. , Cazenave Pierre-André , Pied Sylviane
Published: April 2014

Various components of innate and adaptive immunity contribute to host defenses against Plasmodium infection. We investigated the contribution of NK cells to the immune response to primary infection with Plasmodium yoelii sporozoites in C57BL/6 mice. We found that hepatic and splenic NK cells were activated during infection and displayed different phenotypic and functional properties. The number of hepatic NK cells increased whereas the number of splenic NK cells decreased. Expression of the Ly49 repertoire was modified in the spleen but not in the liver. Splenic and hepatic NK cells have a different inflammatory cytokines profile production. In addition, liver NK cells were cytotoxic to YAC-1 cells and P. yoelii liver stages in vitro but not to erythrocytic stages. No such activity was observed with splenic NK cells from infected mice. These in vitro results were confirmed by the in vivo observation that Rag2−/− mice were more resistant to sporozoite infection than Rag2−/− γ c−/− mice, whereas survival rates were similar for the two strains following blood-stage infection. Thus, NK cells are involved in early immune mechanisms controlling Plasmodium infection, mostly at the pre-erythrocytic stage.
Chlamydia Heat Shock Protein 60 Induces Trophoblast Apoptosis through TLR4
Authors: Equils Ozlem , Lu Daning , Gatter Mary , Witkin Steve S. , Bertolotto Cristina , Arditi Moshe , McGregor James A. , Simmons Charles F. , Hobel Calvin J.
Published: April 2014

Intrauterine infection affects placental development and function, and subsequently may lead to complications such as preterm delivery, intrauterine growth retardation, and preeclampsia; however, the molecular mechanisms are not clearly known. TLRs mediate innate immune responses in placenta, and recently, TLR2-induced trophoblast apoptosis has been suggested to play a role in infection-induced preterm delivery. Chlamydia trachomatis is the etiological agent of the most prevalent sexually transmitted bacterial infection in the United States. In this study, we show that in vitro chlamydial heat shock protein 60 induces apoptosis in primary human trophoblasts, placental fibroblasts, and the JEG3 trophoblast cell line, and that TLR4 mediates this event. We observed a host cell type-dependent apoptotic response. In primary placental fibroblasts, chlamydial heat shock protein 60-induced apoptosis was caspase dependent, whereas in JEG3 trophoblast cell lines it was caspase independent. These data suggest that TLR4 stimulation induces apoptosis in placenta, and this could provide a novel mechanism of pathogenesis for poor fertility and pregnancy outcome in women with persistent chlamydia infection.
Signaling via Platelet-Activating Factor Receptors Accounts for the Impairment of Neutrophil Migration in Polymicrobial Sepsis
Authors: Moreno Susana E. , Alves-Filho José C. , Rios-Santos Fabrício , Silva João S. , Ferreira Sérgio H. , Cunha Fernando Q. , Teixeira Mauro M.
Published: April 2014

Sepsis is a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. Recently, we have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This impairment seems to be mediated by circulating cytokines, chemokines, and NO. Platelet-activating factor (PAF) plays an important role in the orchestration of different inflammatory reactions, including the release of cytokines, chemokines, and free radicals. Using a PAFR antagonist, PCA-4248, and PAFR-deficient mice, we investigated whether signaling via PAFR was relevant for the failure of neutrophils to migrate to the site of infection after lethal sepsis caused by cecum ligation and puncture in mice. In PAFR-deficient mice or mice pretreated with PCA-4248 (5 mg/kg) and subjected to lethal sepsis, neutrophil migration failure was prevented, and bacterial clearance was more efficient. There was also reduced systemic inflammation (low serum cytokine levels), lower nitrate levels in plasma, and higher survival rate. Altogether, the results firmly establish a role for PAFR in mediating the early impairment of neutrophil migration toward the infectious focus. Blockade of PAFR may prevent the establishment of severe sepsis.
Hyaluronan Fragments Act as an Endogenous Danger Signal by Engaging TLR2
Authors: Scheibner Kara A. , Lutz Michael A. , Boodoo Sada , Fenton Matthew J. , Powell Jonathan D. , Horton Maureen R.
Published: April 2014

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Cζ-, and NF-κB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2null mice.
Human Monocytes Recognize Porcine Endothelium via the Interaction of Galectin 3 and α-GAL
Authors: Jin Rongyu , Greenwald Allen , Peterson Mark D. , Waddell Thomas K.
Published: April 2014

Monocytes are one of the key inflammatory cells recruited to xenografts and play an important role in delayed xenograft rejection. Previous studies have demonstrated the ability of monocytes to bind to the major xenoantigen Gal-α(1,3)Gal-β(1,4)GlcNAc-R; however, the receptor that mediates this interaction has yet to be identified. We provide evidence that it is Galectin-3, a ∼30-kDa lectin that recognizes β-galactosides (Gal-β(1–3/4)GlcNAc) and plays diverse roles in many physiological and pathological events. Human monocyte binding is strikingly increased on porcine aortic endothelial cells (PAEC), which express high levels of Gal-α(1,3)Gal-β(1,4)GlcNAc-R, compared with human aortic endothelial cells. Human monocytes obtained from healthy donors bind to Gal-α(1,3)Gal-β(1,4)GlcNAc-R at variable intensities. This variation of binding intensity was consistent and reproducible in individual donors. Galectin-3 is mainly expressed in human monocytes, not lymphocytes. Purified Galectin-3 is able to bind directly to Gal-α(1,3)Gal-β(1,4)GlcNAc-R. Galectin-3 can also be affinity isolated from monocytes (and not lymphocytes) using an Gal-α(1,3)Gal-β(1,4)GlcNAc-R-biotin/streptavidin-bead pull-down system. Soluble Galectin-3 binds preferentially to PAEC vs human aortic endothelial cells, and this binding can be inhibited by lactose, indicating dependence on the carbohydrate recognition domain of Galectin-3. Gal-α(1,3)Gal-β(1,4)GlcNAc-R is at least partly responsible for this phenomenon, as binding decreased after digestion of PAEC with α-galactosidase. Furthermore, monocytes pretreated with a blocking anti-Galectin-3 Ab show decreased adhesion to PAEC when compared with isotype control in a parallel plate flow chamber perfusion assay. Thus, we conclude that Galectin-3 expressed in human monocytes is a receptor for the major xenoantigen (Gal-α(1,3)Gal-β(1,4)GlcNAc-R), expressed on porcine endothelial cells.
Zinc Is Required for FcεRI-Mediated Mast Cell Activation
Authors: Kabu Koki , Yamasaki Satoru , Kamimura Daisuke , Ito Yukitaka , Hasegawa Aiko , Sato Emi , Kitamura Hidemitsu , Nishida Keigo , Hirano Toshio
Published: April 2014

Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon FcεRI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the FcεRI-induced degranulation and cytokine production. We found that Zn was required for FcεRI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-κB, leading to cytokine production, such as IL-6 and TNF-α. These results revealed that Zn was involved in multiple steps of FcεRI-induced MC activation and required for degranulation and cytokine production.
Divergent Signaling Pathways in Phagocytic Cells during Sepsis
Authors: Guo Ren-Feng , Riedemann Niels C. , Sun Lei , Gao Hongwei , Shi Kevin X. , Reuben Jayne S. , Sarma Vidya J. , Zetoune Firas S. , Ward Peter A.
Published: April 2014

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.
TNF-α-Independent IL-8 Expression: Alterations in Bacterial Challenge Dose Cause Differential Human Monocytic Cytokine Response
Authors: Patrone Julia B. , Bish Samuel E. , Stein Daniel C.
Published: April 2014

We examined the effects of different bacterial doses of Neisseria gonorrhoeae on the cytokine response of primary human monocytes. The data indicate that a low multiplicity of infection (MOI) challenge (MOI = 0.1) results in substantial production of IL-8 and other chemokines/cytokines, in the absence of significant TNF-α production. Positive control challenges (MOI = 10) induced levels of IL-8 that were comparable to the low MOI challenges, but now induced significant levels of TNF-α. Induction of IL-8 expression in low MOI challenges was not mediated by an autocrine response as pretreatment of monocytes with neutralizing Abs against TNF-α or IL-1β had no effect on IL-8 expression. IL-8 induction resulting from gonococcal challenge was shown to require NF-κB activation, though this activation was limited by the inoculating dose. These data indicate that IL-8 induction results from direct contact between bacteria and monocytes. Analysis of the overall cytokine profile revealed patterns of expression for growth-regulated oncogene, MCP-1, and IL-6 that were similar to IL-8. Analysis of various MAPKs indicated that low MOI challenges were able to efficiently activate both the ERK and p38 pathways, but in contrast to positive control samples, failed to activate the JNK pathway. A lack of phosphorylated JNK leads to decreased production of AP-1 dimers, transcription factors that are critical for efficient transcription of TNF-α. Therefore, we propose a mechanism where a low MOI gonococcal challenge results in diminished AP-1 activity and TNF-α production while IL-8 levels remain constant.
Role of TLR9 in Anti-Nucleosome and Anti-DNA Antibody Production in lpr Mutation-Induced Murine Lupus
Authors: Lartigue Aurelia , Courville Philippe , Auquit Isabelle , François Arnaud , Arnoult Christophe , Tron Francois , Gilbert Daniele , Musette Philippe
Published: April 2014

Systemic lupus erythematosus is characterized by the production of autoantibodies directed against nuclear Ags, including nucleosome and DNA. TLR9 is thought to play a role in the production of these autoantibodies through the capacity of nuclear immunogenic particles to interact both with BCR and TLR9. To determine the role of TLR9 in SLE, C57BL/6-lpr/lpr-TLR9−/− and TLR9+/+ mice were analyzed. The abrogation of TLR9 totally impaired the production of anti-nucleosome Abs, whereas no difference was observed in the frequency of anti-dsDNA autoantibodies whose titer was strikingly higher in TLR9−/− mice. In addition a higher rate of mesangial proliferation was observed in the kidney of TLR9-deficient animals. These results indicate that in C57BL/6-lpr/lpr mice, TLR9 is absolutely required for the anti-nucleosome Ab response but not for anti-dsDNA Ab production which is involved in mesangial proliferation.
Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII
Authors: Lacroix-Desmazes Sébastien , Wootla Bharath , Dasgupta Suryasarathi , Delignat Sandrine , Bayry Jagadeesh , Reinbolt Joseph , Hoebeke Johan , Saenko Evgueni , Kazatchkine Michel D. , Friboulet Alain , Christophe Olivier , Nagaraja Valakunja , Kaveri Srini V.
Published: April 2014

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.
CD1d Ligands: The Good, the Bad, and the Ugly
Authors: Brutkiewicz Randy R.
Published: April 2014

The MHC class I-like CD1d glycoprotein is a member of the CD1 family of Ag-presenting molecules and is responsible for the selection of NKT cells. A number of ligands that can be presented by CD1d to NKT or other CD1d-restricted T cells have been identified. These include glycolipids from a marine sponge, bacterial glycolipids, normal endogenous glycolipids, tumor-derived phospholipids and glycolipids, and nonlipidic molecules. The presentation of many of these molecules can have immunopotentiating effects, such as serving as an adjuvant against malaria or resulting in a more rapid clearance of certain virus infections. They can also be protective in autoimmune diseases or cancer or can be deleterious. This review will highlight these ligands in a discussion of their potential use against (and role in the pathogenesis of) these diseases.
Cutting Edge: Latecomer CD8 T Cells Are Imprinted with a Unique Differentiation Program
Authors: D’Souza Warren N. , Hedrick Stephen M.
Published: April 2014

Factors that influence T cell responses, such as Ag load, APCs, costimulatory molecules, and cytokines, dramatically change during the course of an immune response. We observed that antiviral CD8 T cells were not recruited from circulation simultaneously, but over a period of 3–4 days. Consequently, locally resident T cells and those that entered secondary lymphoid tissue later were primed in very different environments. The cells recruited later in the response were imprinted with a unique differentiation program, such that their magnitude of proliferation was reduced and their kinetics of expansion was delayed. In addition, we found that the “latecomer” CD8 T cells displayed a unique surface phenotype indicative of reduced stimulation but were not preferentially recruited into the surviving pool of memory cells. This finding demonstrates that the timing of recruitment of individual T cell clones determines the population dynamics of the subsequent immune response.