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1.  Flt3 Ligand Generates Morphologically Distinct Semimature Dendritic Cells in Ovalbumin-sensitized Mice 
Dendritic cells (DCs) are unique antigen presenting cells that are immature prior to their encounter with an antigen. Exposure to allergens induces the maturation of DCs with changes in morphology and presence of dendrites. Here, we demonstrate that the DCs in the lungs of ovalbumin (OVA)-sensitized and challenged mice are more mature owing to their pronounced dendrites than the DCs in the lungs and spleen of PBS-treated mice, which are immature and possess cytoplasmic veils. Intermediate to these two groups are the DCs in the Flt3 ligand-treated group that exhibit comparatively fewer dendrites and cytoplasmic veils and hence are classified as semimature. Presence of large numbers of well developed mitochondria and rough endoplasmic reticulum in myeloid DCs from both lungs and spleen of OVA-sensitized and challenged mice indicate greater functional activity. Additionally, DCs from the OVA-sensitized and challenged mice also exhibit fat and glycogen stores, which are indicative of a mature population. In addition, treatment of the animals with Flt3 ligand attenuated airway hyperresponsiveness to methacholine in OVA-sensitized and challenged mice. These data suggest that morphological features could be indicative of the maturation and distinct functional state of DCs, and this could be associated with underlying mechanisms of Flt3 ligand-induced immunomodulation in allergic asthma.
doi:10.1016/j.yexmp.2006.09.007
PMCID: PMC2745173  PMID: 17182033
Airway hyperresponsiveness; Allergy; Asthma; Cytoplasmic veils; Dendritic cells; Flt3 ligand; Immunomodulation; Morphology of dendritic cells
2.  α-1-Antitrypsin Gene Delivery Reduces Inflammation, Increases T-Regulatory Cell Population Size and Prevents Islet Allograft Rejection 
Molecular Medicine  2011;17(9-10):1000-1011.
Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated “EF”) alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline–injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure–function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.
doi:10.2119/molmed.2011.00145
PMCID: PMC3188864  PMID: 21670848
3.  Unconventional Maturation of Dendritic Cells Induced by Particles from the Laminated Layer of Larval Echinococcus granulosus 
Infection and Immunity  2014;82(8):3164-3176.
The larval stage of the cestode parasite Echinococcus granulosus causes hydatid disease in humans and livestock. This infection is characterized by the growth in internal organ parenchymae of fluid-filled structures (hydatids) that elicit surprisingly little inflammation in spite of their massive size and persistence. Hydatids are protected by a millimeter-thick layer of mucin-based extracellular matrix, termed the laminated layer (LL), which is thought to be a major factor determining the host response to the infection. Host cells can interact both with the LL surface and with materials that are shed from it to allow parasite growth. In this work, we analyzed the response of dendritic cells (DCs) to microscopic pieces of the native mucin-based gel of the LL (pLL). In vitro, this material induced an unusual activation state characterized by upregulation of CD86 without concomitant upregulation of CD40 or secretion of cytokines (interleukin 12 [IL-12], IL-10, tumor necrosis factor alpha [TNF-α], and IL-6). When added to Toll-like receptor (TLR) agonists, pLL-potentiated CD86 upregulation and IL-10 secretion while inhibiting CD40 upregulation and IL-12 secretion. In vivo, pLL also caused upregulation of CD86 and inhibited CD40 upregulation in DCs. Contrary to expectations, oxidation of the mucin glycans in pLL with periodate did not abrogate the effects on cells. Reduction of disulfide bonds, which are known to be important for LL structure, strongly diminished the impact of pLL on DCs without altering the particulate nature of the material. In summary, DCs respond to the LL mucin meshwork with a “semimature” activation phenotype, both in vitro and in vivo.
doi:10.1128/IAI.01959-14
PMCID: PMC4136206  PMID: 24842926
4.  Negative Selection in the Thymus Includes Semimature T Cells 
The thymic medulla plays a key role in negative selection (self-tolerance induction) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+8− thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells reaching the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullary T cells displays unique requirements for tolerance induction; depending upon the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.
PMCID: PMC2196120  PMID: 9016875
5.  DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori–specific immune tolerance, and asthma protection  
The Journal of Clinical Investigation  2012;122(3):1082-1096.
Persistent colonization with the gastric bacterial pathogen Helicobacter pylori causes gastritis and predisposes infected individuals to gastric cancer. Conversely, it is also linked to protection from allergic, chronic inflammatory, and autoimmune diseases. We demonstrate here that H. pylori inhibits LPS-induced maturation of DCs and reprograms DCs toward a tolerance-promoting phenotype. Our results showed that DCs exposed to H. pylori in vitro or in vivo failed to induce T cell effector functions. Instead, they efficiently induced expression of the forkhead transcription factor FoxP3, the master regulator of Tregs, in naive T cells. Depletion of DCs in mice infected with H. pylori during the neonatal period was sufficient to break H. pylori–specific tolerance. DC depletion resulted in improved control of the infection but also aggravated T cell–driven immunopathology. Consistent with the mouse data, DCs infiltrating the gastric mucosa of human H. pylori carriers exhibited a semimature DC-SIGN+HLA–DRhiCD80loCD86lo phenotype. Mechanistically, the tolerogenic activity of H. pylori–experienced DCs was shown to require IL-18 in vitro and in vivo; DC-derived IL-18 acted directly on T cells to drive their conversion to Tregs. CD4+CD25+ Tregs from infected wild-type mice but not Il18–/– or Il18r1–/– mice prevented airway inflammation and hyperresponsiveness in an experimental model of asthma. Taken together, our results indicate that tolerogenic reprogramming of DCs ensures the persistence of H. pylori and protects against allergic asthma in a process that requires IL-18.
doi:10.1172/JCI61029
PMCID: PMC3287234  PMID: 22307326
6.  Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassical Macrophage Activation  
Infection and Immunity  2005;73(1):308-316.
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-κB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.
doi:10.1128/IAI.73.1.308-316.2005
PMCID: PMC538969  PMID: 15618167
7.  In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates 
The Journal of Experimental Medicine  2011;208(12):2477-2488.
Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys.
Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.
doi:10.1084/jem.20111242
PMCID: PMC3256968  PMID: 22025302
8.  Amplification of Autoimmune Response through Induction of Dendritic Cell Maturation in Inflamed Tissues1 
Dendritic cells (DCs) are essential in T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans in type 1 diabetes. In this study, we investigated T cell induction of intra-islet DC maturation during the progression of the disease in both autoimmune-prone NOD and resistant C57BL/6 mice. We demonstrated steady-state capture and retention of unprocessed β cell-derived proteins by semimature intra-islet DCs in both mouse strains. T cell-mediated intra-islet inflammation induced an increase in CD40 and CD80 expression and processing of captured Ag by resident DCs without inducing the expression of the p40 subunit of IL-12/23. Some of the CD40high intra-islet DCs up-regulated CCR7, and a small number of CD40high DCs bearing unprocessed islet Ags were detected in the pancreatic lymph nodes in mice with acute intra-islet inflammation, demonstrating that T cell-mediated tissue inflammation augments migration of mature resident DCs to draining lymph nodes. Our results identify an amplification loop during the progression of autoimmune diabetes, in which initial T cell infiltration leads to rapid maturation of intra-islet DCs, their migration to lymph nodes, and expanded priming of more autoreactive T cells. Therapeutic interventions that intercept this process may be effective at halting the progression of type 1 diabetes.
doi:10.4049/jimmunol.0803543
PMCID: PMC3057894  PMID: 19234153
9.  Systemic Administration of Tolerogenic Dendritic Cells Ameliorates Murine Inflammatory Arthritis 
The expression of various cell surface molecules and the production of certain cytokines are important mechanisms by which dendritic cells (DC) are able to bias immune responses. This paper describes the effects of the inflammatory cytokine tumor necrosis factor (TNF)-α on DC phenotype and function. TNF-α treatment resulted in upregulation of MHC class II and CD86 in the absence of increased cell surface CD40 and CD80 or the production of IL-12. Additionally TNF-α treated cells were able to bias T cell responses towards an anti-inflammatory profile. On a note of caution this tolerogenic phenotype of the DC was not stable upon subsequent TLR-4 ligation as a 4 hour pulse of the TNF-α treated DC with lipopolysaccharide (LPS) resulted in the restoration of IL-12 production and an enhancement of their T cell stimulatory capacity which resulted in an increased IFN-γ production. However, TNF-α treated DC, when administered in vivo, were shown to ameliorate disease in collagen induced arthritis, an experimental model of inflammatory joint disease. Mice receiving TNF-α treated DC but not LPS matured DC had a delayed onset, and significantly reduced severity, of arthritis. Disease suppression was associated with reduced levels of collagen specific IgG2a and decreased inflammatory cell infiltration into affected joints. In summary the treatment of DC with TNF-α generates an antigen presenting cell with a phenotype that can reduce the pro-inflammatory response and direct the immune system towards a disease modifying, anti-inflammatory state.
doi:10.2174/1874312900802010071
PMCID: PMC2627532  PMID: 19156221
10.  IL-10-dependent partial refractoriness to Toll-like receptor stimulation modulates gut mucosal dendritic cell function 
European Journal of Immunology  2008;38(6):1533-1547.
The default response of the intestinal immune system to most antigens is the induction of immunological tolerance, which is difficult to reconcile with the constant exposure to ligands for TLR and other pattern recognition receptors. We showed previously that dendritic cells (DC) from the lamina propria of normal mouse intestine may be inherently tolerogenic and here we have explored how this might relate to the expression and function of Toll-like receptors (TLR). Lamina propria (LP) DC showed higher levels of TLR 2, 3, 4 and 9 protein expression than spleen and MLN DC, with most TLR-expressing DC in the gut being CD11clo, class II MHClo, CD103–, CD11b– and F4/80–. TLR expression by lamina propria DC was low in the upper small intestine and higher in distal small intestine and colon. Freshly isolated lamina propria DC expressed some CD40, CD80, CD86 and functional CCR7. These were up-regulated on CD11clo, but not on CD11chi LP DC by stimulation via TLR. However, there was little induction of IL-12 by either subset in response to TLR ligation. This was associated with constitutive IL-10 production and was reversed by blocking IL-10 function. Thus, IL-10 may maintain LP DC in a partially unresponsive state to TLR ligation, allowing them to have a critical role in immune homeostasis in the gut.
doi:10.1002/eji.200737909
PMCID: PMC2988418  PMID: 18461564
Dendritic cells; Mucosa; Tolerance; IL-10; TLR
11.  Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae 
Bioscience Reports  2012;32(Pt 6):549-557.
Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.
doi:10.1042/BSR20120066
PMCID: PMC3497726  PMID: 22861139
CCM1; mitochondria; moonlighting protein; splicing; stability of 15S rRNA; yeast.; aI4, COX1 fourth intron; bI4, COB fourth intron; COB, cytochrome b; COX1, cytochrome c oxidase subunit 1; DB, dilution buffer; HRP, horseradish peroxidase; mtDNA, mitochondrial DNA; ORF, open reading frame; pAb, polyclonal antibody; PPR, pentatricopeptide repeat; qPCR, quantitative PCR; RT, reverse transcription; SD, synthetic defined; SMDC, sudden mitochondrial deprivation of Ccm1p; SPA, staphylococcal protein A; WB, washing buffer; YEP, yeast extract peptone
12.  IL-1β-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells1 
Maturation resistance and tolerogenic properties can be conferred on human and murine dendritic cells (DC), -crucial regulators of T cell responses, by exposure to rapamycin (RAPA), a ‘tolerance-sparing’ immunosuppressive agent. Mechanisms underlying this acquired unresponsiveness, typified by diminished functional responses to TLR or CD40 ligation, have not been identified. We report that in vitro and in vivo conditioning of murine myeloid (m) DC with RAPA elicits the de novo production of IL-1β by otherwise phenotypically immature DC. Interestingly, IL-1β production promotes overexpression of the transmembrane form of the IL-1R family member, IL-1R-like 1, also know as ST2 on RAPA-conditioned DC (RAPA-DC). ST2 is the recently-identified receptor for IL-33, a cytokine favoring Th type 2 (Th2) responses. In addition, transmembrane ST2, or ST2L, has been implicated as a potent negative regulator of TLR signaling. RAPA-DC generated from ST2−/− mice exhibited higher levels of costimulatory molecules (CD86) than wild-type RAPA-DC. Consistent with its regulatory function, IL-1β-induced ST2L expression suppressed the responsiveness of RAPA-DC to TLR or CD40 ligation. Thus, as a result of their de novo production of IL-1β, RAPA-DC upregulate ST2L and become refractory to pro-inflammatory, maturation-inducing stimuli. This work identifies a novel mechanism through which a clinically-important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.
PMCID: PMC2796738  PMID: 18566370
Dendritic Cells; Cytokines Receptors; Cytokine; Tolerance/Suppression/Anergy; Signal Transduction
13.  Ligation of CD40 Stimulates the Induction of Nitric-oxide Synthase in Microglial Cells* 
The Journal of biological chemistry  2001;276(48):44527-44533.
The present study was undertaken to investigate the role of CD40 ligation in the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 microglial cells and primary microglia. Ligation of CD40 alone by either cross-linking antibodies against CD40 or a recombinant CD40 ligand (CD154) was unable to induce the production of NO in BV-2 microglial cells. The absence of induction of NO production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggests that a signal transduced by the ligation of CD40 alone is not sufficient to induce NO production. However, CD40 ligation markedly stimulated interferon-γ (IFN-γ)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-γ-induced production of NO. This stimulation of NO production was accompanied by stimulation of the iNOS protein and mRNA. In addition to BV-2 glial cells, CD40 ligation also stimulated IFN-γ-mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mechanism of induction/stimulation of iNOS, we investigated the roles of nuclear factor κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ), transcription factors responsible for the induction of iNOS. IFN-γ alone was able to induce the activation of NF-κB as well as C/EBPβ. However, CD40 ligation alone induced the activation of only NF-κB but not of C/EBPβ, suggesting that the activation of NF-κB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPβ is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Δp65) and C/EBPβ (ΔC/EBPβ) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-γ and CD40 ligand. Stimulation of IFN-γ-mediated activation of NF-κB but not of C/EBPβ by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-γ-treated BV-2 microglial cells through the stimulation of NF-κB activation. This study illustrates a novel role for CD40 ligation in stimulating the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.
doi:10.1074/jbc.M106771200
PMCID: PMC2041871  PMID: 11551948
14.  Plasmacytoid Dendritic Cell Number and Responses to Toll-Like Receptor 7 and 9 Agonists Vary in HIV Type 1-Infected Individuals in Relation to Clinical State 
Abstract
In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4+ T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4+ cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.
doi:10.1089/aid.2012.0200
PMCID: PMC3581027  PMID: 23131038
15.  TLR9, TACI and CD40 synergize in causing B cell activation 
Background
B cells receive activating signals from T cells via CD40, from microbial DNA via TLR9, and from dendritic cells (DCs) via transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). TLR9 and CD40 ligation augment TACI driven B cell activation, but only the mechanism of synergy between CD40 and TACI has been explored. Synergy between CD40 and TLR9 in B cell activation is controversial.
Objective
To examine the mechanisms by which TLR9 modulates CD40- and TACI-mediated activation of B cells, and to determine whether all three receptors synergize to activate B cells.
Methods
Naïve mouse B cells and human peripheral blood mononuclear cells (PBMCs) were stimulated with combinations of anti-CD40, CpG and APRIL in the presence of IL-4. Proliferation was measured by 3H-thymidine incorporation. Immuniglobulin production was measured by ELISA. Class switch recombination (CSR) was examined by measuring mRNA for germ line transcripts, activation-induced cytidine deaminase (AICDA) and mature Ig transcripts. Plasma cell differentiation was examined by syndecan-1/CD138 staining and mRNA expression of B lymphocyte-induced maturation protein 1 (Blimp-1).
Results
TLR9 synergized with CD40 and TACI in driving CSR and inducing IgG1 and IgE secretion by naïve murine B cells, and synergized with TACI in driving B cell proliferation and plasma cell differentiation. All three receptors synergized together in driving mouse B cell proliferation, CSR, plasma cell differentiation and IgG1 and IgE secretion. TLR9 synergized with CD40 and TACI in driving IgG secretion in IL-4 stimulated human B cells.
Conclusion
Signals from TLR9, TACI and CD40 are integrated to promote B cell activation and differentiation.
Clinical Implications
Stimulation of B cells via CD40, TLR9 and TACI results in their optimal activation. Defects in these pathways may co-operate to impair B cell immunoglobulin production in patients with common variable immunodeficiency (CVID).
doi:10.1016/j.jaci.2011.04.052
PMCID: PMC3164940  PMID: 21741080
TLR9; CD40; TACI; CSR; plasma cell; immunoglobulin; B cells
16.  A Live and Inactivated Chlamydia trachomatis Mouse Pneumonitis Strain Induces the Maturation of Dendritic Cells That Are Phenotypically and Immunologically Distinct  
Infection and Immunity  2005;73(3):1568-1577.
The intracellular bacterial pathogen Chlamydia trachomatis is a major cause of sexually transmitted disease worldwide. While protective immunity does appear to develop following natural chlamydial infection in humans, early vaccine trials using heat-killed C. trachomatis resulted in limited and transient protection with possible enhanced disease during follow-up. Thus, immunity following natural infection with live chlamydia may differ from immune responses induced by immunization with inactivated chlamydia. To study this differing immunology, we used murine bone marrow-derived dendritic cells (DC) to examine DC maturation and immune effector function induced by live and UV-irradiated C. trachomatis elementary bodies (live EBs and UV-EB, respectively). DC exposed to live EBs acquired a mature DC morphology; expressed high levels of major histocompatibility complex (MHC) class II, CD80, CD86, CD40, and ICAM-1; produced elevated amounts of interleukin-12 and tumor necrosis factor alpha; and were efficiently recognized by Chlamydia-specific CD4+ T cells. In contrast, UV-EB-pulsed DC expressed low levels of CD40 and CD86 but displayed high levels of MHC class II, ICAM-1, and CD80; secreted low levels of proinflammatory cytokines; and exhibited reduced recognition by Chlamydia-specific CD4+ T cells. Adoptive transfer of live EB-pulsed DC was more effective than that of UV-EB-pulsed DC at protecting mice against challenge with live C. trachomatis. The expression of DC maturation markers and immune protection induced by UV-EB could be significantly enhanced by costimulation of DC ex vivo with UV-EB and oligodeoxynucleotides containing cytosine phosphate guanosine; however, the level of protection was significantly less than that achieved by using DC pulsed ex vivo with viable EBs. Thus, exposure of DC to live EBs results in a mature DC phenotype which is able to promote protective immunity, while exposure to UV-EB generates a semimature DC phenotype with less protective potential. This result may explain in part the differences in protective immunity induced by natural infection and immunization with whole inactivated organisms and is relevant to rational chlamydia vaccine design strategies.
doi:10.1128/IAI.73.3.1568-1577.2005
PMCID: PMC1064943  PMID: 15731055
17.  Transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) enhances CD40 driven plasma cell differentiation 
Background
Transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) is a receptor used by B-cell activating factor of the TNF family (BAFF) and a proliferation inducing ligand (APRIL) to induce isotype switching independently of CD40 and is mutated in patients with common variable immunodeficiency (CVID).
Objective
To determine whether TACI and CD40 cooperate in inducing class switch recombination (CSR) and immunoglobulin production.
Methods
Naïve mouse B cells were stimulated with suboptimal concentrations of anti-CD40+IL-4 in the presence or absence of APRIL or anti-TACI. IgG1 and IgE production was measured by ELISA. mRNA for Cγ1 and Cε germ line transcripts, activation-induced cytidine deaminase (AICDA) and mature γ1 and ε transcripts were measured by RT-PCR. Plasmablasts were enumerated by syndecan-1/CD138 staining. Interferon regulatory factor 4 (IRF4), B lymphocyte-induced maturation protein 1 (Blimp1) and IL-6 mRNA expression was measured by quantitative PCR.
Results
TACI ligation enhanced IgG1 and IgE secretion by naïve murine B cells stimulated by anti-CD40+IL-4, with little effect on B cell proliferation or CSR. In contrast, TACI ligation of anti-CD40+IL-4 stimulated B cells induced a significant increase in syndecan-1/CD138 positive cells. TACI ligation caused a modest, but significant increase in the expression of IRF4, with no detectable change in Blimp1 expression.
Conclusion
TACI and CD40 signaling converge to promote B cell differentiation into plasmabalsts.
Clinical implications
Our data suggest that TACI dysfunction could contribute to the impaired antibody response to T dependent antigens in CVID.
CAPSULE SUMMARY
This work shows that CD40 and TACI cooperate to promote B cell differentiation into plasma cells and increase immunoglobulin production.
doi:10.1016/j.jaci.2007.06.012
PMCID: PMC2121612  PMID: 17689597
CD40; TACI; CVID; plasma cell; Immunoglobulin; B cells
18.  Evidence for defective transmembrane signaling in B cells from patients with Wiskott-Aldrich syndrome. 
Journal of Clinical Investigation  1992;90(4):1396-1405.
B lymphocytes from patients expressing the X chromosome-linked immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), fail to produce antibodies in response to stimulation with polysaccharides and other type-2 T cell-independent antigens. To investigate whether this abnormality reflects a defect in the signal transduction cascade normally triggered by ligation of surface immunoglobulin (sIg) on B cells, we have examined early signaling events induced by anti-Ig antibody stimulation of EBV B lymphoblastoid cell lines from WAS patients and healthy controls. Despite the expression of comparable levels of sIg and sIgM on WAS and control EBV B cells, WAS cells failed to manifest the increased proliferation in response to anti-Ig treatment observed in the control cell lines. WAS and control EBV B cells also differed in the magnitude of the change in cytosolic free calcium ([Ca2+]i) induced by sIg ligation; WAS cells showed either markedly diminished or no changes in [Ca2+]i levels whereas control EBV B cells consistently showed increases in [Ca2+]i. Anti-Ig-induced changes in inositol phosphate release were also markedly reduced in WAS compared with control cells. As protein tyrosine phosphorylation is thought to represent a proximal event in the activation of B cells, inducing increases in [Ca2+]i by virtue of tyrosine phosphorylation of phospholipase C (PLC)-gamma, profiles of protein tyrosine phosphorylation and expression of tyrosine-phosphorylated PLC-gamma 1 were compared between WAS and normal EBV B cells before and after sIg cross-linking. These studies revealed that in addition to defective mobilization of Ca2+, the WAS cells manifested little or no increase in tyrosine phosphorylation of PLC-gamma 1 or other intracellular proteins after sIg ligation. Together these results indicate the association of WAS with a defect in the coupling of sIg to signal transduction pathways considered prerequisite for B cell activation, likely at the level of tyrosine phosphorylation. The abnormalities observed in these early transmembrane signaling events in WAS EBV B cells may play a role not only in the nonresponsiveness of WAS patient B cells to certain T independent antigens, but also in the genesis of some of the other cellular deficits exhibited by these patients.
Images
PMCID: PMC443185  PMID: 1401074
19.  Differential bone marrow stem cell mobilization by G-CSF injection or arterial ligation in baboons 
Background
Bone marrow stem cells (BMSCs) are mobilized in response to ischemic attacks, e.g. myocardial infarction, to repair the damage, or by cytokines, e.g. granulocyte colony-stimulating factor (G-CSF), which is used to harvest BMSCs for autologous transplantation. In order to optimize BMSC mobilization strategy for cardiovascular repair, we investigated whether BMSCs mobilized by G-CSF share the same subtype profile as that by ischemia in a nonhuman primate model.
Methods and results
We subjected 5 baboons to subcutaneous G-CSF injection and 5 baboons to femoral artery ligation. Blood BMSCs were measured by surface antigens; functional differentiation to endothelial cells (ECs) was assessed by colony forming capacity, expression of mature EC antigens and tube-like formation. The number of circulating CD34+/CD45RA- cells spiked on day 3 post-stimulation in both groups. While the number of CD34+ cells released by artery ligation was 2-fold lower by comparison with the number released by G-CSF administration, significantly more CD133+/KDR+/CXCR4+/CD31+ cells were detected in the baboons that underwent artery ligation. After culture in endothelial growth medium, mononuclear cells from baboons with artery ligation formed more EC colonies and more capillary-like tubes (p<0.05), expressed higher vWF and phagocytosed more Dil-Ac-LDL (p<0.05).
Conclusions
While G-CSF and artery ligation can mobilize BMSCs capable of differentiating into ECs, BMSCs mobilized by the artery ligation simulating in vivo ischemic attacks have higher potential for vascular differentiation. Our findings demonstrate that different mobilization forces release different sets of BMSCs that may have different capacity for cardiovascular differentiation.
doi:10.1111/j.1582-4934.2008.00405.x
PMCID: PMC3032010  PMID: 20187303
Bone marrow stem cells; nonhuman primate; endothelial progenitor cells
20.  Angiogenesis in rheumatoid arthritis is directly fostered by TLR5 ligation and indirectly through IL-17 induction 
Arthritis and rheumatism  2013;65(8):2024-2036.
Objective
In this study we focus on examining the impact of TLR5 on rheumatoid arthritis (RA) endothelial cell function and collagen induced arthritis (CIA) vascularization.
Methods
Endothelial migration and tube formation were employed to demonstrate the direct role of TLR5 ligation in angiogenesis. CIA mice were treated with TLR5 agonist, flagellin to document the effect of TLR5 ligation in RA pathology. CIA vascularization was examined by histology and joint cytokine levels and spleen TH-17 cells were quantified by ELISA and FACS analysis. Development of TH-17 cells by TLR5 ligation was validated in RA peripheral blood mononuclear cells.
Results
Ligation of TLR5 to endogenous ligands expressed in RA synovial fluid contributes to endothelial infiltration and tube formation. Further, post onset treatment with flagellin, exacerbates CIA joint inflammation while in the control mice, disease activity reaches the plateau phase. We show that TLR5 enhanced disease severity is due to TH-17 cell differentiation and CIA joint vascularization. When the underlying mechanism was examined in RA peripheral blood mononuclear cells, we found that ligation of myeloid TLR5 and their production of TH-17 promoting cytokines was necessary for TH-17 cell polarization. Additionally we demonstrate that blockade of IL-17 cascade can markedly reduce endothelial migration activated by flagellin condition media suggesting that TLR5 ligation can mediate RA angiogenesis either directly through attracting endothelial cells or indirectly by fostering TH-17 cell development.
Conclusion
Our data demonstrate a novel role for TLR5 in RA angiogenesis hence TLR5 may be a promising new target for RA treatment.
doi:10.1002/art.37992
PMCID: PMC3729873  PMID: 23666857
RA; TLR5; angiogenesis; CIA; TH-17 cells
21.  Mincle and human B cell function 
Journal of autoimmunity  2012;39(4):315-322.
C-type lectin receptors are pattern recognition receptors that are critical for autoimmunity and the immune response. Mincle is a C-type lectin receptor expressed by a variety of antigen presenting cells including macrophages, neutrophils, dendritic cells and B cells; a variety of stimuli including stress are known to induce the expression of Mincle. Mincle is an FcRγ-associated activation receptor that senses damaged cells and upon ligation induces activated macrophages to produce inflammatory cytokines. Recently, while several studies have reported that Mincle plays an important role in macrophage responses to fungal infection its function on B cells remains to be defined. In efforts to elucidate the function of Mincle expressed by B cells, we studied the expression of Mincle on subsets of B cells and analyzed cytokines and synthesized immunoglobulin upon ligation of Mincle. The expression of Mincle on CD27−CD19+ naïve B cells is significantly higher than CD27+CD19+ memory B cells. The stimulation of TLR9 ligand induced Mincle expression on B cells. Furthermore, co-stimulation of TLR9 and Mincle ligand reduced IgG and IgA production from B cells without a significant change in the inflammatory cytokines TNFα, IL-6, IL-8 and IL-10. Our data identifies Mincle as a potentially critical player in human B cell responses.
doi:10.1016/j.jaut.2012.04.004
PMCID: PMC3443704  PMID: 22698596
C-type lectin; Mincle; Toll-like receptor
22.  Porphyromonas gingivalis–dendritic cell interactions: consequences for coronary artery disease 
Journal of Oral Microbiology  2010;2:10.3402/jom.v2i0.5782.
An estimated 80 million US adults have one or more types of cardiovascular diseases. Atherosclerosis is the single most important contributor to cardiovascular diseases; however, only 50% of atherosclerosis patients have currently identified risk factors. Chronic periodontitis, a common inflammatory disease, is linked to an increased cardiovascular risk. Dendritic cells (DCs) are potent antigen presenting cells that infiltrate arterial walls and may destabilize atherosclerotic plaques in cardiovascular disease. While the source of these DCs in atherosclerotic plaques is presently unclear, we propose that dermal DCs from peripheral inflamed sites such as CP tissues are a potential source. This review will examine the role of the opportunistic oral pathogen Porphyromonas gingivalis in invading DCs and stimulating their mobilization and misdirection through the bloodstream. Based on our published observations, combined with some new data, as well as a focused review of the literature we will propose a model for how P. gingivalis may exploit DCs to gain access to systemic circulation and contribute to coronary artery disease. Our published evidence supports a significant role for P. gingivalis in subverting normal DC function, promoting a semimature, highly migratory, and immunosuppressive DC phenotype that contributes to the inflammatory development of atherosclerosis and, eventually, plaque rupture.
doi:10.3402/jom.v2i0.5782
PMCID: PMC3084565  PMID: 21523219
dendritic cells; periodontitis; atherosclerosis; Porphyromonas gingivalis; DC-SIGN
23.  CD40 activation in epithelial ovarian carcinoma cells modulates growth, apoptosis, and cytokine secretion 
Molecular Pathology  2002;55(2):110-120.
Background/Aims: CD40, a member of the tumour necrosis factor (TNF) receptor family, is expressed on a variety of haematopoietic cells and is crucial in orchestrating both humoral and cellular immune responses. CD40 is also expressed on some carcinoma cells, where its function remains largely unknown. This study investigated the effects of CD40 ligation on ovarian carcinoma cell growth and apoptosis and on cytokine production, in addition to the role of the NF-κB and JNK signalling pathways.
Methods: CD40 expression was measured in epithelial ovarian carcinoma (EOC) biopsies by immunohistochemistry and in EOC cell lines by flow cytometry. To examine the effects of CD40 ligation on cell growth recombinant soluble CD40 ligand was used to stimulate EOC cell lines and growth was measured by MMT assays. Cytokine production was measured by enzyme linked immunosorbent assays interleukin 8 (IL-8) gene transcription was estimated by means of reverse transcription polymerase chain reaction. The integrity of the CD40 signalling pathway in those cell lines that did not produce cytokines in response to CD40 ligation was assessed by the detection of the transcription factor NF-κB by an electrophoretic mobility shift assay. To investigate the defect in the NF-κB pathway the phosphorylation status of IκBα was determined by an antibody specific to phosphorylated IκBα and dissociation of the IκBα–p65 complex was assessed by co-immunoprecipitation.
Results: CD40 is expressed in primary ovarian carcinoma biopsies and EOC cell lines. CD40 ligation resulted in growth inhibition in most of these carcinoma cell lines and was also found to promote apoptosis, with this last effect only being evident in early passage EOC cells. CD40 ligation also induced significant IL-6 and IL-8 production in most of the EOC cell lines examined and it was confirmed for IL-8 that this effect was regulated at the transcriptional level. NF-κB activation in response to CD40 ligation was found in three of the EOC cell lines and specific defects in the CD40 induced NF-κB pathway were identified in two cell lines. However, CD40 engagement induced JNK activation in all the EOC cell lines.
Conclusions: These data suggest that the CD40 pathway is functional in ovarian carcinoma cells and highlight the need for further studies to provide insight into the role of CD40 in the carcinogenic process and the possible exploitation of this pathway for novel therapeutic approaches.
PMCID: PMC1187159  PMID: 11950960
CD40; cytokines; ovarian carcinoma
24.  Activation of human B cells by the agonist CD40 antibody CP-870,893 and augmentation with simultaneous toll-like receptor 9 stimulation 
Background
CD40 activation of antigen presenting cells (APC) such as dendritic cells (DC) and B cells plays an important role in immunological licensing of T cell immunity. Agonist CD40 antibodies have been previously shown in murine models to activate APC and enhance tumor immunity; in humans, CD40-activated DC and B cells induce tumor-specific T cells in vitro. Although clinical translation of these findings for patients with cancer has been previously limited due to the lack of a suitable and available drug, promising clinical results are now emerging from phase I studies of the agonist CD40 monoclonal antibody CP-870,893. The most prominent pharmacodynamic effect of CP-870,893 infusion is peripheral B cell modulation, but direct evidence of CP-870,893-mediated B cell activation and the potential impact on T cell reactivity has not been reported, despite increasing evidence that B cells, like DC, regulate cellular immunity.
Methods
Purified total CD19+ B cells, CD19+ CD27+ memory, or CD19+ CD27neg subsets from peripheral blood were stimulated in vitro with CP-870,893, in the presence or absence of the toll like receptor 9 (TLR9) ligand CpG oligodeoxynucleotide (ODN). B cell surface molecule expression and cytokine secretion were evaluated using flow cytometry. Activated B cells were used as stimulators in mixed lymphocyte reactions to evaluate their ability to induce allogeneic T cell responses.
Results
Incubation with CP-870,893 activated B cells, including both memory and naïve B cells, as demonstrated by upregulation of CD86, CD70, CD40, and MHC class I and II. CP-870,893-activated B cells induced T cell proliferation and T cell secretion of effector cytokines including IFN-gamma and IL-2. These effects were increased by TLR9 co-stimulation via a CpG ODN identical in sequence to a well-studied clinical grade reagent.
Conclusion
The CD40 mAb CP-870,893 activates both memory and naïve B cells and triggers their T cell stimulatory capacity. Simultaneous TLR9 ligation augments the effect of CP-870,893 alone. These results provide further rationale for combining CD40 and TLR9 activation using available clinical reagents in strategies of novel tumor immunotherapy.
doi:10.1186/1479-5876-7-93
PMCID: PMC2777861  PMID: 19906293
25.  The inhibitory receptor LILRB4 (ILT3) modulates antigen presenting cell phenotype and, along with LILRB2 (ILT4), is upregulated in response to Salmonella infection 
BMC Immunology  2009;10:56.
Background
Leukocyte Ig-like receptors (LILR) are a family of innate immune receptors with immunomodulatory functions. High-level expression of the receptors LILRB2 (ILT4) and LILRB4 (ILT3) is a feature of tolerogenic antigen presenting cells and has been observed in cancer and transplant situations. There are relatively few studies regarding these receptors in the context of infection and it is not yet clear how LILRB4 exerts its inhibitory effects.
Results
We studied the effects of LILRB4 ligation on antigen presenting cell phenotype, and the expression of LILRB2 and LILRB4 on Salmonella-infected antigen presenting cells. Ligation of LILRB4 throughout in vitro culture of dendritic cells led to an upregulation of the co-stimulatory protein CD86. Alterations in the production of IL-8 and IL-10 by LILRB4-ligated macrophages were also observed. Infection with Salmonella typhimurium or TLR stimulation with Salmonella components led to an upregulation of LILRB2 and LILRB4.
Conclusion
Our results indicate that the inhibitory effects of LILRB4 do not result from a failure to upregulate co-stimulatory proteins. In addition to the high level expression that can render antigen presenting cells tolerogenic, there may be a role for lower level expression and activity of LILRB2 and LILRB4 in response to TLR signalling during an immune response to bacterial infection.
doi:10.1186/1471-2172-10-56
PMCID: PMC2773765  PMID: 19860908

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