Plasmacytoid dendritic cells control dengue and Chikungunya virus infections via IRF7-regulated interferon responses
Upon sensing invading viruses , host cells produce type I interferons ( IFNs ) , leading to the expression of an array of IFN-stimulated genes ( ISGs ) . This first-line response suppresses viral spread by generating an antiviral state within host cells , and supports the initiation of adaptive immunity ( Hoffmann et al . , 2015 ) . Viral sensing may involve non-hematopoietic or hematopoietic cells that are targets of infection . Specific pathogen-associated motifs , such as viral nucleic acids , are recognized by pattern recognition receptors , which can be cytoplasmic ( e . g . retinoic inducible gene-I ( RIG-I ) -like receptors , and NOD-like Receptors ) or endosomal ( e . g . Toll-like receptors; TLRs ) ( Jensen and Thomsen , 2012 ) . Due to the potency of these innate responses , most viruses have evolved mechanisms to subvert or evade pathogen-sensing pathways ( GarciaGarcía-Sastre , 2017 ) . We and others have recently highlighted the existence of alternative or indirect pathogen-sensing mechanisms that circumvents cell-intrinsic viral evasion mechanisms ( Webster et al . , 2016 ) . Such alternative pathways involve the sensing of infected target cells by plasmacytoid dendritic cells ( pDCs ) , a DC subtype specialized in the production of robust level of type I IFNs ( referred herein to as IFN-I ) ( Swiecki and Colonna , 2015 ) . Notably , this was illustrated in the context of dengue virus ( DENV ) , a positive-sense single-stranded RNA virus , which represents global health concerns ( Bhatt et al . , 2013 ) . Recent in vitro work highlighted a newly defined mode of pDC activation , which is mediated primarily by non-infectious immature DENV particles and requires physical cell-cell contact with DENV-infected cells ( Décembre et al . , 2014 ) . Importantly , such cell-cell contact-dependent activation of pDCs has also been reported for several other genetically distant viruses ( Webster et al . , 2016 ) . Consistently , DENV , like some other viruses , does not to directly infect pDCs ( Décembre et al . , 2014; Webster et al . , 2016 ) . We thus aimed to test how such indirect cell-cell sensing of viral pathogens by pDCs , in the absence of pDC infection , engages host mechanisms for in vivo viral clearance . pDCs function as sentinels of viral infection , predominantly via recognition of single-stranded RNA and unmethylated CpG containing DNA by endosomal TLR7 and TLR9 , respectively . Activation of TLR7 or TLR9 results in copious secretion of IFN-I as well as other proinflammatory cytokines ( notably TNFα ) , driven by the transcription factor ( s ) interferon regulatory factor ( IRF ) −7 and nuclear factor-kappa B ( NF-κB ) , respectively ( Swiecki and Colonna , 2015 ) . Interestingly , despite the inherent ability of pDCs to produce both IFN-I and NF-κB-induced cytokines , previous studies have suggested a ‘bifurcated’ pattern of TLR7/9 signaling , where IFN-I production may occur in the absence of NF-κB activation , and vice versa ( Swiecki and Colonna , 2015 ) . The production of IFN-I or pro-inflammatory cytokines may in part be dependent on the sub-cellular compartment in which these TLRs encounter activating signal , as CpG-A accumulates in early endosomes to induce IFN-I whereas CpG-B aggregates in endolysosomes to activate NF-κB ( Swiecki and Colonna , 2015 ) . In the context of viral infection , pDCs exposed to cell-associated Hepatitis C virus produced IFN-I but not NF-κB-dependent cytokines ( Dental et al . , 2012 ) . Despite these observations , the consequences of pDC IFN-I production in the absence of NF-κB induction remain unclear , and have not yet been shown to be sufficient for in vivo viral control . Herein , we show that indirect activation of pDCs by contact with DENV or CHIKV infected cells results in an IRF7-induced IFN response , in the absence of NF-κB inflammatory responses . We developed a novel mouse model where Irf7 expression is pDC-restricted , that is , Irf3-/-;Irf7-/- double knockout mice , with Irf7 expression driven under the pDC-specific promoter Sialic acid binding Ig-like lectin H ( Siglech ) ( Blasius et al . , 2006; Takagi et al . , 2011; Zhang et al . , 2006 ) . We demonstrated that pDC-restricted IRF7-induced signaling is sufficient to achieve in vivo control of DENV and CHIKV infections . We further elucidated that the early pDC IRF7-mediated response accelerates type II IFN ( IFNγ ) responses via natural killer ( NK ) cell activation , thus positioning pDCs as a cell type that regulates the interplay between type I and type II IFN responses in the control of these viral infections , an immune axis that is independent of other sources of type I IFN or pDC-derived NF-κB-induced cytokines .
We previously reported that ex vivo activation of an antiviral response by pDCs requires physical contact with DENV-infected cells ( Décembre et al . , 2014 ) . Prior studies suggested that pDC TLR7/9 signaling differentially induced IRF7 and/or NF-κB , depending on the ligand ( Swiecki and Colonna , 2015 ) . Thus , we determined relative pDC secretion of representative NF-κB-induced ( TNFα ) and IRF7-induced ( IFNα ) cytokines in response to DENV . The sensing of DENV-infected cells by human pDCs failed to induce TNFα secretion ( Figure 1A ) . This was in contrast to the robust TNFα production triggered by direct TLR7 stimuli , including the synthetic ligand imiquimod ( IMQ ) and influenza A [Flu] , which directly activates pDCs as cell-free virus as previously reported ( Décembre et al . , 2014 ) ( Figure 1A ) . pDC activation by DENV , the result of cell-cell transfer of TLR7 ligands , may occur more slowly than direct activation by cell-free stimuli . To assess if differential kinetics of pDC activation underlie the difference in TNFα production , we evaluated pre-treatment of human pDCs with low-dose IFNβ , prior to coculture with infected cells . Such pre-treatment regimens have been previously shown to accelerate both IFNα and TNFα production in response to influenza virus ( Phipps-Yonas et al . , 2008 ) . While IFNβ pretreatment accelerated the kinetics of pDC-derived IFNα ( all stimuli ) and TNFα production ( IMQ/Flu ) , we observed minimal TNFα production in response to DENV-infected cells ( Figure 1B ) . These observations imply that the defect in DENV-mediated TNFα induction by pDCs might be due to the indirect mechanism of stimulation . Furthermore , we showed that pDC TNFα levels in response to IMQ were not diminished in presence of DENV-infected cells ( Figure 1—figure supplement 1A ) . Together , these results indicated that DENV is neither promoting nor preventing the activation of NF-κB-induced TNFα production by pDCs . We extended these findings to mouse pDC activation , utilizing cells derived from either bone marrow ( BM ) or isolated from spleen . We also assessed a broader range of NF-κB-dependent genes . DENV-infected cells induced higher levels of IFNα and interferon-stimulated genes ( ISGs ) as compared to synthetic TLR7/8 agonists ( R848 and IMQ ) ( Figure 1C , Figure 1—figure supplement 1C–D ) , results that were consistent with human pDC findings ( Figure 1A–B ) . Conversely , the TLR7/8 agonist R848 induced a strong NF-κB-dependent gene signature in mouse pDCs , whereas limited induction was observed when using DENV-infected cells ( Figure 1D and Figure 1—figure supplement 1C ) . Together , these results demonstrated that pDC-mediated sensing of DENV-infected cells primarily leads to IFN-I production and a strong ISG response in the absence of NF-κB-mediated inflammatory responses . To address how this IRF7- and IFN-I-restricted response to DENV may position pDCs as critical in the control of viral infections , we generated a unique mouse model where IRF7 signaling is restricted to pDCs . We established a knock-in strain where Irf7 expression is driven by the pDC-specific Siglech promoter ( thus called SiglechIrf7/+ ) ( Figure 2A ) . We next backcrossed these mice onto Irf3-/-;Irf7-/- double knockout mice to generate hemizygous Irf7-expressing animals ( SiglechIrf7/+;Irf3-/-;Irf7-/- referred to as ‘pDC:Irf7+’ mice ) . Use of hemizygous mice preserved one copy of the Siglech gene ( Figure 1—figure supplement 1B ) . Irf3/7 double knockout mice ( referred to as Irf3/7 DKO mice ) , deficient in IFN-I production ( Rudd et al . , 2012; Schilte et al . , 2012 ) were used as comparator negative controls in all experiments . To validate the Irf7 knock-in in pDC:Irf7+ mice , we analyzed IRF7 protein levels in DC subsets . pDCs were the only cell type to retain significant levels of IRF7 protein expression , seen in both pDC:Irf7+ and WT mice , but not in Irf3/7 DKO mice ( Figure 2B ) . To functionally validate the pDC:Irf7+ mice , we assessed IFN-I activity induced upon in vivo treatment with agonists of TLR9 and TLR3 , which are expressed or not by pDCs , respectively ( Swiecki and Colonna , 2015 ) . As expected , we observed IFN-I activity in plasma/spleen of WT mice stimulated by either agonist , whereas little-to-no IFN-I activity was detected in Irf3/7 DKO mice ( Figure 2C–D ) . Consistent with the TLR expression patterns in pDCs ( Swiecki and Colonna , 2015 ) , pDC:Irf7+ mice produced high levels of IFN-I in response to TLR9 , but not TLR3 agonists . Using this model system , we assessed how pDC IRF7-signaling mediates antiviral responses to DENV . First , we purified pDCs from WT , Irf3/7 DKO and pDC:Irf7+ mice , and treated them with TLR7 agonists ( R848/IMQ/cell-free Flu ) or DENV-infected cells . pDC:Irf7+ and WT pDCs produced similar amounts of IFNα ( Figures 2E and 3A ) , confirming the functionality of IRF7 signaling in pDC:Irf7+ mice . We also tested NF-κB-signaling in pDCs from Irf3/7 DKO and pDC:Irf7+ mice induced by the same TLR7 agonists . Confirming independent activation of NF-κB , we observed TNFα secretion levels in both strains to be comparable to WT mice ( Figures 2F and 3B ) . Of note , ISGs previously defined as IRF5-dependent ( e . g . Mx1 , Ifit1 ) ( Lazear et al . , 2013 ) were still upregulated in Irf3/7 DKO pDCs when cocultured with DENV-infected cells or stimulated by other TLR7 agonists ( Figure 1—figure supplement 1D ) . To determine whether IFN-I response to viral stimuli was restored in pDC:Irf7+ mice , animals were infected with DENV systemically ( intravenously , i . v . ) , and IFNα/β expression was assessed . High levels of IFNα were detected in both the spleen and plasma of infected WT mice ( Figure 3C–D ) , but not Irf3/7 DKO mice , in agreement with previous results ( Chen et al . , 2013 ) . IFNβ levels displayed the same pattern of expression ( data not shown ) . pDC:Irf7+ mice had undetectable IFNα/β levels in both the plasma and spleen at all analyzed times post-infection ( p . i . ) ( Figure 3C–D ) . We considered the possibility that the pDC response to DENV may occur in a localized manner , with insufficient type I IFN in plasma or spleen to allow its detection . We thus assessed downstream responses of IFNα/β receptor ( IFNAR ) signaling via qRT-PCR analysis of ISGs in different tissues of DENV-infected mice . The ISGs ( Ifit1 , Usp18 , and Ifit3 ) were selected for their robust expression following by IFN-I stimulation , as compared to type II IFN ( Liu et al . , 2012 ) . Importantly , despite undetectable IFNα/β in DENV-infected pDC:Irf7+ mice , an early induction ( i . e . 18 h p . i . ) of ISG RNAs was observed in all tissues analyzed . By contrast , this early induction was not observed in Irf3/7 DKO mice ( Figure 3E ) . Of note , basal ISG expression levels were lower in Irf3/7 DKO and pDC:Irf7+ mice compared to WT , likely reflecting diminished basal IFNAR signaling in the absence of Irf3 and Irf7 ( Gough et al . , 2012 ) . These results indicate that restricted expression of Irf7 in pDCs is sufficient to generate a systemic ISG response . The pDC:Irf7+ model provided a means to distinguish , in vivo , the role of pDCs in regulating antiviral IFN-I-mediated versus NF-κB-induced response ( s ) to DENV infection . We therefore measured IRF/STAT-dependent ISG expression in multiple organs , and a larger panel of ISGs and NF-κB-dependent transcripts in the liver of DENV-infected animals . DENV infection induced ISGs and NF-κB-dependent transcripts in WT , but not Irf3/7 DKO mice ( Figure 3E–G; expressed as magnitude-independent Z score and Figure 3—figure supplement 1; expressed as magnitude-dependent fold-change ) , confirming previous findings ( Chen et al . , 2013 ) . Strikingly , despite a strong upregulation of ISGs in DENV-infected pDC:Irf7+ mice , we observed minimal induction of NF-κB-dependent inflammation-related transcripts ( Figure 3F–G and Figure 3—figure supplement 1 ) . Based on these in vivo and ex vivo observations , we concluded that the principal signaling pathway downstream of DENV-activated pDCs is IRF7-dependent induction of the IFN-I pathway , which could be wholly segregated from the activation of NF-κB-induced cytokines . We next sought to determine whether pDC IRF7-induced responses are sufficient to protect mice from DENV . Mice were infected i . v . by DENV and viral RNA and titers were assessed in the spleen , the primary site of replication in infected mice . Viral titer and RNA levels were lower in WT mice than either Irf3/7 DKO or pDC:Irf7+ mice ( Figure 4 and Figure 4—figure supplement 1A ) , likely due to the higher IFN-I response . Notably , at 42–72 hr post-infection , pDC:Irf7+ mice displayed significantly reduced levels of DENV titer and RNA in the spleen and plasma compared to Irf3/7 DKO mice ( Figure 4 and Figure 4—figure supplement 1A ) . The higher DENV levels in pDC:Irf7+ mice as compared to Irf3/7 DKO mice at early time points ( i . e . 28 h p . i . ) may reflect an enhanced recruitment of DENV-susceptible cells in the spleen of pDC:Irf7+ mice due to the localized pDC-induced IFN-I response ( Schmid and Harris , 2014; Seo et al . , 2011 ) . Our results indicate that IRF7 expression in pDCs is sufficient to control DENV through the local production of IFN-I . To examine the downstream effector mechanisms initiated by pDC-derived IFN-I , we considered the relative contribution of lymphocyte-derived type II IFN ( IFN-II ) to viral clearance . Notably , an IFN-II response has been observed even in the absence of Irf3 and Irf7 expression in response to DENV infections , and was demonstrated to eventually control DENV infection ( Chen et al . , 2013 ) . To assess IFN-II responses , we measured Ifng and Gbp2 expression , the latter being known to specifically define an IFN-II response ( Hall et al . , 2012 ) . Interestingly , Ifng and Gbp2 up-regulation ( Figure 5B–C ) temporally correlated with the induction of IFN-I-induced ISGs in DENV-infected Irf3/7 DKO mice ( Figure 5A and Figure 4—figure supplement 1B–E ) . Of note , these ISGs including Ifit1/Usp18/Ifit3 respond more highly to IFN-I , but will still be induced to some extent by type II IFN [ ( Chen et al . , 2013; Hall et al . , 2012; Liu et al . , 2012 ) and data not shown] . Therefore , in agreement with these prior studies , IFN-II signaling likely mediates the late induction of a larger set of ISGs observed in Irf3-/-/7-/- mice . The kinetic profile of the type II IFN response was markedly different in pDC:Irf7 +mice as compared to Irf3/7 DKO mice , with Ifng and Gbp2 expression detected as early as 18 h p . i and peaking at 28 h p . i . ( Figure 5B–C ) . This led to the compelling hypothesis that early pDC IRF7-mediated signaling accelerates the IFN-II response , which is known to be part of the eventual control of DENV in mouse models ( Chen et al . , 2013; Costa et al . , 2017; Shresta et al . , 2004 ) . Given that the ex vivo response of pDCs was biased toward IRF7-mediated IFN-I production ( Figures 1 and 3A–B ) , we hypothesized that the early ISG response in pDC:Irf7+ mice was mediated by this pathway . To formally test the dependence of this early ISG response on IFN-I and thus IFNAR signaling , we employed an IFNAR1 blocking antibody and challenged mice with DENV . IFNAR blockade in pDC:Irf7+ mice abrogated this early ISG response ( Figure 5—figure supplement 1A ) and correspondingly increased both DENV viremia in these mice , as readily observed in the spleen ( Figure 5—figure supplement 1C ) and to a lesser extent in the blood ( Figure 5—figure supplement 1D; not significant; likely as the consequence of the limited dynamic range of DENV detection at this time point in blood ) . Of note , consistent with the results of Figure 5 , ISG expression was still detected in the Irf3/7 DKO and pDC:Irf7+ at a later time point ( 72 h p . i . ) ( Figure 5—figure supplement 1A–B ) , further suggesting that the late induction of ISGs is independent of pDC IRF7-mediated IFN-I and likely induced by IFN-II . Along the same line , recent work demonstrates that IRF1-mediated IFN-II can be induced by DENV , independent of Irf3/Irf7 expression ( Carlin et al . , 2017 ) . In accordance , we showed that the IFNAR1-dependent response contributes to the control of DENV in Irf3/7 DKO mice ( Figure 5—figure supplement 1C ) . Together , our results demonstrated that , despite undetectable IFN-I at a systemic level , the early ISG induction and control of DENV by the pDC-IRF7-induced response relies on IFN-I/IFNAR1 signaling . Importantly , we also observed an acceleration of IFN-II signaling as a result of IRF7 expression in pDCs , temporally associated with viral control in DENV-infected pDC:Irf7+ mice . We next aimed at identifying the cell type ( s ) responsible for IFN-II production and its subsequent impact on signaling in infected pDC:Irf7+ mice . We observed that an elevated fraction ( ~12% ) of splenic NK cells produced IFNγ in DENV-infected pDC:Irf7+ mice ( Figure 6A ) . For other cell types , including NKT cells , γδ-T cells , αβ-T cells and neutrophils , IFNγ+ cells represented an extremely small fraction of total splenocytes ( Figure 6A , right panel and S5A-B ) . Interestingly , splenic , but not blood-circulating NK cells , produced IFNγ protein in response to DENV infection , consistent with the absence of upregulation of blood Ifng mRNA ( data not shown ) and limited induction of the activation marker CD69 on blood NK cells ( Figure 6—figure supplement 1C ) . This implies that NK cell response is localized to the spleen , the primary site of DENV replication . In agreement with a localized host response , we showed that the accelerated IFN-II response in DENV-infected pDC:Irf7+ mice was associated with an early recruitment of neutrophils to the spleen ( Figure 6—figure supplement 1D ) as well as maturation of monocytes ( identified as Ly6C++/Lineage- cells ) , as represented by upregulation of MHCII , which did not occur in Irf3/7 DKO mice ( Figure 6—figure supplement 1E ) . Of note , both cell types have been previously shown to be involved in the modulation of NK cell activation ( Costa et al . , 2017; Kang et al . , 2008 ) . As NK cells were the primary producers of IFN-II ( Figure 6A ) , we sought to identify their role during DENV infection in pDC:Irf7+ mice in NK cell depletion studies . An efficient depletion of NK cells ( i . e . reduction by >80% of splenic and blood NK cells , Figure 6B and Figure 6—figure supplement 2A ) lead to a significant increase of both splenic DENV RNA and viral titers in pDC:Irf7+ mice ( Figure 6C ) . This was associated with a decrease in IFNγ mRNA , IFNγ protein and IFN-II ISG signature in the spleen and plasma ( Figure 6D–E ) , along with reduced neutrophil recruitment to the spleen and maturation of monocytes ( Figure 6—figure supplement 2B–C ) . Closing the circle and establishing IFNα responses as upstream , we observed that IFN-I-induced ISG levels were not impacted by NK cell depletion in pDC:Irf7+ mice at 48 h p . i . ( Figure 6E ) . We extended the study to CHIKV , also a positive-sense single-stranded RNA mosquito-borne virus of major concern for human health . We tested whether pDC activation by CHIKV shares the same features as DENV , showing that indeed , human pDCs produced robust IFNα in response to CHIKV only when in contact with CHIKV-infected cells ( Figure 7A ) . Similarly , robust IFNα levels were detected when WT mouse pDCs were co-cultured with CHIKV infected Vero cells or with syngeneic co-cultures of Irf3/7 DKO mouse embryonic fibroblasts ( MEFs ) ( Figure 7B–C ) . By contrast , cell-free CHIKV and CHIKV-infected cells physically separated from pDCs did not induce IFNα secretion ( Figure 7A–B ) . CHIKV-infected cells failed to activate pDCs deficient for Tlr7 , whereas pDCs deficient for Mavs ( i . e . the downstream signaling adaptor of RLR pathway ) produced levels of IFNα comparable to WT pDCs ( Figure 7D and data not shown ) . Comparable to DENV-induced responses , the sensing of CHIKV-infected cells by pDCs led to an IRF7-mediated cytokine production ( IFNα ) , but not NF-κB-induced inflammatory cytokines ( TNFα ) ( Figure 7E ) . Together , this demonstrated that , like DENV , pDC induction by CHIKV required the cell-to-cell sensing of infected cells by TLR7-induced signaling and led to an IRF7-restricted response . We thus tested whether pDC IRF7-induced responses were sufficient to protect mice from CHIKV . Mice were infected s . c . with CHIKV , and as previously reported WT mice showed no clinical symptoms , whereas Irf3/7 DKO mice rapidly succumbed to CHIKV infection ( Figure 7F ) ( Schilte et al . , 2012 ) . By contrast , pDC:Irf7+ mice experienced no overt clinical symptoms with 100% of mice surviving infection ( Figure 7F ) . Early control of viremia was improved in pDC:Irf7+ mice compared to Irf3/7 DKO mice , although reduced as compared to WT mice ( Figure 7G ) . By day 7 p . i . , pDC:Irf7+ mice had cleared CHIKV infections ( data not shown ) . Importantly , there was differential control of CHIKV in pDC:Irf7+ mice depending on the tissue . The initial sites of CHIKV replication ( i . e . the infection site: skin/muscle and muscle ) displayed relatively higher viral titers as compared to more distal sites ( i . e . the spleen and liver ) , where virus was barely detectable at 48 h p . i . in pDC:Irf7+ mice ( Figure 7G ) . It is possible that the pDC antiviral response controls infection once virus spreads systematically , as previously proposed in the context of Herpes Simplex virus infection ( Swiecki et al . , 2013 ) . We established a mouse IFNα Single Molecule Assay ( SiMoA ) ( Rissin et al . , 2010 ) , with a quantification limit at 0 . 1 pg/ml ( i . e . >100 fold increased sensitivity compared to current assays , data not shown ) . Nonetheless plasma IFNα remained undetectable in CHIKV-infected pDC:Irf7+ mice at both 24 and 48 h p . i . ( data not shown ) . Therefore , we validated the dependence on IFN-I-induced signaling for the control of CHIK by pDCs using IFNAR1 blocking antibody . IFNAR blockade in pDC:Irf7+ mice induced lethality and , consistently , greatly increased CHIKV viremia ( Figure 7—figure supplement 1A–B ) . By contrast with results for DENV ( Figure 5—figure supplement 1C ) , IFNAR1-dependent response did contribute to the control of CHIKV in Irf3/7 DKO mice ( Figure 7—figure supplement 1B ) . This discrepancy between DENV and CHIKV infections likely reflects the greater impact of IFN-I signaling in CHIKV versus DENV infection and/or a differential contribution of the alternate IRF-mediated signal transduction . Similar to DENV , NK cell depletion in pDC:Irf7+ mice increased CHIKV titers ( Figure 7—figure supplement 1C , ~10 fold-increase , although not statistically significant ) . Together , these data indicate that IRF7 expression in pDCs is sufficient to control two genetically distant RNA viruses ( CHIKV and DENV ) via the local production of IFN-I and IFNγ secretion by NK cells .
DENV and CHIKV are important mediators of human diseases , causing a significant human and economic burden worldwide . We provide compelling evidence that pDCs are a key cell type in the initiation of antiviral responses to these two distinct RNA viruses . We demonstrate that pDC activation by DENV/CHIKV is characterized by IRF7-prioritized signaling , while passively excluding NF-κB-mediated responses . Despite undetectable levels of systemic IFN-I , pDC IRF7 signaling is critical for the induction of a potent antiviral response . This might reflect a local pDC IFN-I response and is in keeping with the requirement for physical contact with infected cells to activate pDCs , in turn inducing a potent ISG response . Notably , pDC-IRF7 activation accelerates NK-mediated IFN-II induction , which assists in control of viremia . pDCs have previously been demonstrated to be required for the control of several viral infections , including herpes simplex virus ( HSV ) , murine hepatitis virus ( MHV ) , murine cytomegalovirus ( MCMV ) , vesicular stomatitis virus ( VSV ) and lymphocytic choriomeningitis virus ( LCMV ) ( Swiecki and Colonna , 2015 ) . In contrast , others reported that pDCs are dispensable and/or do not contribute to IFN-I production in the case of certain infections , including influenza virus , respiratory syncytial virus ( RSV ) , Newcastle disease virus ( NDV ) and MCMV ( Del Prete et al . , 2015; GeurtsvanKessel et al . , 2008; Jewell et al . , 2007; Kumagai et al . , 2007; Swiecki et al . , 2013 ) , which may be a reflection of viral replication mechanisms , inoculum size , or location ( Swiecki et al . , 2010; Swiecki et al . , 2013 ) . These observations bring the emerging concept that while antiviral response ( i . e . IFN-I production ) in infected cells acts as a first line defense , pDCs may serve as a failsafe , triggered upon systemic infection and therefore failure of viral control ( GarciaGarcía-Sastre , 2017; Tomasello et al . , 2014 ) . This assumption is in line with the multiple inhibitory mechanisms of antiviral sensing by viral proteins expressed by infected cells ( GarciaGarcía-Sastre , 2017 ) . Nonetheless , previous investigations have primarily demonstrated the importance of pDCs in viral control by depletion of pDCs ( Swiecki and Colonna , 2015 ) . Here , we validated a model consisting of a pDC-restricted IFN response , which offers a unique opportunity to define pDC function . This new model also permitted the study of the possible cryptic pDC-mediated control of certain infections . For instance , pDC antiviral function might be masked due to the homeostasis of the innate immunity , including an enhanced response by other cell types when pDCs are depleted . We demonstrated that pDCs are sufficient to initiate early control of viral infections , via induction of IRF7-prone signaling . In this mouse model , the restoration of Irf7 expression in pDCs does not reflect control under the endogenous promoter , as the Irf7 gene is under the control of the pDC-specific Siglech promoter . However , Irf7 overexpression artifacts are avoided in the pDC:IRF7+ model as Irf7 steady-state expression is reduced under the control of the Siglech promoter relative to the Irf7 promoter ( Figure 2B ) . Furthermore , Siglech expression by pDCs is inversely correlated to ISG upregulation in vivo ( Figure 4—figure supplement 1E ) , in agreement with prior study ( Puttur et al . , 2013 ) . Importantly , while systemic IFN-I was not detectable in response to DENV/CHIKV infection , the pDC-IRF7 response rapidly and potently induces a broad array of ISGs in multiple organs , dependent on IFN-I signaling . Therefore , in line with our ex vivo results in this and previous studies ( Décembre et al . , 2014 ) , we propose that pDCs respond in a contact-dependent manner to infected cells , and thus induce highly localized IFN-dependent responses . Importantly , this response is sufficient to impart a large-scale antiviral response , which protects from viral infections . The engagement of TLR7/9 leading to IFN-I production and NF-κB-dependent pro-inflammatory cytokines by pDCs may affect downstream immunological parameters , such as for example , B cell differentiation induced by pDC response to influenza virus ( Jego et al . , 2003 ) . We observed that pDCs fail to respond via NF-κB-mediated inflammatory responses to DENV/CHIKV-infected cells , indicating that pDC TLR7 signaling induced by these viruses primarily leads to IRF7 induction . Consistently , on the whole-organism level , while ectopic IRF7 expression in pDCs fully restores ISG responses , NF-κB responses to DENV were absent in both pDC:Irf7+ and Irf3/7 DKO mice , thus underlining the importance of the IRF7 and dispensability of the NF-κB responses in pDC-mediated control of DENV and CHIKV infections . Our results also imply that NF-κB induction by these viruses in other cell types likely requires cross-talk between IRF3 and NF-κB ( Freaney et al . , 2013; Iwanaszko and Kimmel , 2015 ) . While pDC IFN-I responses were important in early control of both DENV and CHIKV infections in vivo , substantial differences were observed in the overall outcome of infections . CHIKV infections were invariably lethal in the Irf3/7 DKO genetic background , and pDC IFN-I responses provided 100% protection to this lethal phenotype . By contrast , DENV infections were well-controlled even in Irf3/7 DKO mice by 72 h p . i . Notably , pDC IFN-I responses lead to substantial control of DENV viremia at time points intermediate between WT and Irf3/7 DKO mice ( ~42–48 hr ) . In our study , the lethal phenotype in CHIKV infection in Irf3/7 DKO mice correlated with substantially higher viral titers , as compared to DENV levels . Interestingly , blockade of IFN-I signaling in Irf3/7 DKO mice only increased viremia in situations where lower basal viral titers were observed: for example , DENV infections in all organs , or CHIKV viral titers in the liver . We thus suggest that the persistence of IFN-I-mediated viral control under these conditions depends on IRF5 or IRF1 signaling ( Carlin et al . , 2017; Chen et al . , 2013; Lazear et al . , 2013; Rudd et al . , 2012 ) , as control of flaviviral infections is still contingent on IFNAR signaling in the absence of Irf3/Irf7 expression . The contribution of these alternate IRF proteins to the antiviral response may only be revealed in case of relatively low viremia . Eventual control of DENV in the Irf3/7 DKO background depended on the induced IFNγ response ( Chen et al . , 2013 ) . In agreement , we observe an IFN-II response in DENV-infected Irf3/7 DKO mice peaking at ~42 h p . i . , which is largely absent in WT mice . Importantly , the antiviral IFN-II response is accelerated in pDC:Irf7+ mice relative to Irf3/7 DKO mice . These pDC-regulated IFN-II responses are consistent with prior work showing a defect in type II IFN activation in a pDC-deficient mouse model ( Guillerey et al . , 2012 ) . We demonstrated that IFNγ production in DENV-infected pDC:Irf7+ mice is largely mediated by NK cells in the spleen , the primary site of DENV replication . These activated NK cells mediate downstream IFN-II responses that contribute to control of DENV viremia , in accordance with a recent report ( Costa et al . , 2017 ) . A similar decrease in viremia was observed in CHIKV-infected pDC:Irf7+ mice depleted of NK cells , although these results did not reach statistical significance . DENV may be more sensitive to type II IFN signaling ( relative to type I IFN ) compared to CHIKV , as DENV infections cause higher lethality in Ifngr-/- compared to Ifnar-/- mice , a situation that is reversed in the context of CHIKV infection ( Gardner et al . , 2012; Shresta et al . , 2004 ) . Importantly , IFNγ production by NK cells was not observed in the blood , a site of low DENV viremia , likely indicating a necessity for NK cells to be closely apposed to DENV-infected cells and/or DENV-activated cells to achieve NK cell activation of IFN-II responses . We also observed an association with other immune cell parameters , including a pDC IRF7-mediated influx of neutrophils and monocyte activation in the spleens of DENV-infected mice . While some reports suggested that type I IFN directly contributes to NK cell response , NF-κB-dependent cytokines are well-known to regulate IFNγ production by NK cells ( Schulthess et al . , 2012 ) . Along the same lines , in vitro studies suggested that pDCs act in concert with monocytes to potentiate NK-cell-mediated IFNγ production in the context of HCV and DENV infection ( Costa et al . , 2017; Zhang et al . , 2013 ) . Future studies are required to determine how DENV/CHIKV-activated pDCs can directly augment NK cell IFNγ responses , or whether pDC-produced type I IFN acts through other cell types and attendant NF-κB-dependent cytokine responses for this amplification of type II induction . The ability to segregate NF-κB and IRF7 responses may have potential use in developing novel therapies . Notably , NF-κB-dependent cytokines have been associated with disease severity in both DENV and CHIKV infection ( Chen et al . , 2007; Dupuis-Maguiraga et al . , 2012 ) . This is juxtaposed with pDC-derived IFN responses supporting viral clearance while avoiding possible inflammatory responses . As arboviral infections tend to be acute , rather than chronic diseases , this study provides an important indication of how early viral control may be mediated by one of the key drivers of the IFN response , the plasmacytoid dendritic cell .
Huh7 . 5 . 1 cells ( Zhong et al . , 2005 ) ( RRID:CVCL_E049 ) , Vero cells ( CLS Cat# 605372/p622_VERO , RRID:CVCL_0059 ) , and BHK-21 cells ( ATCC Cat# CCL-10 , RRID:CVCL_1915 ) were maintained in DMEM ( Dulbecco's modified Eagle medium; Life Technologies ) supplemented with 10% FBS , 100 units ( U ) /ml penicillin , 100 mg/ml streptomycin , 2 mM L-glutamine ( Life Technologies ) . Cells were maintained at 37°C in 5% CO2 . MEFs were maintained in supplemented RPMI growth media . The work involved cell line , with routinely verify status for contamination ( e . g . Mycoplasma ) . Cell culture reagents included the TLR7 agonist ( IMQ; imiquimod ) and TLR7/8 agonist ( R848 ) ( Invivogen , San Diego , CA ) ; TLR7 antagonist , IRS661 ( 5’-TGCTTGCAAGCTTGCAAGCA-3’ ) synthesized on a phosphorothioate backbone ( MWG Biotech , Ebersberg , Germany ) ; TLR3 agonist ( poly-I:C ) ( Invivogen ) ; TLR9 agonist ( CpG ) ( Invivogen ) ; recombinant IFNβ ( PBL Interferon Source , Piscataway , NJ ) . General reagents included: paraformaldehyde ( PFA ) and brefeldin A ( BFA ) ( Sigma-Aldrich , St Louis , MO ) ; Ficoll-Hypaque ( GE Healthcare Life Sciences , Piscataway , NJ ) . Viral stocks of the prototypic DENV-2 strain New Guinea C ( NGC ) ( AF038403 ) were produced using in vitro RNA transcripts prepared from DENV-2 infectious plasmid clone pDVWS601 plasmid ( Pryor et al . , 2001 ) . Briefly , plasmid was linearized with XbaI ( New England Biolabs , Ipswich , MA ) and RNA transcripts were produced and purified using mMESSAGE mMACHINE T7 Kit ( Ambion/Thermo Fisher Scientific , Waltham , MA ) . RNA transcripts were introduced into BHK-21 cells by electroporation ( using 100 µg RNA to transfect 8 × 107 cells , with 4 × 106 cells per electroporation ) . Electroporation was performed in 0 . 4 cm cuvettes ( Bio-Rad , Hercules , CA ) with a Gene Pulser system ( Bio-rad ) , using a square-wave pulse ( 280 V , 25 ms ) . Virus-containing supernatants were collected at 3–5 days post-electroporation and clarified through a 0 . 2 µm filter ( Corning Inc , Corning , NY ) . Viral supernatants were concentrated by ultracentrifugation at 75000 x g for 2 hr at 4°C , resuspended in DMEM growth media , and frozen at −80°C until titration and/or use . Viral stocks were titered on Huh7 . 5 . 1 cells , as described below , and adjusted to 2 × 107 focus-forming units ( ffu ) /mL . CHIKV-21 viral stocks , used for all in vivo infections , were prepared from clinical samples ( Schilte et al . , 2010 ) . Briefly , CHIKV-21 was propagated in C6/36 mosquito cells , and infectious supernatants were harvested , clarified through a 0 . 2 µm filter , and frozen at −80°C until titration and/or use . Recombinant CHIKV-GFP , which expresses GFP under a subgenomic promoter ( CHIKV-GFP 5’ ) , was generated from a full-length infectious cDNA clone provided by S . Higgs ( Vanlandingham et al . , 2005 ) and was used for all CHIKV in vitro mouse studies . Briefly , CHIKV-GFP 5’ plasmid was linearized with NotI ( New England Biolabs ) and RNA transcripts were produced and purified using mMESSAGE mMACHINE SP6 Kit ( Ambion ) . RNA transcripts were introduced into BHK-21 cells by electroporation ( using 1 µg RNA to transfect 5 × 106 cells ) . Virus-containing supernatants were collected when the viral cytopathic effect reached 75% of transfected cells , clarified through a 0 . 2 µm filter ( Corning Inc ) , and frozen at −80°C until titration and/or use . Viral stocks were titered on Vero cells , as described below . DENV infectivity titers in concentrated culture supernatants or mouse organ homogenates were determined by end-point dilution using Huh 7 . 5 . 1 cells ( Décembre et al . , 2014 ) . Foci forming units ( ffu ) were detected 72 hr after infection . Briefly , Huh7 . 5 . 1 cells were fixed with 4% PFA and permeabilized by incubation for 7 min in PBS containing 0 . 1% Triton . Cells were then blocked in PBS containing 3% BSA for 15 min and incubated for 1 hr with mouse anti-E glycoprotein ( clone 3H5 ) hybridoma supernatant diluted in PBS containing 1% BSA . After three washes with PBS , cells were incubated 1 hr with secondary Alexa 555-conjugated anti-mouse antibody . Virally-infected foci of E-positive cells were determined using a Zeiss Axiovert 135 microscope . CHIKV infectivity titers in culture supernatants or mouse organ homogenates were titrated as TCID50 endpoint on Vero cells using a standard procedure . Serial 10-fold dilutions of supernatants were added in six replicates in 96-well plates seeded with 104 Vero cells . The cytopathic effect was scored 5 days after infection and the titers were calculated by determining the last dilution giving 50% of wells with cells displaying a cytopathic effect . Results were expressed as TCID50/mL . Viral stocks of Influenza A Virus ( FluAV , H1N1/New Caledonia/2006 ) were produced as previously described ( de Chassey et al . , 2013 ) and kindly provided by Dr V . Lotteau ( CIRI , Lyon , France ) . All mice , previously backcrossed on a C57BL/6 background , were bred and maintained at the Institut Pasteur ( Paris , France ) and the Plateau de Biologie Expérimentale de la Souris ( PBES , Lyon , France ) with Authorization Agreement C 69 123 0303 . All animal studies were performed in accordance with the European Union guidelines for approval of the protocols by the Institutional Committees on Animal Welfare of the Institut Pasteur ( Paris , France , OLAW assurance #A5476-01 ) and by the local ethics committee Rhône-Alpes d’Ethique pour l’Expérimentation Animale ( Lyon , France , Authorization Agreement C2EA-15 ) . Housing conditions for mice in the PBES were as follows . Mice were housed under group conditions in individually ventilated cages , from 1 to 8 single-sex mice per cage . Specific-pathogen free ( SPF ) housing was maintained , with regular sanitary status testing of sentinel mice and bedding for standard mouse pathogens . Mice were fed LASQCdiet Rod16-R ( Lasvendi ) , and temperatures were maintained at 22 ± 2°C . All mice were treatment- and drug-naive at the time of experimental manipulation . Manipulations including and following viral infections were carried out under animal BSL-3 ( A3 ) conditions; mice were anesthetized with xylazine/ketamine ( 20 mg/kg and 100 mg/kg by body weight ) for all A3 manipulations . Similar housing conditions were maintained at the Institut Pasteur: SPF housing , 1–5 single-sex mice per cage , and manipulations carried out under animal BSL3 . All in vivo CHIKV experiments were performed at the Institut Pasteur . Irf3-/-Irf7-/- double knockout ( referred to as Irf3/7 DKO ) mice were generated and kindly provided by Dr T . Taniguchi ( University of Tokyo , Tokyo , Japan ) ( Honda et al . , 2005 ) . cardif-/- mice were generated by Dr J . Tschopp , and Tlr7-/- mice were provided by Dr S . Akira ( Osaka University , Osaka , Japan ) . Knock-in SiglechIrf7+ mice were generated by Ozgene Pty Ltd company . Next , knock-in SiglechIrf7+mice were backcrossed onto the Irf3/7 DKO background strain for approximately 10 generations to generate SiglechIrf7+Irf3/7 DKO breeding stocks , referred to as ‘Union Irf3/7 DKO’ . As the SiglechIrf7+ transgene replaced the endogenous Siglech gene , to preserve Siglech expression and functionality in experimental mice , ‘Union Irf3/7 DKO’ mice were bred with Irf3/7 DKO mice to generate SiglechIrf7/+ Irf3/7 DKO ( ‘pDC:Irf7+’ ) F1 mice . For in vivo synthetic agonist experiments , 100 µg of CpG 2216 ( Invivogen , tlrl-hodna ) +DOTAP ( Roche ) or 10 µg of poly-I:C ( Invivogen , tlrl-pic ) were injected intravenously ( i . v . ) into 8- to 12-week-old mice . For DENV in vivo experiments , 8- to 16-week-old mice were infected systemically with DENV . 1–3 litters of mice of each genotype were used per experiment; each litter was aged within 3 weeks of all others in the same experiment . Mice were randomly assorted into treatment groups by the experimenter . 2 × 106 ffu of DENV was injected i . v . in the retro-orbital sinus using a 31-gauge 0 . 3 mL insulin syringe ( Becton-Dickinson , Franklin Lakes , NJ ) for all experiments . In Figure 3—figure supplement 1 , an additional group of mice were i . v . infected with 2 × 105 ffu DENV , as indicated . DMEM growth media was used as a negative injection control . For CHIKV experiments , 8- to 12-week-old adult mice were infected with 106 pfu ( plaque-forming units ) of CHIKV-21 sub-cutaneously in the right flank . For IFNAR blocking studies , a single dose ( 800 µg/mouse ) of anti-IFNAR1 antibody ( Leinco Technologies , Fenton , MO; clone MAR1-5A3 ) or IgG1 isotype control was administered i . p . 24 hr ( CHIKV ) or 3 hr ( DENV ) before infection . For NK cell depletion , a single dose ( 800 µg and 200 µg for CHIKV and DENV experiments , respectively ) of α-NK1 . 1 antibody ( BioXCell , West Lebanon , NH; clone PK136 ) or IgG2A isotype control was injected i . p . 24 hr before infection . Mice cohorts , representing individual experiments , are provided in Supplementary file 1 , with mice age and sex . Solid tissues were collected from mice and cell suspensions were prepared by pressing tissues through 40 µm cell strainers ( Becton-Dickinson ) or homogenates were prepared with the TissueLyser II ( Qiagen , Hilden , Germany ) or GentleMACs ( Miltenyi , Bergisch-Gladbach , Germany ) systems . Blood was collected by cardiac puncture ( end-point ) or retro-orbital bleeds and anticoagulated with ethylaminediamine tetra-acetic acid ( EDTA ) prior to centrifugation to collect formed elements ( ‘blood’ ) or plasma . For viral titers and protein ( ELISA ) analysis , tissues were homogenized in PBS , and for qRT-PCR analysis , tissues were homogenized in guanidinium thiocyanate citrate buffer ( GTC ) ( Dreux et al . , 2012 ) . Virus from tissue homogenates was titrated on Vero cells ( CHIKV ) or Huh7 . 5 . 1 cells ( DENV ) . Results are expressed as median culture infective dose TCID50/g ( CHIKV ) or ffu/g ( DENV ) . IFNα/β activity from mouse tissue homogenates or plasma was quantified using Luciferase assay ( Promega , Madison , WI ) . Briefly , type I IFN levels were determined by incubating the reporter cell line LL171 with diluted plasma or tissue homogenates ( in RPMI 1640 medium supplemented with 10% FCS , 2 mM L-glutamine , 50 mM β-mercaptoethanol ) for 8 hr . Type I IFN activity was calculated using serial dilutions of a recombinant standard ( IFNα4; PBL Interferon Source , Piscataway , NJ ) . Cytokine protein measurements from tissue culture supernatants , plasma , and tissue homogenates were performed by ELISA using commercially available kits: human IFNα and mouse IFNα/IFNβ ( PBL Interferon Source ) , human TNFα and mouse TNFα/IL6/IFNγ ( eBioscience/Thermo Fisher Scientific , Waltham , MA ) . When indicated , mouse IFNα protein levels were determined using a newly developed mouse IFNα assay on the Quanterix SiMoA platform using capture and detection antibodies obtained from eBioscience ( capture: Mouse IFNα Platinum ELISA capture antibody; detection: Mouse IFNα Platinum ELISA detection antibody ) . Recombinant mouse IFNα2 ( eBioscience ) was used as a standard . RNAs were isolated from plasma or tissue homogenates prepared in guanidinium thiocyanate citrate buffer ( GTC; Sigma-Aldrich ) by phenol/chloroform extraction procedure as previously ( Dreux et al . , 2012 ) . Reverse transcription was performed using the random hexamer-primed High-Capacity cDNA reverse transcription kit ( Applied Biosystems , Foster City , CA ) and quantitative PCR was carried out using the Powerup Sybr Green Master Mix ( Applied Biosystems ) . For DENV and ISG analysis in mouse spleen , liver , and blood , absolute numbers of transcripts were generally normalized to the geometric mean of β-actin , hprt1 , and 18S housekeeping gene transcript numbers . For plasma samples lacking housekeeping transcripts , qRT-PCR was controlled by the addition of xenogeneic carrier RNAs encoding xef1α ( xenopus transcription factor 1α ) in vitro transcripts in plasma diluted in GTC buffer . The sequences of the primers used in analysis are described in Supplementary file 2 . Mouse pDCs were isolated from male and female mice from 8 to 22 weeks of age , of the indicated genotypes . pDCs were isolated from splenocytes/bone marrow via negative selection using the pDC isolation kit II ( Miltenyi ) or from bone marrow via positive selection using PDCA1-biotin antibody ( Miltenyi ) followed by anti-biotin microbead ( Miltenyi ) selection . Human pDCs were isolated from PBMCs derived from leukapheresis of healthy adult human volunteers , obtained according to procedures approved by the ‘Etablissement Français du sang’ ( EFS ) Committee . PBMCs were isolated using Ficoll-Hypaque density centrifugation . Human pDCs were positively selected from PBMCs using BDCA-4-magnetic beads ( Miltenyi ) , as we previously reported ( Décembre et al . , 2014 ) . After isolation , pDCs were cultured with Vero , Huh7 . 5 . 1 , BHK-21 , or Irf3/7 DKO primary MEF cells in RPMI cell growth media ( 10% FBS , 100 U/ml penicillin , 100 mg/ml streptomycin , 2 mM L-glutamine , non-essential amino acids , 1 mM sodium pyruvate and 0 . 05 mM β-mercaptoethanol ) in 96-well round bottom plates at 37°C , as previously described ( Décembre et al . , 2014 ) , using 8 × 104 mouse pDCs when cocultured or 1 × 104 human pDCs . Cocultured cells were either naive , previously infected or infected 24 hr post-coculture , using a multiplicity of infection of 5 and 2 for CHIKV and DENV , respectively . Cell culture supernatants were collected at 22–24 hr after the beginning of coculture with the infected cells . For flow cytometry , FACs sorting , and magnetic cell isolation , biotin , BV421 , Pacific Blue , FITC , PE , PE-Cy7 , APC , AlexaFluor-647 , AlexaFluor-700 , APC-eFluor780 or PerCP-Cy5 . 5 conjugates of the following anti-mouse antibodies were used ( clone in parentheses ) : Siglec-H ( 440 c ) , B220 ( RA3-6B2 ) , CD11c ( N418 ) , CD11b ( M1/70 ) , CD8a ( 53–6 . 7 ) , CD2 ( RM2-5 ) , CD19 ( 1D3 ) , CD4 ( GK1 . 5 ) , CD3 ( 17A2 , 500A2 ) , NK1 . 1 ( PK136 ) , CD69 ( H1 . 2F3 ) , Ly6C ( HK1 . 4 ) , Ly6G ( 1A8 ) , PDCA-1 ( JF05-1C2 . 4 . 1 ) , CD25 ( 3C7 ) , CD49b ( DX5 , HMα2 ) , TER-119 ( TER-119 ) , TCR-γδ ( UC7-13D5 ) , TCR-β ( H57-597 ) , IFNγ ( XMG1 . 2 ) , IRF7 ( MNGPKL ) ( Miltenyi; eBioscience; Becton-Dickinson; Biolegend , San Diego , CA ) . Dylight-405 and Dylight-680 streptavidin conjugates ( Life Technologies ) were used to detect biotinylated antibodies . Intracellular and extracellular staining was performed as follows . Isolated splenocytes and blood cells were depleted for erythrocytes by incubation at room temperature in red blood cell lysis buffer ( 1 . 5 M NH4Cl , 100 mM NaHCO3 , 10 mM Na2EDTA , pH 7 . 4 ) and cells were washed in FACS buffer ( PBS + 2 mM EDTA +5% FCS ) . For extracellular staining , cell surface marker staining was performed with antibodies diluted in FACS buffer , followed by fixation using 4% PFA . For intracellular staining , cells were incubated for 3 hr at 37°C in RPMI cell growth media + 10 µg/mL BFA , and cell surface marker staining was performed in FACS Buffer +10 µg/mL BFA . Fixation , permeabilization , and intracellular staining ( IFNγ ) was performed using the Cytofix/Cytoperm kit ( Becton-Dickinson ) . Statistical analysis was performed using PRISM v7 . 03 software ( Graphpad , La Jolla , CA ) ; only biological replicates representing separate mice and/or human donors are presented as data herein . Technical replicates , representing repeated measurements or treatments of the same cellular populations , were averaged prior to analysis . Mouse experiments were designed to detect a two-fold difference in means of parametric data at 95% confidence ( n ≥ 4 per condition , assumed CV = 0 . 3 ) ; however , larger sample sizes were used as litter numbers permitted . Viral titers and cytokine levels were considered non-parametric due to the presence of many data points at the assay detection limits; therefore , non-parametric Kruskal-Wallis tests with Dunn’s multiple comparison corrections were performed on this data . Viral RNA levels and transcript fold changes , as log-normal parametric data , were log-transformed prior to analysis using parametric tests . Kinetic analyses of ex vivo pDC cytokines ( Figure 1A–B ) were analyzed by one-way ANOVA parametric tests of AUC ( area under the curve ) . Parametric tests were: unpaired Student’s t test for comparisons of two conditions , one- or two-way ANOVA with a Holm-Sidak multiple comparison correction for comparison of multiple conditions . Statistical analysis of survival curves was performed using log-rank ( Mantel-Cox ) tests .
Type I interferon ( IFN-I ) responses are critical for the control of RNA virus infections , however , many viruses , including Dengue ( DENV ) and Chikungunya ( CHIKV ) virus , do not directly activate plasmacytoid dendritic cells ( pDCs ) , robust IFN-I producing cells . Herein , we demonstrated that DENV and CHIKV infected cells are sensed by pDCs , indirectly , resulting in selective IRF7 activation and IFN-I production , in the absence of other inflammatory cytokine responses . To elucidate pDC immunomodulatory functions , we developed a mouse model in which IRF7 signaling is restricted to pDC . Despite undetectable levels of IFN-I protein , pDC-restricted IRF7 signaling controlled both viruses and was sufficient to protect mice from lethal CHIKV infection . Early pDC IRF7-signaling resulted in amplification of downstream antiviral responses , including an accelerated natural killer ( NK ) cell-mediated type II IFN response . These studies revealed the dominant , yet indirect role of pDC IRF7-signaling in directing both type I and II IFN responses during arbovirus infections .
Viruses , like the ones responsible for the tropical diseases dengue and chikungunya , are parasites of living cells . As they cannot multiply on their own , these microbes need to infect a host cell and hijack its machinery to make more of themselves . When a cell is invaded , it can sense the viral particles , and defend itself by releasing antiviral molecules . Some of these molecules , such as interferons , also help recruit immune cells that can fight the germs . However , viruses often evolve mechanisms to escape being detected by the cell they occupy . Plasmacytoid dendritic cells are a rare group of immune cells , and they are able to detect when another cell is infected by the dengue virus . When they are in close physical contact with an invaded cell , these sentinels can recognize immature viral particles and release large amounts of antiviral molecules . However , it is unclear how important plasmacytoid dendritic cells are in clearing a viral infection . Here , Webster , Werneke et al . confirmed that plasmacytoid dendritic cells were able to sense cells infected by dengue , but also by chikungunya . When this happened , the dendritic cells primarily produced interferon , rather than other defense molecules . In addition , mice were genetically engineered so that the production of interferon was restricted to the plasmacytoid dendritic cells . When infected with dengue or chikungunya , the modified rodents resisted the diseases . These results show that , even though they are only a small percentage of all immune cells , plasmacytoid dendritic cells have an outsize role as first responders and as coordinators of the immune response . Finally , Webster , Werneke et al . showed that when low doses of interferon are added , , the plasmacytoid dendritic cells respond more quickly to cells infected by dengue . Together these findings could potentially be leveraged to create new treatments to fight dengue . These would be of particular interest because interferons are not as damaging to the body compared to other types of defense molecules . The issue is timely since climate change is allowing the mosquitos that transmit dengue and chikungunya to live in new places , exposing more people to these serious infections .