IFN-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes is promoted by NK cells via MIP-1β and LFA-1

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE(2), and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC-induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56(dim) NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56(bright) NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1β, RANTES, IFN-γ, and TNF-α via RNA-IC-mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1β secretion and LFA-mediated cell-cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC-induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC-stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK-pDC axis in IFN-α-driven autoimmune diseases such as SLE should be investigated.     

TRAIL(+) human plasmacytoid dendritic cells kill tumor cells in vitro: mechanisms of imiquimod- and IFN-α-mediated antitumor reactivity

Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered cytotoxic activity. We and others have previously shown that TLR7/8- and TLR9-stimulated plasmacytoid DCs (pDCs) isolated from human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a comparatively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-α/β-dependent fashion and, notably, TRAIL expression on pDCs could be induced by IFN-α stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-α-activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-α-activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ and IFN-α exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply that tumor immunity in patients receiving adjuvant IMQ and/or IFN-α may involve the active participation of cytotoxic pDCs.     

Hepatitis C virus fails to activate NF-κB signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.     

Distinctive TLR7 signaling, type I IFN production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host

Why cross-species transmissions of zoonotic viral infections to humans are frequently associated with severe disease when viruses responsible for many zoonotic diseases appear to cause only benign infections in their reservoir hosts is unclear. Sooty mangabeys (SMs), a reservoir host for SIV, do not develop disease following SIV infection, unlike nonnatural HIV-infected human or SIV-infected rhesus macaque (RM) hosts. SIV infections of SMs are characterized by an absence of chronic immune activation, in association with significantly reduced IFN-α production by plasmacytoid dendritic cells (pDCs) following exposure to SIV or other defined TLR7 or TLR9 ligands. In this study, we demonstrate that SM pDCs produce significantly less IFN-α following ex vivo exposure to the live attenuated yellow fever virus 17D strain vaccine, a virus that we show is also recognized by TLR7, than do RM or human pDCs. Furthermore, in contrast to RMs, SMs mount limited activation of innate immune responses and adaptive T cell proliferative responses, along with only transient antiviral Ab responses, following infection with yellow fever vaccine 17D strain. However, SMs do raise significant and durable cellular and humoral immune responses comparable to those seen in RMs when infected with modified vaccinia Ankara, a virus whose immunogenicity does not require TLR7/9 recognition. Hence, differences in the pattern of TLR7 signaling and type I IFN production by pDCs between primate species play an important role in determining their ability to mount and maintain innate and adaptive immune responses to specific viruses, and they may also contribute to determining whether disease follows infection.     

Synthesis and immunological activities of novel Toll-like receptor 7 and 8 agonists

Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.     

Role of Autophagy in Innate Viral Recognition

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes via Toll-like receptors (TLRs) upon endocytosis of virions. Yet, pDC responses to certain single-stranded RNA viruses occur only following live viral infection. In our recent study, we presented evidence that the recognition of such viruses by TLR7 requires autophagy. We speculate that the requirement for autophagy in viral recognition reflects the necessity for transportation of cytosolic viral replication intermediates into the lysosome where TLR7 is activated. In addition, autophagy was found to be required for pDCs to produce type I interferon (IFN) in response to both ssRNA and dsDNA viruses. These results indicated that autophagy plays a key role in mediating virus detection and IFNα secretion in pDCs, and suggest that cytosolic replication intermediates of ssRNA viruses serve as pathogen signatures recognized by TLR7.

Addendum to:

Autophagy-Dependent Viral Recognition by Plasmacytoid Dendritic Cells

H.K. Lee, J.M. Lund, B. Ramanathan, N. Mizushima and A. Iwasaki

Science 2007; In press     

Patients with chronic hepatitis B infection display deficiency of plasmacytoid dendritic cells with reduced expression of TLR9

Chronic hepatitis B virus (HBV) infection is a complex interaction between replicating noncytopathic virus and dysregulatory host antiviral immunity. Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immunity via secreting type I interferons. Toll-like receptor (TLR) 9 is involved in major pattern recognition receptors expressed in pDCs. The frequency of pDCs and TLR9 expression in peripheral blood mononuclear cells (PBMC) was determined, using flow cytometry. IFN-α production by PBMC was evaluated in vitro in the presence of cytidine phosphate guanosine (CpG) with/without pDCs. The correlation between TLR9, pDCs frequency and viral load was also evaluated. TLR9 expression in pDCs in chronic HBV patients was significantly (∼50%) reduced, supported by ∼70% reduction of TLR9 mRNA, in comparison to healthy controls, correlating with the impairment of IFN-α production in vitro. Furthermore, pDCs frequency in these patients was substantially reduced (∼30%), inversely correlating with serum ALT levels and HBV viral load. HBsAg and HBcAg were detected by immunohistochemistry in pDCs in chronic HBV patients. We conclude that HBV infection results in reduced frequency of circulating pDCs and their functional impairment via inhibiting the expression of TLR9. These data may provide useful information in both basic research and clinical treatment of chronic HBV infection.     

Role of autophagy in innate viral recognition

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes via Toll-like receptors (TLRs) upon endocytosis of virions. Yet, pDC responses to certain single-stranded RNA viruses occur only following live viral infection. In our recent study, we presented evidence that the recognition of such viruses by TLR7 requires autophagy. We speculate that the requirement for autophagy in viral recognition reflects the necessity for transportation of cytosolic viral replication intermediates into the lysosome where TLR7 is activated. In addition, autophagy was found to be required for pDCs to produce type I interferon (IFN) in response to both ssRNA and dsDNA viruses. These results indicated that autophagy plays a key role in mediating virus detection and IFNalpha secretion in pDCs, and suggest that cytosolic replication intermediates of ssRNA viruses serve as pathogen signatures recognized by TLR7.     

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.     

Innate immune responses in human monocyte-derived dendritic cells are highly dependent on the size and the 5' phosphorylation of RNA molecules

Recognition of viral genetic material takes place via several different receptor systems, such as retinoic acid-inducible gene I-like receptors and TLRs 3, 7, 8, and 9. At present, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human immune cells has been limited. In this study, we generated bacteriophage 6 and influenza A virus-specific ssRNA and dsRNA molecules ranging from 58 to 2956 nt. In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently upregulated the expression of IFN (IFN-α, IFN-β, and IFN-λ1) and proinflammatory (TNF-α, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as with dsRNA molecules. Dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to stimulate innate immune responses. Such a difference was not detected for long ssRNAs. RNA-induced cytokine responses correlated well with IFN regulatory factor 3 phosphorylation, suggesting that IFN regulatory factor 3 plays a major role in both ssRNA- and dsRNA-activated responses in human moDCs. We also found that IFN gene expression was efficiently stimulated following recognition of short dsRNAs by retinoic acid-inducible gene I and TLR3 in human embryonic kidney 293 cells, whereas ssRNA-induced responses were less dependent on the size of the RNA molecule. Our data suggest that human moDCs are extremely sensitive in recognizing foreign RNA, and the responses depend on RNA size, form (ssRNA versus dsRNA), and the level of 5' phosphorylation.     

The double-stranded RNA bluetongue virus induces type I interferon in plasmacytoid dendritic cells via a MYD88-dependent TLR7/8-independent signaling pathway

Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.     

Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates

Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.     

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.     

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.     

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.     

Immature cell populations and an erythropoiesis gene-expression signature in systemic juvenile idiopathic arthritis: implications for pathogenesis

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.