Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction.     

Ribavirin Contributes to Hepatitis C Virus Suppression by Augmenting pDC Activation and Type 1 IFN Production

Ribavirin is used as a component of combination therapies for the treatment of chronic hepatitis C virus (HCV) infection together with pegylated interferon and/or direct-acting antiviral drugs. Its mechanism of action, however, is not clear. Direct antiviral activity and immunomodulatory functions have been implicated. Plasmacytoid dendritic cells (pDCs) are the principal source of type 1 interferon during viral infection. The interaction of pDCs with HCV-infected hepatocytes is the subject of intense recent investigation, but the effect of ribavirin on pDC activation has not been evaluated. In this study we showed that ribavirin augments toll-like receptors 7 and 9-mediated IFNα/β expression from pDCs and up-regulated numerous interferon-stimulated genes. Using the H77S.3 HCV infection and replication system, we showed that ribavirin enhanced the ability of activated pDCs to inhibit HCV replication, correlated with elevated induction of IFNα. Our findings provide novel evidence that ribavirin contributes to HCV inhibition by augmenting pDCs-derived type 1 IFN production.     

Functional Limitations of Plasmacytoid Dendritic Cells Limit Type I Interferon,T Cell Responses and Virus Control in Early Life

Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8⁺ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.     

Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells In Vitro and In Vivo

Background

Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro.

Methods & Findings

Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production

Conclusions

This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR^−/−) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-β in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-β production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-β production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-β mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-β by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.     

Absence of Siglec-H in MCMV Infection Elevates Interferon Alpha Production but Does Not Enhance Viral Clearance

Plasmacytoid dendritic cells (pDCs) express the I-type lectin receptor Siglec-H and produce interferon α (IFNα), a critical anti-viral cytokine during the acute phase of murine cytomegalovirus (MCMV) infection. The ligands and biological functions of Siglec-H still remain incompletely defined in vivo. Thus, we generated a novel bacterial artificial chromosome (BAC)-transgenic “pDCre” mouse which expresses Cre recombinase under the control of the Siglec-H promoter. By crossing these mice with a Rosa26 reporter strain, a representative fraction of Siglec-H⁺ pDCs is terminally labeled with red fluorescent protein (RFP). Interestingly, systemic MCMV infection of these mice causes the downregulation of Siglec-H surface expression. This decline occurs in a TLR9- and MyD88-dependent manner. To elucidate the functional role of Siglec-H during MCMV infection, we utilized a novel Siglec-H deficient mouse strain. In the absence of Siglec-H, the low infection rate of pDCs with MCMV remained unchanged, and pDC activation was still intact. Strikingly, Siglec-H deficiency induced a significant increase in serum IFNα levels following systemic MCMV infection. Although Siglec-H modulates anti-viral IFNα production, the control of viral replication was unchanged in vivo. The novel mouse models will be valuable to shed further light on pDC biology in future studies.     

Differential activation of human monocyte-derived and plasmacytoid dendritic cells by West Nile virus generated in different host cells

Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNV^Vero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNV^Vero to induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNV^C7/10), while mDCs responded similarly to WNV^Vero or WNV^C7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNV^Vero but not WNV^C7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNV^C7/10 than in those infected with WNV^Vero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.     

Hepatitis C Virus Core Protein Inhibits Interferon Production by a Human Plasmacytoid Dendritic Cell Line and Dysregulates Interferon Regulatory Factor-7 and Signal Transducer and Activator of Transcription (STAT) 1 Protein Expression

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.     

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     

Slc15a4, a Gene Required for pDC Sensing of TLR Ligands,Is Required to Control Persistent Viral Infection

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.     

Interferon Regulatory Factor-1 Protects from Fatal Neurotropic Infection with Vesicular Stomatitis Virus by Specific Inhibition of Viral Replication in Neurons

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1^−/− mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1^−/− mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.     

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.     

HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.     

EBI2 Is a Negative Regulator of Type I Interferons in Plasmacytoid and Myeloid Dendritic Cells

Epstein-Barr virus induced receptor 2 (EBI2), a Gα_i-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b⁺ myeloid cells. Activation of EBI2^−/− pDCs and CD11b⁺ cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, in vivo challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2^−/− mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b⁺ cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.