Fever range temperature promotes TLR4 expression and signaling in dendritic cells

Fever improves survival and shortens disease duration in microbial infections. However, the mechanisms of these beneficial responses still remain elusive. Toll-like receptors (TLRs) play important roles in sensing microbes invading and therefore we hypothesized that fever range temperature may enhance responsiveness of dendritic cells (DCs) to lipopolysaccharide (LPS) by promoting TLR4 expression and signaling. In this study, we found that pretreatment of DCs with 39.5 degrees C temperature can up-regulate TLR4 expression in DCs and enhances LPS-induced DC production of interleukins (IL) IL-6, IL-10 and IL-12 but not tumor necrosis factor alpha (TNF-alpha). Blockade of the autocrine action of IL-10 could increase LPS-induced TNF-alpha and IL-12 production in DCs. Further experiments confirmed that TLR4 ligation activates extracellular signal-regulated kinase (ERK), p38, and nuclear factor-kappaB pathways more potently in DCs pretreated with 39.5 degrees C. We conclude that fever range temperature can promote TLR4 expression and signaling in DCs, leading to enhancement of immune responses to inflammatory stimuli. These results might reveal a possible mechanistic explanation for the significance of fever in activating innate immune responses.     

Snapin promotes HIV‐1 transmission from dendritic cells by dampening TLR8 signaling

HIV‐1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV‐1 somehow evades detection by the pattern‐recognition receptor (PRR) Toll‐like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV‐1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV‐1 trans‐infection of CD4⁺ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV‐1 with TLR8⁺ early endosomes, triggered a pro‐inflammatory response, and inhibited trans‐infection of CD4⁺ T cells. Snapin inhibited TLR8 signaling in the absence of HIV‐1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV‐1 to promote transmission.     

Human metapneumovirus glycoprotein G inhibits TLR4-dependent signaling in monocyte-derived dendritic cells

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells (DCs) are potent APCs and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived DCs (moDCs), as downregulation of its expression by small interfering RNA significantly blocked hMPV-induced chemokine and type I IFN expression. Similar results were found in bone marrow-derived DCs from TLR4-deficient mice. moDCs infected with a virus lacking G protein expression produced higher levels of cytokines and chemokines compared with cells infected with wild-type virus, suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as infection of moDCs with recombinant hMPV lacking G protein inhibited LPS-induced production of cytokine and chemokines significantly less than did wild-type virus, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.     

Regulatory T cells interfere with glutathione metabolism in dendritic cells and T cells

Naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) suppress proliferation of CD4(+)CD25(-) effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.     

TLR4 signaling in cancer cells promotes chemoattraction of immature dendritic cells via autocrine CCL20

Toll-like receptors (TLRs) are involved in the production of inflammatory mediators upon specific ligands stimuli. Chemokines are important inflammatory mediators capable of chemoattracting diverse immune cells. In addition to normal immune cells, the expression of TLRs and chemokines has been detected in various tumor cells. However, the roles of TLRs and chemokines expressed by tumor cells in the processes of tumor progression and immune escape have not been fully elucidated. Here we report that TLR4 ligation by lipopolysaccharide (LPS) significantly promotes CT-26 colon cancer cells to produce chemokine CCL20 via activation of TLR4 signaling pathways. We find that LPS treatment of CT-26 cells can significantly increase the chemoattraction of immature dendritic cells (DC) by the autocrine CCL20. Our studies suggest that TLR4 expressed by tumor cells may be involved in the induction of chemokines like CCL20, providing a potential linkage between chronic inflammation and tumor immune escape.     

Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

Polymorphonuclear neutrophil-derived ectosomes interfere with the maturation of monocyte-derived dendritic cells

Polymorphonuclear neutrophils (PMNs) are a key component of the innate immune system. Their activation leads to the release of potent antimicrobial agents through degranulation. Simultaneously, PMNs release cell surface-derived microvesicles, so-called ectosomes (PMN-Ect). PMN-Ect are rightside-out vesicles with a diameter of 50-200 nm. They expose phosphatidylserine in the outer leaflet of their membrane and down-modulate monocyte/macrophage-activation in vitro. In this study, we analyzed the effects of PMN-Ect on maturation of human monocyte-derived dendritic cells (MoDCs). Intriguingly, exposing immature MoDCs to PMN-Ect modified their morphology, reduced their phagocytic activity, and increased the release of TGF-beta1. When immature MoDCs were incubated with PMN-Ect and stimulated with the TLR4 ligand LPS, the maturation process was partially inhibited as evidenced by reduced expression of cell surface markers (CD40, CD80, CD83, CD86, and HLA-DP DQ DR), inhibition of cytokine-release (IL-8, IL-10, IL-12, and TNF-alpha), and a reduced capacity to induce T cell proliferation. Together these data provide evidence that PMN-Ect have the ability to modify MoDC maturation and function. PMN-Ect may thus represent an as yet unidentified host-factor influencing MoDC maturation at the site of injury, thereby possibly impacting on downstream MoDC-dependent immunity.     

Virus or TLR agonists induce TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells

Among dendritic cells, plasmacytoid dendritic cells (PDC) represent a functionally distinct lineage. Regarding innate immunity, PDC secrete large amounts of type I IFN upon viral exposure or stimulation by microbial products such as unmethylated CpG-motif containing oligo-DNA due to their selective expression of TLR7 and TLR9. We asked whether they could acquire cytotoxic functions during the early phases of infection or after activation with TLR7 or TLR9 agonists. In the present study, we describe a human PDC cell line called GEN2.2, derived from leukemic PDC, that shares most of the phenotypic and functional features of normal PDC. We show that after contact with the influenza virus, GEN2.2, as well as normal PDC, acquires TRAIL and killer activity against TRAIL-sensitive target cells. Moreover, we show that activation of GEN2.2 cells by CpG-motif containing oligo-DNA or R848 also induces TRAIL and endows them with the ability to kill melanoma cells. Therefore, PDC may represent a major component of innate immunity that could participate to the clearance of infected cells and tumor cells. This phenomenon could be relevant for the efficacy of TLR7 or TLR9 agonists in the therapy of infectious disease and cancer.     

Alkaline phosphatase activity can interfere with the enzymatic determination of triglycerides

Unlike human plasma, rat plasma was found to contain substantial quantities of alkaline phosphatase. The large amount of phosphatase activity in rat plasma interferes with assay methods that employ orthophosphoric acid esters as substrates or reaction intermediates. Although these methods are effective when applied to human plasma samples, they cannot be used with rodent plasma.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

Spontaneous renal allograft acceptance associated with "regulatory" dendritic cells and IDO

MHC-mismatched DBA/2 renal allografts are spontaneously accepted by C57BL/6 mice by poorly understood mechanisms, but both immune regulation and graft acceptance develop without exogenous immune modulation. Previous studies have shown that this model of spontaneous renal allograft acceptance is associated with TGF-beta-dependent immune regulation, suggesting a role for T regulatory cells. The current study shows that TGF-beta immune regulation develops 30 days posttransplant, but is lost by 150 days posttransplant. Despite loss of detectable TGF-beta immune regulation, renal allografts continue to function normally for >200 days posttransplantation. Because of its recently described immunoregulatory capabilities, we studied IDO expression in this model, and found that intragraft IDO gene expression progressively increases over time, and that IDO in "regulatory" dendritic cells (RDC) may contribute to regulation associated with long-term maintenance of renal allografts. Immunohistochemistry evaluation confirms the presence of both Foxp3+ T cells and IDO+ DCs in accepted renal allografts, and localization of both cell types within accepted allografts suggests the possibility of synergistic involvement in allograft acceptance. Interestingly, at the time when RDCs become detectable in spleens of allograft acceptors, approximately 30% of these mice challenged with donor-matched skin allografts accept these skin grafts, demonstrating progression to "true" tolerance. Together, these data suggest that spontaneous renal allograft acceptance evolves through a series of transient mechanisms, beginning with TGF-beta and T regulatory cells, which together may stimulate development of more robust regulation associated with RDC and IDO.     

Activation of plasmacytoid dendritic cells with TLR9 agonists initiates invariant NKT cell-mediated cross-talk with myeloid dendritic cells

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

Flavivirus activation of plasmacytoid dendritic cells delineates key elements of TLR7 signaling beyond endosomal recognition

TLR7 senses RNA in endosomal compartments. TLR7 expression and signaling have been demonstrated in plasmacytoid and myeloid dendritic cells, B cells, and T cells. The regulation of TLR7 signaling can play a crucial role in shaping the immune response to RNA viruses with different cellular tropisms, and in developing adjuvants capable of promoting balanced humoral and cell-mediated immunity. We used unique characteristics of two ssRNA viruses, dengue virus and influenza virus, to delineate factors that regulate viral RNA-human TLR7 signaling beyond recognition in endosomal compartments. Our data show that TLR7 recognition of enveloped RNA virus genomes is linked to virus fusion or uncoating from the endosome. The signaling threshold required to activate TLR7-type I IFN production is greater than that required to activate TLR7-NF-kappaB-IL-8 production. The higher order structure of viral RNA appears to be an important determinant of TLR7-signaling potency. A greater understanding of viral RNA-TLR7 activity relationships will promote rational approaches to interventional and vaccine strategies for important human viral pathogens.     

MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties

Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.     

TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity

Microbial molecular patterns engage TLRs and activate dendritic cells and other accessory cells. Follicular dendritic cells (FDCs) exist in resting and activated states, but are activated in germinal centers, where they provide accessory function. We reasoned that FDCs might express TLRs and that engagement might activate FDCs by up-regulating molecules important for accessory activity. To test this hypothesis, TLR4 expression on FDCs was studied in situ with immunohistochemistry, followed by flow cytometry and RT-PCR analysis. TLR4 was expressed on FDC reticula in situ, and flow cytometry indicated that TLR4 was expressed on surface membranes and TLR4 message was readily apparent in FDCs by RT-PCR. Injecting mice or treating purified FDCs with LPS up-regulated molecules important for accessory activity including, FDC-Fc gammaRIIB, FDC-ICAM-1, and FDC-VCAM-1. Treatment of purified FDCs with LPS also induced intracellular phospho-IkappaB-alpha, indicating NF-kappaB activation, and that correlated with increased Fc gammaRIIB, ICAM-1, and VCAM-1. FDCs in C3H/HeJ mice were not activated with LPS even when mice were reconstituted with C3H/HeN leukocytes, suggesting that engagement of FDC-TLR4 is necessary for activation. Moreover, activated FDCs exhibited increased accessory activity in anti-OVA recall responses in vitro, and the FDC number could be reduced 4-fold if they were activated. In short, we report expression of TLR4 on FDCs for the first time and that engagement of FDC-TLR4 activated NF-kappaB, up-regulated expression of molecules important in FDC accessory function, including Fc gammaRIIB, ICAM-1, and VCAM-1, as well as FDC accessory activity in promoting recall IgG responses.     

Preextinction viral RNA can interfere with infectivity

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.