Inhibition Of Dendritic Cell Activation Research Articles

Inhibition of Dendritic Cell Activation and Modulation of T Cell Polarization by the Platelet Secretome.

Platelet transfusions are a frequently administered therapy for especially hemato-oncological patients with thrombocytopenia. Next to their primary function in hemostasis, currently there is increased attention for the capacity of platelets to affect the function of various cells of the immune system. Here, we investigate the capacity of platelets to immuno-modulate monocyte-derived dendritic cells (moDC) as well as primary dendritic cells and effects on subsequent T cell responses. Platelets significantly inhibited pro-inflammatory (IL-12, IL-6, TNFα) and increased anti-inflammatory (IL-10) cytokine production of moDCs primed with toll-like receptor (TLR)-dependent and TLR-independent stimuli. Transwell assays and ultracentrifugation revealed that a soluble factor secreted by platelets, but not microvesicles, inhibited DC activation. Interestingly, platelet-derived soluble mediators also inhibited cytokine production by human ex vivo stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFNγ+ Th1 phenotype by moDCs. Overall, these results show that platelets are able to inhibit the pro-inflammatory properties of DCs, and may even induce an anti-inflammatory DC phenotype, with decreased T cell priming capacity by the DC. The results of this study provide more insight in the potential role of platelets in immune modulation, especially in the context of platelet transfusions.

The mechanisms of hydroxychloroquine in rheumatoid arthritis treatment: Inhibition of dendritic cell functions via Toll like receptor 9 signaling

Hydroxychloroquine (HCQ) is one of the most commonly prescribed immune-suppressants in treating rheumatoid arthritis (RA). Our previous research showed that HCQ suppressed RA development by inhibiting T follicular helper (Tfh) cells directly. Dendritic cells (DCs) serve as the link between innate and acquired immunity. Whether HCQ suppressed Tfh cell through DCs was not clear. In current study, we found that HCQ efficiently inhibited CD86, chemokine (C-X-C motif) receptor 4 (CXCR4) expression and interferon-α (IFN-α) secretion of healthy donor derived purified DCs stimulated by RA patient serum. To mimic RA, collagen-induced arthritis (CIA) mouse model was used and treated with HCQ daily for fifty-four days prior to sacrifice. We found HCQ inhibited DC maturation and migration to lymph nodes (LNs), manifested as down-regulated expression of CD40, CD80, CD86, MHCII (I-Aq) on LN DCs. In addition, HCQ reduced the level of chemokine receptor 7 (CCR7) and L-selectin on peripheral blood DCs and diminished percentage of LN DCs. Of note, HCQ only inhibited CpG ODN 1826-induced IL-12 secretion by bone marrow DCs (BMDCs) stimulated by various toll like receptor (TLR) agonists. Mechanistically, HCQ down-regulated the expression of TLR9 not only in healthy donor PBMC-derived DCs stimulated by RA patient serum, but also in LN DCs of CIA mice and CpG-activated BMDCs. Furthermore, arthritis scores in TLR9−/− mice were much lower than that in wild type mice with impaired maturity and migration capability of DCs. Collectively, activation of DCs contributes to the pathogenesis of RA and HCQ shows protective effects on RA by inhibition of DC activation via blocking TLR9.

Tolerogenic dendritic cells in organ transplantation.

Dendritic cells (DCs) are specialized cells of the innate immune system that are characterized by their ability to take up, process and present antigens (Ag) to effector T cells. They are derived from DC precursors produced in the bone marrow. Different DC subsets have been described according to lineage-specific transcription factors required for their development and function. Functionally, DCs are responsible for inducing Ag-specific immune responses that mediate organ transplant rejection. Consequently, to prevent anti-donor immune responses, therapeutic strategies have been directed toward the inhibition of DC activation. In addition however, an extensive body of preclinical research, using transplant models in rodents and nonhuman primates, has established a central role of DCs in the negative regulation of alloimmune responses. As a result, DCs have been employed as cell-based immunotherapy in early phase I/II clinical trials in organ transplantation. Together with in vivo targeting through use of myeloid cell-specific nanobiologics, DC manipulation represents a promising approach for the induction of transplantation tolerance. In this review, we summarize fundamental characteristics of DCs and their roles in promotion of central and peripheral tolerance. We also discuss their clinical application to promote improved long-term outcomes in organ transplantation.

Leishmania amazonensis-Induced cAMP Triggered by Adenosine A2B Receptor Is Important to Inhibit Dendritic Cell Activation and Evade Immune Response in Infected Mice.

Differently from others Leishmania species, infection by the protozoan parasite L. amazonensis is associated with a lack of antigen-specific T-cell responses. Dendritic cells (DC) are essential for the innate immune response and for directing the differentiation of T-helper lymphocytes. Previously, we showed that L. amazonensis infection impairs DC activation through the activation of adenosine A2B receptor, and here, we evaluated the intracellular events triggered by this receptor in infected cells. To this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Our results show, for the first time, that L. amazonensis increases the production of cAMP and the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in infected DC by a mechanism dependent on the A2B receptor. Furthermore, L. amazonensis impairs CD40 expression and IL-12 production by DC, and the inhibition of adenylate cyclase, phosphoinositide 3-kinase (PI3K), and ERK1/2 prevent these effects. The increase of ERK1/2 phosphorylation and the inhibition of DC activation by L. amazonensis are independent of protein kinase A (PKA). In addition, C57BL/6J mice were inoculated in the ears with metacyclic promastigotes, in the presence of PSB1115, an A2B receptor antagonist. PSB1115 treatment increases the percentage of CD40+ DC on ears and draining lymph nodes. Furthermore, this treatment reduces lesion size and tissue parasitism. Lymph node cells from treated mice produce higher levels of IFN-γ than control mice, without altering the production of IL-10. In conclusion, we suggest a new pathway used by the parasite (A2B receptor → cAMP → PI3K → ERK1/2) to suppress DC activation, which may contribute to the decrease of IFN-γ production following by the deficiency in immune response characteristic of L. amazonensis infection.

Dendritic cell function and pathogen-specific T cell immunity are inhibited in mice administered levonorgestrel prior to intranasal Chlamydia trachomatis infection.

The growing popularity of levonorgestrel (LNG)-releasing intra-uterine systems for long-acting reversible contraception provides strong impetus to define immunomodulatory properties of this exogenous progestin. In initial in vitro studies herein, we found LNG significantly impaired activation of human dendritic cell (DCs) and their capacity to promote allogeneic T cell proliferation. In follow-up studies in a murine model of intranasal Chlamydia trachomatis infection, we analogously found that LNG treatment prior to infection dramatically reduced CD40 expression in DCs isolated from draining lymph nodes at 2 days post infection (dpi). At 12 dpi, we also detected significantly fewer CD4+ and CD8+ T cells in the lungs of LNG-treated mice. This inhibition of DC activation and T cell expansion in LNG-treated mice also delayed chlamydial clearance and the resolution of pulmonary inflammation. Conversely, administering agonist anti-CD40 monoclonal antibody to LNG-treated mice at 1 dpi restored lung T cell numbers and chlamydial burden at 12 dpi to levels seen in infected controls. Together, these studies reveal that LNG suppresses DC activation and function, and inhibits formation of pathogen-specific T cell immunity. They also highlight the need for studies that define in vivo effects of LNG use on human host response to microbial pathogens.

Abatacept Inhibition of T Cell Priming in Mice by Induction of a Unique Transcriptional Profile That Reduces Their Ability to Activate Antigen-Presenting Cells.

To determine at the phenotypic, functional, and transcriptional levels whether abatacept, a CTLA-4Ig molecule that binds with high affinity to CD80/86 on antigen-presenting cells (APCs) and is used to treat rheumatoid arthritis, induces a state of immunologic tolerance in T cells and dendritic cells in mice. We investigated the capacity of abatacept to regulate the development of antigen-specific immunologic tolerance in vivo using murine models of priming and tolerance to generate highly purified antigen-specific T cell populations and CD11c+ APCs. These were combined with detailed immunologic and full genome transcriptional analyses. We found that abatacept inhibited T cell activation, but did not render T cells anergic or lead to the generation of Treg cells. However, it induced a sustained inhibition of T cell activation due to the inability of these cells to progress through the cell cycle following T cell receptor stimulation. We also observed that this state was accompanied by an inhibition of dendritic cell activation due to their reduced licensing by T cells. This study provides detailed insight into the mode of action of abatacept, demonstrating that its effectiveness is not due to the induction of T cell tolerance, but rather to a sustained inhibition of T cell activation that results in reduced functionality of APCs, with significant implications for its clinical application.

The JAK Inhibitor Ruxolitinib Impairs Dendritic Cell Migration Via Off-Target Inhibition of Rock

Introduction: Ruxolitinib is a JAK inhibitor that was recently approved for treatment of primary and secondary MF and shows impressive symptom control by suppression of inflammation. The observed effects are irrespective of the JAK mutational status. Ruxolitinib is also a promising drug for treatment of acute and chronic GvHD (Spoerl et al., Blood 2014 and Zeiser et al., Leukemia, in press). We recently demonstrated that the immune-modulatory effects of ruxolitinib might at least in part be due to an inhibitory effect on dendritic cell (DC) biology (Heine et al., Blood 2014). Dendritic cells are important antigen-presenting cells, which mature and migrate from the periphery into draining lymph nodes upon antigen contact to prime T cells. This process follows chemokine gradients and requires cytoskeletal rearrangement to allow cell movement along the gradients. Members of the family of small GTPases are critically involved in cell chemotaxis, migration and formation of the immunological synapse allowing DC-T cell interaction. The aim of this study was to define in detail the impact of the JAK inhibitor ruxolitinib on DC migration.Methods: CD14+ cells isolated from human buffy coats were differentiated for seven days in the presence of GM-CSF and IL-4 to moDCs and finally matured with LPS. Murine bone marrow-derived DCs (bmDCs) were generated by flushing bone marrow from femura and tibiae of mice and subsequent plating of the cells in medium containing GM-CSF followed by final maturation by LPS. Migration of DCs was analyzed in Transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards CCL19 gradients. Adhesion of cells was tested on different substrates and phenotypic DC marker checked by flow cytometry. Signaling events were analyzed by Western Blot to evaluate changes in phosphorylation levels of relevant proteins of the RhoA/ROCK-pathway.Results: 2D-migration of ruxolitinib-exposed DC on fibronectin is dose-dependently reduced in vitro. By analyzing the migratory phenotype of human moDCs within 3D-collagen gels, ruxolitinib-exposed DCs are still able to sense chemokine gradients as they form lamellipodia at the leading edge of the cell, whereas the retraction of the uropod is clearly inhibited. As a consequence, cell velocity, accumulated and euclidean distance are all significantly reduced by ruxolitinib. These effects are already seen 30 minutes after ruxolitinib exposure. Notably (and in line with the morphological chemokine-sensing by lamellipodia formation), the surface expression of the CCL19-sensing chemokine receptor CCR7 is not altered by ruxolitinib. Moreover, we could not detect any changes of integrin expression or the adhesion of mature DC to fibronectin or collagen as a potential reason for lowered migration due to increased "stickiness" of the DCs. Additional in vivo studies could show that the JAK inhibitor potently reduces homing of bmDCs into draining lymph nodes in mice. Notably, the impaired DC migration is independent of JAK inhibition, as siRNA knockdown experiments revealed that DCs with JAK1, JAK2 or JAK1/2 knockdown migrate appropriately in vitro applying again the 3D-migration system. On a molecular level we could show a reduced phosphorylation of myosin-light chain phosphatase 1 (MYPT1), a direct target of the Rho-associated protein kinase (ROCK) in ruxolitinib-treated moDC upon CCL19 stimulation. Interestingly, the known inhibition of DC activation and maturation seen after treatment with ruxolitinib could not be observed if cells are incubated with the ROCK inhibitor Y-27632. Whereas pharmacological inhibition of ROCK mimicked the migration phenotype seen in moDC exposed to ruxolitinib.Conclusion: RhoA/ROCK pathway is critical for cell migration, as ROCK is a downstream effector of RhoA and leads to stabilization of the actin cytoskeleton via cofilin and acto-myosin II contraction by the myosin light chain. The observed reduction of ROCK activity may reveal an important novel mechanism of ruxolitinib-induced inhibition of DC migration. Our current efforts focus on the validation of ROCK as off-target JAK1/2-independent target kinase of ruxolitinib as potential mediator of the observed DC inhibitory effects, which may at least in part also explain the potential therapeutic impact of ruxolitinib for treatment of GvHD or autoimmunity. DisclosuresNo relevant conflicts of interest to declare.

Chapter Nine - Vitamin D Effects on Lung Immunity and Respiratory Diseases

Our understanding of vitamin D metabolism and biological effects has grown exponentially in recent years and it has become clear that vitamin D has extensive immunomodulatory effects. The active vitamin D generating enzyme, 1α-hydroxylase, is expressed by the airway epithelium, alveolar macrophages, dendritic cells, and lymphocytes indicating that active vitamin D can be produced locally within the lungs. Vitamin D generated in tissues is responsible for many of the immunomodulatory actions of vitamin D. The effects of vitamin D within the lungs include increased secretion of the antimicrobial peptide cathelicidin, decreased chemokine production, inhibition of dendritic cell activation, and alteration of T-cell activation. These cellular effects are important for host responses against infection and the development of allergic lung diseases like asthma. Epidemiological studies do suggest that vitamin D deficiency predisposes to viral respiratory tract infections and mycobacterial infections and that vitamin D may play a role in the development and treatment of asthma. Randomized, placebo-controlled trials are lacking but ongoing.

Dendritic Cells in Innate and Adaptive Immune Responses against Influenza Virus

Dendritic cells (DC) are major players in both innate and adaptive immune responses against influenza virus. These immune responses, as well as the important interface between the innate and adaptive systems, are orchestrated by specialized subsets of DC, including conventional steady-state DC, migratory DC and plasmacytoid DC. The characteristics and efficacy of the responses are dependent on the relative activity of these DC subsets, rendering DC crucial for the development of both naïve and memory immune responses. However, due to their critical role, DC also contribute to the immunopathological processes observed during acute influenza, such as that caused by the pathogenic H5N1 viruses. Therein, the role of different DC subsets in the induction of interferon type I, pro-inflammatory cytokine and chemokine responses is important for the outcome of interaction between the virus and host immune defences. The present review will present current knowledge on this area, relating to the importance of DC activity for the induction of efficacious humoral and cell-mediated immune responses. This will include the main viral elements associated with the triggering or inhibition of DC activation. Finally, the current knowledge on understanding how differences in various vaccines influence the manner of immune defence induction will be presented.

The Autographa californica nuclear polyhedrosis virus AcNPV induces functional maturation of human monocyte-derived dendritic cells

The initiation of an adaptive immune response is critically dependent on the activation of dendritic cells (DCs). Therefore, vaccination strategies targeting DCs have to ensure a proper presentation of the immunogen as well as an activation of DCs to accomplish their full maturation. Viral vectors can achieve gene delivery and a subsequent presentation of the expressed immunogen, however, the immunization efficiency may be hampered by an inhibition of DC activation. Here we report that the insect born Autographa californica nuclear polyhedrosis virus (AcNPV), which is already used for genetic immunization, is able to activate human monocyte-derived DCs. This activation induces the production of tumor necrosis factor alpha (TNF-α), an up-regulation of the surface molecules CD83, CD80, CD86, HLA-DR and HLA-I and increases the T cell stimulatory capacity of DCs. Thus, AcNPV represents a promising vector for vaccine trials.

Oncoprotein hides tumor

An oncogenic fusion protein hijacks a transcription factor, and uses it to help its tumor avoid immune detection, according to a study by Nabarro and colleagues on page 1399. This is the first description of an oncoprotein that can both transform cells and shut down the immune response.The protein, PAX3-FKHR, results from a reciprocal chromosomal translocation in a rare form of a childhood muscle cancer, alveolar rhabdomyosarcoma (ARMS). Through the combined action of the PAX3 DNA-binding domain and the FKHR transcriptional activation domain, the oncoprotein binds and strongly activates PAX3 target genes such as IGF2. This activity confers rapid and invasive tumor growth. Figure PAX3-FKHR induction (top) turns on STAT3 and helps tumors to hide. Now, this group finds that expression of PAX3-FKHR also significantly dampens immune activation, in part by reducing surface levels of class I MHC on transfected cells. A microarray screen revealed that expression of the fusion protein repressed numerous genes, 40% of which are known targets of STAT1 and/or STAT3, including class I MHC and the cytokine IL-10. Recent studies have shown that overactive STAT3 contributes to tumor immune evasion by stifling inflammation and preventing crosstalk between innate and adaptive immune systems. STAT3 also acts as a transcriptional coactivator with FKHR. The group now show that PAX3-FKHR also binds to STAT3, but not to STATs 2,4, and 6. Media from cells expressing the fusion protein contained at least one inhibitory cytokine, IL-10, and prevented dendritic cell (DC) activation. Mouse xenografts expressing PAX3-FKHR had decreased inflammatory responses because the numbers of both activated DCs and infiltrating neutrophils and macrophages were reduced. This inhibition of DC activation and immune cell recruitment might help the tumor establish an immunoinhibitory environment in which to hide. Determining which target genes help the PAX3-FKHRSTAT3 complex to create this environment will require further experiments.

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation.

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.