Maturation Of Dendritic Cells In Response Research Articles

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Development of Anti-CD32b Antibodies with Enhanced Fc Function for the Treatment of B and Plasma Cell Malignancies

The sole inhibitory Fcγ receptor CD32b (FcγRIIb) is expressed throughout B and plasma cell development and on their malignant counterparts. CD32b expression on malignant B cells is known to provide a mechanism of resistance to rituximab that can be ameliorated with a CD32b-blocking antibody. CD32b, therefore, represents an attractive tumor antigen for targeting with a monoclonal antibody (mAb). To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. Their complementarity-determining regions (CDR) bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated to enhance activation of FcγRIIIa on immune effector cells. The NVS32b mAbs selectively target CD32b+ malignant cells and healthy B cells but not myeloid cells. They mediate potent killing of opsonized CD32b+ cells via antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) as well as complement-dependent cytotoxicity (CDC). In addition, NVS32b CDRs block the CD32b Fc-binding domain, thereby minimizing CD32b-mediated resistance to therapeutic mAbs including rituximab, obinutuzumab, and daratumumab. NVS32b mAbs demonstrate robust antitumor activity against CD32b+ xenografts in vivo and immunomodulatory activity including recruitment of macrophages to the tumor and enhancement of dendritic cell maturation in response to immune complexes. Finally, the activity of NVS32b mAbs on CD32b+ primary malignant B and plasma cells was confirmed using samples from patients with B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. The findings indicate the promising potential of NVS32b mAbs as a single agent or in combination with other mAb therapeutics for patients with CD32b+ malignant cells.

Oxidative Stress Enhances Dendritic Cell Responses to Plasmodium falciparum.

Malaria is a highly inflammatory disease caused by the protozoan parasite Plasmodium During the blood stage of infection, patients exhibit fever with high levels of inflammatory cytokines in their blood. However, when cells of the immune system are incubated with the parasite in vitro, their cytokine response is low. In particular, human primary dendritic cells (DCs) respond to Plasmodium falciparum-infected erythrocytes by upregulating maturation markers and chemokines but lack a substantial cytokine response. Because oxidative stress is a trigger of inflammatory cytokines in malaria and synergizes with P. falciparum to induce IL-1β secretion by macrophages, we assessed whether oxidative stress has an impact on DC maturation and function in response to P. falciparum Using xanthine oxidase, a reactive oxygen species- (ROS) producing enzyme that is increased during malaria, we observed that exposure to extracellular ROS potentiated DC maturation in response to the parasite. Xanthine oxidase-derived ROS increased parasite-induced cytokine secretion and CD80 surface expression in DCs. This enhanced maturation phenotype boosted the DCs' ability to prime autologous naive CD4+ T cells, resulting in higher T cell proliferation in vitro. Xanthine oxidase-derived ROS did not have an effect on the cytokines produced by primed T cells. We propose that oxidative stress during malaria contributes to the inflammatory response by enhancing the magnitude of DC and CD4+ T cell responses without changing the quality.

Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation

Previous in vivo studies established that inactivated Francisella tularensis immune complexes (mAb-iFt) are a more protective vaccine against lethal tularemia than iFt alone. Subsequent in vitro studies revealed enhanced DC maturation marker expression with mAb-iFt stimulation. The goal of this study was to determine the mechanism of enhanced DC maturation. Multiparameter analysis of surface marker expression and cytokine secretion demonstrates a requirement for FcγR signaling in enhanced DC maturation. MyD88 was also found to be essential for heightened DC maturation, implicating MyD88-dependent TLRs in DC maturation. Upon further study, we discovered that TLRs 2 & 4 drive cytokine secretion, but surprisingly TLR9 is required for DC maturation marker upregulation. These studies reveal a separation of DC cytokine and maturation marker induction pathways and demonstrate that FcγR-TLR/MyD88 synergy underlies the enhanced dendritic cell maturation in response to the mAb-iFt vaccine.

Epigenetically modulated FOXM1 suppresses dendritic cell maturation in pancreatic cancer and colon cancer.

Forkhead box transcription factor M1 (FOXM1) is a proliferation‐associated transcription factor involved in tumorigenesis through transcriptional regulation of its target genes in various cells, including dendritic cells (DCs). Although previous work has shown that FOXM1 enhances DC maturation in response to house dust mite allergens, it is not known whether FOXM1 affects DC maturation in the context of tumor‐specific immunity. In this study, we examined the central role of FOXM1 in regulating bone marrow‐derived dendritic cell (BMDC) maturation phenotypes and function in pancreatic cancer and colon cancer. FOXM1 retarded maturation phenotypes of BMDCs, inhibited promotion of T‐cell proliferation, and decreased interleukin‐12 (IL‐12) p70 in tumor‐bearing mice (TBM). Notably, FOXM1 expression was epigenetically regulated by dimethylation on H3 lysine 79 (H3K79me2), a modification present in both tumor cells and BMDCs. Increased H3K79me2 enrichment was observed at the FOXM1 promoter in both BMDCs from TBM, and in BMDCs from wild‐type mice cultured with tumor‐conditioned medium that mimics the tumor microenvironment (TME). Furthermore, inhibition of the H3K79 methyltransferase DOT1L not only decreased enrichment of H3K79me2, but also downregulated expression of FOXM1 and partially reversed its immunosuppressive effects on BMDCs. Furthermore, we found that FOXM1 upregulated transcription of Wnt family number 5A (Wnt5a) in BMDCs in vitro; we also observed that exogenous Wnt5a expression abrogated BMDC maturation phenotypes by inhibiting FOXM1 and H3K79me2 modification. Therefore, our results reveal that upregulation of FOXM1 by H3K79me2 in pancreatic cancer and colon cancer significantly inhibits maturation phenotypes and function of BMDCs through the Wnt5a signaling pathway, and thus provide novel insights into FOXM1‐based antitumor immunotherapy.

Mycobacterium tuberculosis ESAT6 Drives the Activation and Maturation of Bone Marrow-Derived Dendritic Cells via TLR4-Mediated Signaling.

6-kDa early secretory antigenic target (ESAT6), a virulent factor of Mycobacterium tuberculosis, is involved in immune regulation. However, the underlying mechanism behind the activation and maturation of dendritic cells (DCs) by ESAT6 remains unclear. In this study, we investigated the effect on TLRs signaling on the regulation of ESAT6-induced activation and maturation of DCs. ESAT6 induced production of IL-6, TNF-α, and IL-12p40 in bone marrow-derived dendritic cells (BMDCs) from wild-type and TLR2-deficient mice, with this induction abolished in TLR4-deficient cells. NF-κB is essential for the ESAT6-induced production of the cytokines in BMDCs. TLR4 was also required for ESAT6-induced activation of NF-κB and MAPKs in BMDCs. ESAT6 additionally upregulated the expression of surface molecules CD80, CD86, and MHC-II, and also promoted the ability of CD4+ T cells to secrete IFN-γ via the TLR4-dependent pathway. Our findings suggest that TLR4 is critical in the activation and maturation of DCs in response to ESAT6.

Effect of calcineurin inhibition on phenotypic maturation of dendritic cells in an in-vitro model of invasive aspergillosis in lung transplant recipients

BackgroundInvasive aspergillosis in lung transplant recipients on immunosuppression has high morbidity and mortality. The calcineurin inhibitor FK506 inhibits the calcineurin–NFAT (nucleated factor of activated T cells) axis, which impairs the innate response to fungal infection. Immature dendritic cells (DCs) have a crucial immune signalling role by phagocytosing antigen, and then maturing and stimulating the T cell response. We investigated the effect of FK506 on phenotypic maturation of DCs in response to Aspergillus fumigatus infection. MethodsPeripheral blood mononuclear cells from healthy volunteers were negatively isolated by density gradient centrifugation (Ficoll, GE Healthcare, Amersham, UK) and were differentiated into DCs with molgramostim (GM-CSF) and interleukin 4. Day 5 cells were matured with interferon (IFN) γ. Day 7 cells were treated with FK506 or inoculated with swollen conidia of A fumigatus (multiplicity of infection 1:1). Cells were then stained with PE-bound anti-CD83 (a late maturation marker) and PerCP-Cy5.5-bound anti-CD209 (a DC-specific marker) and analysed by imaging flow cytometry (ImageStream, Amnis, Seattle, USA). Statistical analysis was performed with Graphpad Prism (version 6.0). Findings5000 cells per condition were analysed. The subset of DCs was identified by gating for CD209 positivity. We demonstrated upregulation of CD83 on the surface of DCs with IFNγ stimulation (26 228 mean fluorescence units [SE 1462] vs 12 534 [799], p<0·0001) A fumigatus infection of unstimulated DCs (29 888 [1393] vs 12 534 [799], p<0·0001), and A fumigatus infection of IFNγ-stimulated DCs (36 778 [1356] vs 26 228 [1462], p<0·0001). CD83 mean fluorescence intensity was reduced with FK506 treatment of IFNγ-stimulated DCs (26 228 [1462] vs 20 219 [846], p=0·0004), A fumigatus-infected unstimulated DCs (29 888 [1393] vs 24 289 [1253], p=0·0028), and A fumigatus-infected, IFN γ-stimulated DCs (36 778 [1356] vs 30 159 [1279], p=0·0004), but unchanged for unstimulated, uninfected DCs (12 534 [799] vs 11 942 [762], p=0·5921). InterpretationBoth A fumigatus infection and IFNγ stimulation promote phenotypic maturation of DCs in vitro, and treatment with FK506 inhibits maturation in this context. These findings suggest that FK506 has an inhibitory effect on innate antigen presentation to T cells and that it might impair the adaptive immune response to invasive aspergillosis in lung transplant recipients. FundingMedical Research Council Chain-Florey Clinical Research Fellowship.

Polyphenol administration impairs T-cell proliferation by imprinting a distinct dendritic cell maturational profile.

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs-QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs-QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs-QP-exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs-QP administration suppresses antigen-specific T-cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti-inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

Chicken bone marrow-derived dendritic cells maturation in response to infectious bursal disease virus

Infectious bursal disease virus (IBDV) is highly contagious disease which easily lead to immunosuppression and a decreased response to vaccinations in young chicken. Since dendritic cells (DCs) are crucial to induce immunity and their maturation and functions are influenced by microbial and environmental stimuli, we investigated the effects of inactivated IBDV and IBDV on chicken DC activation and maturation. Chicken bone marrow-derived dendritic cells (chBM-DCs) cultured in complete medium (including recombinant chicken: granulocyte-macrophage colony-stimulating factor and interleukin 4) expressed high levels of MHC-II and the putative CD11c. After LPS or virus stimulation, chBM-DCs displayed the typical morphology of DCs. In addition, stimulation by LPS or viruses significantly elevated chBM-DCs surface expression levels of CD40 and CD86 molecules, as well as the ability to induce T-cell proliferative response, compared to the non-stimulated chBM-DCs. Interestingly, inactive IBDV showed stronger ability to up-regulate expression levels of CD40 and CD86 molecules and stimulate naive T cells proliferation than live IBDV. These results revealed that live viruses infection impaired DC maturation and functions, probably explaining why chickens infected with IBDV fails to trigger an effective specific immune response or develop immune memory.

AN IMMUNE SURVEILLANCE MODEL TO MONITOR THE RESPONSE TO NEOADJUVANT CHEMOTHERAPY TREATMENT IN BREAST CANCER PATIENTS

AN IMMUNE SURVEILLANCE MODEL TO MONITOR THE RESPONSE TO NEOADJUVANT CHEMOTHERAPY TREATMENT IN BREAST CANCER PATIENTS

Vibrio cholerae ghosts (VCG) exert immunomodulatory effect on dendritic cells for enhanced antigen presentation and induction of protective immunity.

BackgroundWe previously showed that the Vibrio cholerae ghost platform (VCG; empty V. cholerae cell envelopes) is an effective delivery system for vaccine antigens promoting the induction of substantial immunity in the absence of external adjuvants. However, the mechanism by which these cell envelopes enhance immunity and stimulate a predominantly Th1 cellular and humoral immune response has not been elucidated. We hypothesized that the immunostimulatory ability of VCG involves dendritic cell (DC) activation.ObjectiveThe aims of this study were: a) to investigate the ability of DCs [using mouse bone marrow-derived DCs (BMDCs) as a model system] to take up and internalize VCGs; b) to evaluate the immunomodulatory effect of internalized VCGs on DC activation and maturation and their functional capacity to present chlamydial antigen to naïve and infection-sensitized CD4+ T cells and; c) to evaluate the ability of VCGs to enhance the protective immunity of a chlamydial antigen.ResultsVCGs were efficiently internalized by DCs without affecting their viability and modulated DC-mediated immune responses. VCG-pulsed DCs showed increased secretion of proinflammatory cytokines and expression of co-stimulatory molecules associated with DC maturation in response to stimulation with UV-irradiated chlamydial elementary bodies (UV-EBs). Furthermore, this interaction resulted in effective chlamydial antigen presentation to infection-sensitized but not naïve CD4+ T cells and enhancement of protective immunity.ConclusionsThe present study demonstrated that VCGs activate DCs leading to the surface expression of co-stimulatory molecules associated with DC activation and maturation and enhancement of protective immunity induced by a chlamydial antigen. The results indicate that the immunoenhancing activity of VCG for increased T-cell activation against antigens is mediated, at least in part, through DC triggering. Thus, VCGs could be harnessed as immunomodulators to target antigens to DCs for enhancement of protective immunity against microbial infections.

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.

RelB and the aryl hydrocarbon receptor: dendritic cell tolerance at the epithelial interface

RelB and the aryl hydrocarbon receptor: dendritic cell tolerance at the epithelial interface

Resveratrol Prevents Dendritic Cell Maturation in Response to Advanced Glycation End Products

Advanced glycation end products (AGEs), generated through nonenzymatic glycosylation of proteins, lipids, and nucleic acids, accumulate in the body by age thus being considered as biomarkers of senescence. Senescence is characterized by a breakdown of immunological self-tolerance, resulting in increased reactivity to self-antigens. Previous findings suggest that AGE and its receptor RAGE may be involved in the pathogenesis of autoimmune reactions through dendritic cell (DC) activation. The aim of this study was to investigate whether resveratrol, a polyphenolic antioxidant compound with tolerogenic effects on DCs, was able to counteract the mechanisms triggered by AGE/RAGE interaction on DCs. By immunochemical and cytofluorimetric assays, we demonstrated that in vitro pretreatment of human monocyte-derived DCs with resveratrol prevents DC activation in response to glucose-treated albumin (AGE-albumin). We found that resveratrol exerts an inhibitory effect on DC surface maturation marker and RAGE up-regulation in response to AGE-albumin. It also inhibited proinflammatory cytokine expression, allostimulatory ability upregulation, mitogen-activated protein (MAP) kinases, and NF-κB activation in AGE-albumin-stimulated DCs. We suggest that resveratrol, by dismantling AGE/RAGE signaling on DCs may prevent or reduce increased reactivity to self-molecules in aging.

TNF-α and Tumor Lysate Promote the Maturation of Dendritic Cells for Immunotherapy for Advanced Malignant Bone and Soft Tissue Tumors

BackgroundDendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli.MethodsThirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-γ and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy.ResultsApproximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL-12 showed significant elevation at one and three months after DC-based immunotherapy.ConclusionsAlthough TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors.

Impaired dendritic cell maturation in response to pandemic H1N109 influenza virus

BackgroundInfection with pandemic A/H1N1/2009 influenza virus led to hospitalisation of patients not expected to be at risk of severe disease from seasonal influenza infection. ObjectivesWe sought to establish whether (i) DC maturation was compromised in patients experiencing severe pandemic influenza infection, (ii) the pandemic virus differed from seasonal influenza virus in its ability to induce DC maturation and (iii) there was an associated inability to activate memory B cells or induce antibody. Study designPeripheral blood mononuclear (PBMCs) cells were sampled from individuals with confirmed acute pandemic A/H1N1/2009 influenza infection or from healthy vaccinated controls. DCs were differentiated from the PBMC and tested for their ability to mature following stimulation with pandemic virus, seasonal H3N2 influenza virus or LPS. Serum samples from the patients were used to assess seroconversion to influenza and the levels of influenza specific memory B cells in PBMC were also determined. ResultsDCs obtained from all individuals exhibited negligible maturation marker upregulation when exposed to pandemic A/H1N1/2009 virus but showed a strong response to the seasonal H3N2 virus and LPS. Robust levels of memory B cell were obtained in both groups and patients seroconverted to the virus. ConclusionsOverall, the ability of patient's DC to mature in response to different stimuli was no different to that of control subjects DCs. Importantly, panH1N109 virus failed to induce substantial DC maturation in any individual, contrasting with seasonal virus, but this did not result in failure to mount memory B cell and antibody responses to the virus.

Dendritic Cells Maturation in Immunosuppressive Agents In Vitro

Introduction: The most important antigen-presenting cells (APCs) are dendritic cells (DCs), which present antigen to T cells. The state of maturation of DCs is crucial for induction of a T-cell lymphocyte response. It was noted that immature DCs play an important role in peripheral tolerance, whereas mature DCs induce a complete immune response. We have studied the effect of immunosuppressive agents: rapamycin and cyclosporine A on DC maturation in response to LPS. We have compared their effect when DCs were differentiated in an environment of immunosuppressive agents and then were activated with LPS or they were administered simultaneously with LPS. Methods: Human peripheral blood monocytes were induced by using cytokines: IL-4 and GM-CSF, in the direction of DCs in the presence of rapamycin and cyclosporine A or without drugs. Then the ability of these cells to undergo activation by LPS was evaluated. Additionally, DCs, which had been generated without drugs were activated with LPS simultaneously with rapamycin or cyclosporine A. DCs were identified by the expression of surface molecules: CD11c, HLA-DR (MHC II), CD86 and the absence of CD3, CD4, CD14, CD16, CD19, CD20, CD56 (lin-). Results: We have observed a decrease in the percentage of LPS-activated DC when these cells were differentiated in the presence of immunosuppressive agents. The lowest percentage of DC was noted in an environment of rapamycin. We have also noted a decrease in the percentage of LPS-activated DC when rapamycin or cyclosporine A was administered simultaneously with LPS. The lowest percentage of DC was noted in an environment of cyclosporine A. Conclusions: We have shown that the environment of immunosuppressive agents: rapamycin and cyclosporine A impairs DC maturation in response to LPS.

Sphingosine analog AAL-R enhances TLR7-mediated dendritic cell activation via p38 MAPK and type I IFN signaling pathways (180.12)

Abstract Sphingosine analogs display immunosuppressive activities and are therapeutically used for the treatment of autoimmune diseases. Here, we investigated the effects of the sphingosine analog AAL-R (FTY720 derivative) on dendritic cell (DC) response to toll-like receptor (TLR) stimulation. AAL-R inhibited DC maturation in response to TLR3 or TLR4 activation, representing its previously known immunosuppressive action. In contrast, AAL-R increased TLR7-mediated DC responses by increasing the expression of MHC-I and co-stimulatory molecules and type I interferon (IFN), and by enhancing the capacity of DCs to induce CD8+ T cell proliferation. Importantly, the stimulatory activity of AAL-R was dependent on type I IFN signaling, since type I IFN receptor-deficient DCs failed to respond to AAL-R. Further, AAL-R activated p38 MAPK signaling to increase type I IFN synthesis and TLR7-mediated DC maturation. Thus, our results indicate that AAL-R’s regulatory action is strongly affected by the form of pathogenic molecular patterns and is stimulatory when DCs are treated with a TLR7 agonist. These findings enhance our understanding of sphingosine regulation of host immune responses particularly in pathogenic infections.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Escherichia coli K1 Induces the Upregulation of CD47 in Myeloid Dendritic Cells

Escherichia coli K1 is one of the most common pathogens that cause meningitis in neonates. Studies have shown that a high degree of bactremia is required for E. coli K1 to cross the blood‐brain barrier, indicating that the bacterium must evade host immune defense mechanisms and survive in the blood. Dendritic cells (DCs) are the most potent antigen presenting cells that play a crucial role in initiation and modulation of specific immune responses. Nonetheless, very little is known about the interaction of E. coli K1 with DCs. Here, our studies demonstrated that the interaction of OmpA+ E. coli K1 with DCs enhances the expression of CD47, an integrin associated molecule reported to induce tolerogenicity in immune cells like T cells. Significant increase in the expression of TSP‐1, a natural ligand for CD47, on the surface of DCs was also observed. In contrast, OmpA− E. coli, which promotes the maturation of DCs, failed to upregulate the expression of CD47 and TSP‐1 in DCs. Pretreatment of DCs with CD47 blocking antibodies led to maturation of DCs substantiating the role of CD47 in OmpA+ E. coli K1 mediated tolerogenicity. Furthermore, OmpA+ E. coli K1 infected DCs failed to present antigen to T cells. The present study identified an important and novel mechanism by which E. coli K1 makes DCs tolerogenic by upregulating the expression of CD47. Targeting CD47 might help in restoring maturation of DCs in response to E. coli K1 leading to efficient antigen presentation thus promoting clearance of bacteria.