Self‐priming determines high type I IFN production by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are responsible for the robust and immediate production of type I IFNs during viral infection. pDCs employ TLR7 and TLR9 to detect RNA and CpG motifs present in microbial genomes. CpG‐A was the first synthetic stimulus available that induced large amounts of IFN‐α (type I IFN) in pDCs. CpG‐B, however, only weakly activates pDCs to produce IFN‐α. Here, we demonstrate that differences in the kinetics of TLR9 activation in human pDCs are essential for the understanding of the functional difference between CpG‐A and CpG‐B. While CpG‐B quickly induces IFN‐α production in pDCs, CpG‐A stimulation results in delayed yet maximal IFN‐α induction. Constitutive production of low levels of type I IFN in pDCs, acting in a paracrine and autocrine fashion, turned out to be the key mechanism responsible for this phenomenon. At high cell density, pDC‐derived, constitutive type I IFN production primes pDCs for maximal TLR responsiveness. This accounts for the high activity of higher structured TLR agonists that trigger type I IFN production in a delayed fashion. Altogether, these data demonstrate that high type I IFN production by pDCs cannot be simply ascribed to cell‐autonomous mechanisms, yet critically depends on the local immune context.     

The tyrosine kinase inhibitor dasatinib suppresses cytokine production by plasmacytoid dendritic cells by targeting endosomal transport of CpG DNA

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)‐α in response to nucleic acids from viruses and damaged self‐cells through Toll‐like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN‐α production by pDCs are instrumental in elucidating the mechanisms behind IFN‐α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN‐α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG‐A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG‐A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN‐α. This study suggests that dasatinib suppresses IFN‐α production by pDCs by inhibiting SFK‐dependent pathways and SFK‐independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC‐related inflammatory disorders.     

Hematopoietic and nonhematopoietic cells promote Type I interferon‐ and TLR7‐dependent monocytosis during low‐dose LCMV infection

Release of inflammatory monocytes from the bone marrow (BM) into the blood is an important physiological response to infection, but the mechanisms regulating this phenomenon during viral infection are not completely defined. Here, we show that low‐dose infection with lymphocytic choriomeningitis virus (LCMV) caused rapid, transient inflammatory monocytosis that required type I interferon (IFN) and Toll‐like receptor (TLR) 7 signaling. Type I IFN and TLR7 signals were critical for induction of IFN‐stimulated gene expression and CCR2 ligand upregulation in the BM microenvironment in response to LCMV infection. Experiments utilizing BM chimeric mice demonstrated that type I IFN and TLR7 signaling on either hematopoietic or nonhematopoietic cells was sufficient to initiate monocytosis in response to LCMV infection. BM plasmacytoid dendritic cells (pDCs) generated type I IFN directly ex vivo, suggesting that pDCs are a hematopoietic contributor of type I IFN in the BM early during LCMV infection. Overall, we describe novel roles for type I IFN and TLR7 signaling in nonhematopoietic cells and BM pDCs in directing IFN‐stimulated gene and CCR2 ligand expression in the BM to initiate an increase in blood inflammatory monocytes during viral infection.     

SHP-2 phosphatase negatively regulates the TRIF adaptor protein-dependent type I interferon and proinflammatory cytokine production

The Toll-like receptor 3 (TLR3) and TLR4-signaling pathway that involves the adaptor protein TRIF activates type I interferon (IFN) and proinflammatory cytokine expression. Little is known about how TRIF pathway-dependent gene expression is regulated. SH2-containing protein tyrosine phosphatase 2 (SHP-2) is a widely expressed cytoplasmic tyrosine phosphatase. Here we demonstrate that SHP-2 negatively regulated TLR4- and TLR3-activated IFN-beta production. SHP-2 inhibited TLR3-activated but not TLR2-, TLR7-, and TLR9-activated proinflammatory cytokine IL-6 and TNF-alpha production. SHP-2 inhibited poly(I:C)-induced cytokine production by a phosphatase activity-independent mechanism. C-terminal domain of SHP-2 directly bound TANK binding kinase (TBK1) by interacting with the kinase domain of TBK1. SHP-2 deficiency increased TBK1-activated IFN-beta and TNF-alpha expression. TBK1 knockdown inhibited poly(I:C)-induced IL-6 production in SHP-2-deficient cells. SHP-2 also inhibited poly(I:C)-induced activation of MAP kinase pathways. These results demonstrate that SHP-2 specifically negatively regulate TRIF-mediated gene expression in TLR signaling, partially through inhibiting TBK1-activated signal transduction.     

CCK8 negatively regulates the TLR9‐induced activation of human peripheral blood pDCs by targeting TRAF6 signaling

Plasmacytoid dendritic cells (pDCs) are specialized in rapid and massive secretion of type I interferon in response to foreign nuclei acids. Combined with their antigen presentation capacity, this powerful functionality enables pDCs to orchestrate innate and adaptive immune responses. Cholecystokinin octapeptide (CCK8) is a potent immunomodulator, whose role in pDCs function is unknown. In this study, we found that two different cholecystokinin receptors, CCK1R and CCK2R, are expressed on human peripheral blood pDCs. Exogenous CCK8 was able to modulate the TLR‐induced activation of pDCs, including phenotypic maturation, IFN‐α synthesis and secretion, and could also regulate the potential of pDCs to induce adaptive immune responses in vitro. CCK8 inhibited TLR9‐induced activation of tumor‐necrosis factor receptor‐associated factor 6, which is an important adapter protein in activation of interferon‐regulatory factor (IRF)5 and IRF7, possibly through CCK2R, by evoking the activity of protein kinase (PK)A and reducing the activity of PKC. All these results indicate that CCK8 can inhibit the TLR9‐induced phenotypic maturation and activation of pDCs, acting through CCK2R by modulating the tumor‐necrosis factor receptor‐associated factor 6 signaling pathways.     

Human plasmacytoid dendritic cells elicit a Type I Interferon response by sensing DNA via the cGAS‐STING signaling pathway

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN‐driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self‐DNA and has been linked to TLR9‐independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS‐STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS‐STING pathway and that targeting this pathway could be of therapeutic interest.     

Interleukin receptor-associated kinase-4 deficiency impairs Toll-like receptor-dependent innate antiviral immune responses

BACKGROUND: Engagement of all known Toll-like receptors (TLRs) causes the production of inflammatory cytokines, including TNF-alpha, whereas in humans, engagement of TLRs 3, 7, 8, and 9 also induces type I IFNs. IRAK-4 is a critical effector in signaling by TLRs and the IL-1 receptor, which share homology in their intracellular domain and recruit IRAK-4 via the adaptor myeloid differentiation factor 88 (MyD88). Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections but have so far not been reported to be susceptible to viral infection. Blood cells from these patients are impaired in their ability to make TNF-alpha in response to activation by TLRs. A recent report has described concomitant impairment of type I IFN production after activation of TLRs 7, 8, and 9, but not TLR3. OBJECTIVES: We sought to evaluate the role of IRAK-4 in TLR-induced production of the type I IFN, IFN-alpha, in humans. METHODS: We examined TLR-induced production of TNF-alpha and IFN-alpha in PBMCs from an IRAK-4-deficient patient, his heterozygous carrier parents, and normal controls. RESULTS: TNF-alpha production in response to TLR agonists was severely impaired in the patient. IFN-alpha production induced by TLR7, TLR8, and TLR9, as well as TLR3 agonists, was low or absent. CONCLUSIONS: IRAK-4 plays an important role in the production of type I IFN, as well as TNF-alpha, induced by all TLRs, including TLR3. CLINICAL IMPLICATIONS: IRAK-4 may play a broader role in human innate antiviral immunity than previously appreciated.     

Human and rhesus plasmacytoid dendritic cell and B-cell responses to Toll-like receptor stimulation

Interferon-α (IFN-α) produced at high levels by human plasmacytoid dendritic cells (pDCs) can specifically regulate B-cell activation to Toll-like receptor (TLR) 7/8 stimulation. To explore the influence of IFN-α and pDCs on B-cell functions in vivo, studies in non-human primates that closely resemble humans in terms of TLR expression on different subsets of immune cells are valuable. Here, we performed a side-by side comparison of the response pattern between human and rhesus macaque B cells and pDCs in vitro to well-defined TLR ligands and tested whether IFN-α enhanced B-cell function comparably. We found that both human and rhesus B cells proliferated while pDCs from both species produced high levels of IFN-α in response to ligands targeting TLR7/8 and TLR9. Both human and rhesus B-cell proliferation to TLR7/8 ligand and CpG class C was significantly increased in the presence of IFN-α. Although both human and rhesus B cells produced IgM upon stimulation, only human B cells acquired high expression of CD27 associated with plasmablast formation. Instead, rhesus B-cell differentiation and IgM levels correlated to down-regulation of CD20. These data suggest that the response pattern of human and rhesus B cells and pDCs to TLR7/8 and TLR9 is similar, although some differences in the cell surface phenotype of the differentiating cells exist. A more thorough understanding of potential similarities and differences between human and rhesus cells and their response to potential vaccine components will provide important information for translating non-human primate studies into human trials.     

Sca‐1 expression defines developmental stages of mouse pDCs that show functional heterogeneity in the endosomal but not lysosomal TLR9 response

Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity and were shown to be identical to previously described natural interferon (IFN)‐α‐producing cells. Here, we describe two functionally distinct pDC subpopulations that are characterized by the differential expression of stem cell antigen‐1 (Sca‐1; Ly‐6A/E). Sca‐1^? pDCs are mainly found in the BM, appear first during development, show a higher proliferative activity, and represent the more precursor phenotype. Sca‐1⁺ pDCs are mostly located in secondary lymphoid organs and represent a later developmental stage. Sca‐1^? pDCs give rise to an Sca‐1⁺ subset upon activation or in response to endogenous type I IFN. Interestingly, in contrast to Sca‐1^? pDCs, Sca‐1⁺ pDCs are defective in IFN‐α production upon endosomal TLR9 stimulation, whereas lysosomal signaling via TLR9 is functional in both subsets. Gene expression analysis revealed that osteopontin is strongly upregulated in Sca‐1^? pDCs. These data provide evidence for the molecular basis of the observed functional heterogeneity, as the intracellular isoform of osteopontin couples TLR9 signaling to IFN‐α expression. Taken together, our results indicate that Sca‐1^? pDCs are an early developmental stage of pDCs with distinct innate functions representing the true murine natural IFN‐α‐producing cells.     

Antimicrobial peptides and self-DNA in autoimmune skin inflammation

Toll-like receptor (TLR)-mediated detection of viral nucleic acids and production of type I interferons (IFNs) by plasmacytoid dendritic cells (pDCs) are key elements of antiviral defense. By contrast, inappropriate recognition of self-nucleic acids with induction of IFN responses in pDCs can lead to autoimmunity. In this review we describe how pDC responses to self-DNA are normally avoided and focus on our recent finding that in psoriasis, a common autoimmune disease of the skin, these barriers can be breached by the cationic antimicrobial peptide LL37. LL37 binds extracellular self-DNA fragments into aggregated particles that enter pDCs and trigger robust IFN responses by activating endosomal TLR9 as if they were viruses. We also describe the mechanisms that normally control production and activity of LL37 in human skin and propose that the persistent overexpression of LL37 in psoriasis leads to uncontrolled IFN responses that drive autoimmune skin inflammation.     

Plasmocytoid dendritic cell deficit of early response to toll-like receptor 7 agonist stimulation in multiple sclerosis patients

Plasmacytoid dendritic cells (pDCs), an important immunoregulatory population, are characterized by vigorous secretion of type I interferons (IFNs) in response to toll-like receptor (TLR) 7 and 9 stimulation. We studied the function of pDCs in multiple sclerosis (MS) patients by analysis of TLR7 responses. We assessed a pDC secretion pattern of cytokines in the short term PBMC cultures stimulated with TLR7 agonist. pDCs sorted from PBMCs of both MS patients and controls were used to assess TLR7 expression profile. TLR7 induced signaling in pDCs has been analyzed with intracellular flow cytometry. We have identified a clinically correlated significant decrease of the TLR7-induced IFN-alfa (IFNa) secretion by pDCs from MS patients. This deficit has been accompanied by insufficient intracellular phosphorylation of protein kinase Akt and a decrease of the TLR7 gene expression in MS pDCs. Our results demonstrated a selective pDC deficit in MS supporting a relationship between pDCs and mechanisms of MS.     

Subcutaneous allergen immunotherapy restores human dendritic cell innate immune function

Background We recently reported that human blood dendritic cells from allergic subjects have impaired IFN‐α production following toll‐like receptor 9 (TLR9)‐dependent innate immune stimulation. It is not known how subcutaneous allergen immunotherapy (SCIT) affects dendritic cell immune responses. Objective The aim of this study is to determine how SCIT affects human dendritic cell function. Methods Peripheral blood mononuclear cell (PBMC) and plasmacytoid dendritic cells (pDCs) were isolated from the blood of seven dust mite allergic subjects at baseline and upon reaching a standard SCIT maintenance dose that included dust mite and other aeroallergens. Cells were stimulated with various adaptive and innate immune receptor stimuli, or media alone for 20 h with secreted cytokine levels determined by ELISA. A portion of the cells were used to measure intracellular signalling proteins by flow cytometry. Humoral immune responses were measured from plasma. Results SCIT resulted in a threefold increase in PBMC production of IFN‐α in response to CpG at 100 nm (P=0.015) and at 500 nm (P=0.015), n=7. The predominant cell type known to produce IFN‐α in response to CpG (CpG ODN‐2216) and other TLR9 agonists is the pDC. As expected, a robust innate immune response from isolated pDCs was re‐established among allergic subjects undergoing SCIT resulting in a fivefold increase in IFN‐α production in response to CpG at 500 nm (P=0.046), n=7. In contrast, IL‐6 production was unaffected by SCIT (P=0.468). Consistent with published reports, IgG4 blocking antibody increased 10‐fold with SCIT (P=0.031), n=7. There was no significant increase in the frequency of pDCs or the expression of TLR9 that would account for the rise in IFN‐α production. Conclusions Allergen immunotherapy increases dendritic cell TLR9‐mediated innate immune function, which has previously been shown to be impaired at baseline in allergic subjects. Cite this as: J. R. Tversky, A. P. Bieneman, K. L. Chichester, R. G. Hamilton and J. T. Schroeder, Clinical & Experimental Allergy, 2010 (40) 94–102.     

IRF‐5 and NF‐κB p50 co‐regulate IFN‐β and IL‐6 expression in TLR9‐stimulated human plasmacytoid dendritic cells

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.     

Altered innate immune response of plasmacytoid dendritic cells in multiple sclerosis

Plasmacytoid dendritic cells (pDCs) are of crucial importance in immune regulation and response to microbial factors. In multiple sclerosis (MS), pDCs from peripheral blood showed an immature phenotype, but its role in susceptibility to MS is not determined. Because infectious diseases are established triggers of exacerbations in MS, in this study we have characterized the expression of Toll‐like receptors (TLR) and the maturation and functional properties of peripheral blood pDCs from clinically stable, untreated MS patients in response to signals of innate immunity. After stimulation of TLR‐9, interferon (IFN)‐α production by pDCs was significantly lower in MS (n = 12) compared to healthy controls (n = 9). In an allogenic two‐step co‐culture assay we found an impaired effect of TLR‐9 stimulation on IFN‐γ expression of autologous naive T cells in MS patients (n = 4). In peripheral blood mononuclear cells, TLR‐9 stimulation with type A CpG ODN resulted in a higher expression of TLR‐1, ‐2, ‐4, ‐5 and ‐8 in MS patients (n = 7) compared with healthy controls (n = 11). These findings suggest an altered innate immune response to microbial stimuli in MS patients and may help understanding of why common infectious agents trigger MS attacks.