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Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA

Highlights Authors d TLR9, MyD88, STING, and IRF7 are required for IFN-I Ren-Huan Xu, Eric B. Wong, Daniel expression in the dLN Rubio, ..., Shinu John, Mark Shlomchik, Luis J. Sigal d Inflammatory monocytes are the major producers of IFN-I in the dLN Correspondence + [email protected] d TLR9, MyD88, and IRF7 are required in CD11c cells for iMo recruitment to dLNs In Brief d STING-IRF7 are required in iMos for IFN-a and STING-NF-kB How different pathogen-sensing for IFN-b expression in iMos mechanisms contribute to the expression of IFN-I in vivo is unclear. Sigal and colleagues report that in lymph nodes of ectromelia-virus-infected mice, CD11c+ cells use TLR9-MyD88-IRF7 to recruit inflammatory monocytes (iMos). In turn, infected iMos express IFN-a and IFN-b through STING-IRF7 and STING-NF-kB, respectively.

Xu et al., 2015, Immunity 43, 1148–1159 December 15, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.immuni.2015.11.015 Immunity Article

Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA Virus Infection

Ren-Huan Xu,1,5 Eric B. Wong,1,6 Daniel Rubio,1,7 Felicia Roscoe,1 Xueying Ma,1 Savita Nair,1 Sanda Remakus,1 Reto Schwendener,2 Shinu John,3 Mark Shlomchik,4 and Luis J. Sigal1,6,* 1Immune Development and Host Defense Program, The Research Institute at Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA 2Institute of Molecular Cancer Research, University Zurich, 8057 Zurich, Switzerland 3Department of Laboratory Medicine, Yale University, New Haven, CT 06520, USA 4Department of Immunology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, USA 5Present address: Novavax Inc., 20 Firstfield Road, Gaithersburg, MD 20878, USA 6Present address: Department of Microbiology and Immunology, Thomas Jefferson University, BLSB 730, 233 South 10th Street, Philadelphia, PA 19107, USA 7Present address: GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2015.11.015

SUMMARY and activate innate and adaptive immune cells (Wu and Chen, 2014). Toll-like receptor 9 (TLR9), its adaptor MyD88, the PAMPs in the cell microenvironment are recognized by downstream transcription factor interferon regulato- transmembrane PRRs that reside in the plasma and endosomal ry factor 7 (IRF7), and type I interferons (IFN-I) are all membranes, such as Toll-like receptors (TLRs)—for example, required for resistance to infection with ectromelia vi- TLR9 recognizes double-stranded DNA in endosomes (Ah- rus (ECTV). However, it is not known how or in which mad-Nejad et al., 2002; Hemmi et al., 2000). PAMPs in the cells these effectors function to promote survival. cytosol, on the other hand, are sensed by cytosolic PRRs—for instance, RIG-I-like receptors (RLRs) detect cytosolic viral RNA Here, we showed that after infection with ECTV, species (Goubau et al., 2013; Vabret and Blander, 2013), the TLR9-MyD88-IRF7 pathway was necessary in + whereas DNA-dependent activator of IFN-regulatory factors CD11c cells for the expression of proinflammatory (DAI), interferon-activated 204 (Ifi204, IFI16 in humans), cytokines and the recruitment of inflammatory mono- and cyclic-GMP-AMP (cGAMP) synthase (cGAS, encoded by cytes (iMos) to the draining lymph node (dLN). In the M21d1) respond to cytosolic DNA (Wu and Chen, 2014). dLN, the major producers of IFN-I were infected To signal, PRRs use adapters. The adapters for TLRs are MyD88 iMos, which used the DNA sensor-adaptor STING and TIR-domain-containing adaptor-inducing IFN-b (TRIF); for to activate IRF7 and nuclear factor kB (NF-kB) RLRs, mitochondrial antiviral-signaling protein (MAVS); and for signaling to induce the expression of IFN-a and DNA-sensing PRRs, STING (Wu and Chen, 2014). Additional IFN-b, respectively. Thus, in vivo, two pathways of signaling steps downstream of these adapters link PRRs to the DNA pathogen sensing act sequentially in two activation of several transcription factors, most frequently IRF3, IRF7, and NF-kB. These transcription factors induce the expres- distinct cell types to orchestrate resistance to a viral sion of IFN-I and pro-inflammatory cytokines (Wu and Chen, disease. 2014). Soon after breaching epithelia, many pathogenic INTRODUCTION rapidly spread through afferent lymphatics to the regional drain- ing lymph nodes (dLNs), from where they disseminate through The ability of the innate to sense infection is efferent lymphatics to the bloodstream, ultimately reaching their essential to mount innate and adaptive immune responses (Iwa- target organs (Flint et al., 2009; Virgin, 2007). Ectromelia virus, an saki and Medzhitov, 2010; Wu and Chen, 2014). It is well estab- (a genus of large DNA viruses), is the causative lished that pathogen recognition receptors (PRRs) recognize agent of mousepox, the mouse homolog of human smallpox (Es- pathogen-associated molecular patterns (PAMPs) to activate teban and Buller, 2005). After infection through the skin of the innate immune signaling pathways (Iwasaki and Medzhitov, footpad, ECTV spreads lympho-hematogenously to cause sys- 2010). PAMPs are typically microbial nucleic acids and other temic disease. Indeed, ECTV was the virus used to describe macromolecules with repetitive structures common to patho- this form of dissemination (Fenner, 1948) and is used as the text- gens but not usually encountered in uninfected cells (Schenten book paradigm of lympho-hematogenous spread (Flint et al., and Medzhitov, 2011). PRR-initiated signaling cascades after 2009; Virgin, 2007). virus infection culminate in the expression of type I interferon During lympho-hematogenous dissemination, a swift anti-viral (IFN-I), pro-inflammatory cytokines, and chemokines that recruit innate response in the dLN can play a major role in restricting

1148 Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. A B C B6 Tmem173gt Myd88-/- Mavs-/- 100 10 **** B6 **** 80 gt ** 8

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Figure 1. TLR9, MyD88, and STING Are Critical for Resistance to Mousepox and the Efficient Induction of IFN-I in Lymph Nodes Mice were infected with 3,000 PFUs of ECTV in the footpad. (A) Survival of the indicated mice. (B) Virus loads in the livers of the indicated mice at 7 dpi as determined by plaque assay. (C) Liver sections of the indicated mice at 7 dpi stained with H&E (top) or immunostained with anti-ECTV Ab (bottom). (D) Expression of IFN-I in the dLNs of the indicated mice at 2.5 dpi as determined by RT-qPCR. (E) Survival of the indicated mice. (F) Expression of IFN-I in the dLNs of the indicated mice at 2.5 dpi as determined by RT-qPCR. Data are shown either as individual mice with mean ± SEM or as mean ± SEM. Each panel displays data from one experiment with five mice per group and is representative of three similar experiments. For all, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. viral spread and deterring disease (Fang et al., 2008; Junt et al., well as of MAVS- and STING-driven pathways, in resistance 2007; Kastenmu¨ ller et al., 2012). It is therefore important to un- to mousepox and IFN-I expression in vivo during acute ECTV derstand how different mechanisms of virus sensing contribute infection. We found that after infection with ECTV in the footpad, to this process. Here we dissect the contribution of PRRs to mice deficient in MyD88 (Myd88À/À) or with inactive STING the antiviral response to ECTV in the dLN. We show that the (Tmem173gt), but not those lacking MAVS (MavsÀ/À), were sus- TLR9-MyD88-IRF7 axis is necessary in CD11c+ cells for the che- ceptible to lethal mousepox (Figure 1A). Nevertheless, death mokine-driven recruitment of inflammatory monocytes (iMos) to occurred in 100% of Myd88À/À but in only 80% Tmem173gt the dLN, but not directly essential for IFN-I production. Once mice, a difference that was reproducible and highly significant iMos are infected, they use STING-IRF7 and STING-NF-kB path- (p < 0.0001) suggesting a more profound impairment in the ways to induce the expression of IFN-a and IFN-b, respectively. absence of MyD88 than in the absence of STING signaling. Collectively, our work shows that the TLR9-MyD88-IRF7 and Consistently, virus loads (Figure 1B) and pathology (Figure 1C) STING-IRF7 or STING-NF-kB pathways have non-redundant, in the livers of Myd88À/À and Tmem173gt, but not of MavsÀ/À, complementary, and sequential roles in IFN-I expression in the mice were significantly higher than in B6 mice. Notably, the virus dLNs and in resistance to a highly pathogenic viral disease. titers were significantly lower (p < 0.01) and the liver pathology was somewhat milder in Tmem173gt than in Myd88À/À mice. RESULTS We next performed reverse transcriptase quantitative polymer- ase chain reaction (RT-qPCR) on RNA obtained from the dLNs TLR9-MyD88 and STING Are Critical for Resistance to at 2.5 days post infection (dpi). We found that Myd88À/À and Mousepox and the Efficient Induction of IFN-I in Lymph Tmem173gt but not MavsÀ/À mice expressed significantly lower Nodes levels of ‘‘early’’ IFN-b and ‘‘late’’ IFN-a5 than B6 mice. However, The TLR adaptor MyD88 is required for the inherent resistance of Myd88À/À mice expressed significantly lower levels of IFN-b (p < B6 mice to mousepox (Rubio et al., 2013; Sutherland et al., 0.0001) and IFN-a5 (p < 0.0001) than Tmem173gt mice (Fig- 2011), whereas TRIF is not necessary (data not shown). We per- ure 1D). Thus, MyD88 and STING, but not MAVS, are both critical formed experiments to identify the specific role of MyD88, as adapters for resistance to lethal ECTV infection and the efficient

Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. 1149 À À A B icantly lower in Myd88 / /B6 and Tmem173gt/B6 than in / 25 IFNβ 20 IFNα5 150 IFNβ 150 IFNα5 B6 B6 chimeras. Expression of IFN-b and IFN-a5 was also *** *** significantly lower (p < 0.05 for IFN-b and p < 0.01 for IFN-a5) 20 ** ** * *** 15 À/À gt 100 100 in Myd88 /B6 than in Tmem173 /B6 chimeras (Figure 2A). 15 10 In another approach, Vav1-Cre+ Myd88fl/fl mice, which specif- 10 50 50 5 ically lack MyD88 in hematopoietic cells, expressed significantly 5 Relative to naïve À fl/fl Relative to naïve less IFN-I than Vav1-Cre Myd88 controls (Figure 2B). More- 0 0 0 0 À fl + fl/fl fl/fl fl/fl fl/fl fl/fl fl/ over, Vav1-Cre Myd88 but not Vav1-Cre Myd88 mice suc- →B6 →B6 gt →B6 0/0 gt →B6 0/0 B6→B6 B6→B6 cumbed to mousepox with similar kinetics to constitutive - Myd88 + Myd88 - Myd88 + Myd88 À/À Myd88 Myd88 Myd88 mice (Figure 2C). Consequently, both MyD88 and Tmem173 Tmem173 STING in bone-marrow-derived cells are essential for the effi- Vav1-CreVav1-Cre Vav1-CreVav1-Cre C cient expression of IFN-I during ECTV infection in vivo. More- 100 over, MyD88 in hematopoietic cells is essential for resistance 80 to mousepox. Vav1-Cre- Myd88fl/fl 60 Vav1-Cre+ Myd88fl/fl ** 0/0 Infected Inflammatory Monocytes Are Responsible for 40 Myd88 **

Survival (%) Most of the IFN-I Expressed in the dLNs 20 In several infection models, plasmacytoid dendritic cells (pDCs) 0 0 102030 sense infection through TLR9-MyD88 to become the major pro- dpi ducers of IFN-I (Colonna et al., 2004; Ito et al., 2005). However, B6 mice depleted of pDCs with the anti-BST2 mAb 927 (Fig- Figure 2. IFN-I Expression Requires MyD88 and STING in Bone- ure S1A; Blasius et al., 2006) did not differ significantly from Marrow-Derived Cells Mice were infected with 3,000 PFUs of ECTV in the footpad. mice treated with control rat IgG in terms of IFN-I expression in (A) Expression of IFN-I in the dLNs of the indicated bone marrow chimeras at the dLNs at 2.5 dpi (Figure S1B) and of virus loads in the liver 2.5 dpi. at 7 dpi (Figure S1C). Consistent with these findings, they did (B) Expression of IFN-I in the dLNs of the indicated mice at 2.5 dpi. p values are not show any differences in IFN-I expression in the dLNs at 5 fl/fl compared to Vav1-Cre- Myd88 . dpi and were resistant to lethal mousepox (not shown). Hence, (C) Survival of indicated mice. pDCs are neither the major producers of IFN-I in the dLNs nor When applicable, data are shown as mean ± SEM. Each panel displays data essential for the resistance of B6 mice to mousepox after infec- from one experiment with five mice per group and is representative of three similar experiments except for (A), which was performed twice. For all, *p < tion with ECTV. 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Having found that pDCs are not the major producers of IFN-I in the dLNs, we attempted to identify the relevant bone-marrow- derived cells. In initial experiments analyzing the response to expression of IFN-I in the dLNs in vivo. However, mice are signif- ECTV in the dLNs at 2.5 dpi, we found an increase in CD11b+ icantly more susceptible to mousepox with absent MyD88 than cells that also stained with the anti-Ly6C+G mAb Gr1 (Figure 3A, with deficient STING. gate 1) but at lower levels than typical neutrophils (Figure 3A, Next, we investigated several molecules upstream of MyD88 gate 2). The absolute number of gate 1 cells peaked in the and STING that could be required for resistance to mousepox dLNs at 3 dpi (Figure 3B). At 5 dpi they had disappeared, most and/or IFN-I expression in the dLNs. As before, mice deficient likely because at this time after infection the dLNs were almost in TLR9 (Tlr9À/À), but not in other TLRs, were susceptible to acellular (not shown). The cells in gate 1 stained with anti-Ly6C mousepox (Rubio et al., 2013; Samuelsson et al., 2008; Suther- but not anti-Ly6G mAbs (Figure 3C) and were mononuclear (Fig- land et al., 2011). In contrast, mice deficient in the IL-1 receptor ure 3D), indicating that they were not neutrophils. These cells (Il1rÀ/À), which also uses MyD88 as its adaptor (Muzio et al., were MHC II+ and CD11c+ and expressed the monocyte or 1997), and mice deficient in the cytosolic DNA sensor DAI macrophage markers CD64 and F4/80 (Gautier et al., 2012) but (Zbp1À/À)(Ishii et al., 2008), which is thought to signal through were negative or low for the DC core markers CD26, CD117 (c- STING, were resistant (Figure 1E and not shown). Moreover, at Kit), CD135 (Flt3), and BTLA (Figure 3E; Miller et al., 2012), sug- 2.5 dpi, mice deficient in TLR9, but not in TLR2, IL-1R, or DAI, ex- gesting that they were inflammatory monocytes (iMos) and not pressed significantly lower IFN-I in the dLNs than B6 mice (Fig- dendritic cells (DCs). Hereafter, we will refer to the cells in gate ure 1F and not shown). Thus, among those we tested, the only 1 as iMos. PRR upstream of MyD88 that is required for IFN-I expression Using ECTV-EGFP (Fang et al., 2008), we found that in the in the dLNs during ECTV infection is TLR9. Further, DAI is not dLNs, ECTV preferentially infected myeloid and B cells (Fig- the critical sensor for STING. ure S2), which are mostly MHC II+. To identify the cell types that produce IFN-I in the dLNs, we determined IFN-I expression IFN-I Expression Requires MyD88 and STING in in sorted cell populations from the dLNs at 2.5 dpi. We found that Bone-Marrow-Derived Cells IFN-I expression segregated with MHC II+ cells and, among Next, we sought to identify the cells that produce IFN-I during these cells, iMos but not B cells expressed IFN-I (Figure 3F). infection. To distinguish the role of bone-marrow-derived We also sorted iMos, B cells, and the rest of the cells in the versus parenchymal cells, we used bone marrow chimeras. dLNs of ECTV-infected mice at 2.5 dpi. As compared to the Expression of IFN-b and IFN-a5 in the dLNs at 2.5 dpi was signif- rest of the cells, iMos expressed significantly higher levels of

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Figure 3. Infected Inflammatory Monocytes Are Responsible for Most of the IFN-I Expressed in the dLNs (A) Representative flow cytometry plots depicting the gating and frequency of a population of CD11b+Gr1+ (gate 1) cells in the popliteal LNs of an uninfected mouse and in the dLNs of an infected mouse at 2.5 dpi with ECTV. A second gate (gate 2) with higher expression of the molecules (presumably neutrophils) is also shown. (B) Number of cells in gate 1 at the indicated dpi. p values are compared to day 0. Data are displayed as the mean ± SEM of five mice per group in one experiment, which is representative of three similar experiments. (C) At 2.5 dpi, gate 1 cells were analyzed for expression of the indicated molecules using specific mAbs (shaded) or isotype Ab as control (open). Data displayed as a representative sample of five mice per group. The experiment was repeated three times. (D) At 2.5 dpi, gate 1 (left) and gate 2 (right) cells were sorted and stained with Giemsa. Data are representative of two experiments each with pools of five mice per group. (E) As in (C). Gate 1 cells are now referred as inflammatory monocytes (iMos). (F) IFN-I expression in unsorted cells or the indicated sorted cells from uninfected LNs or dLNs at 2.5 dpi. Data, displayed as mean ± SEM of pooled cells from five mice per group, are representative of two similar experiments. p values are not shown because the data correspond to two technical replicates. (G) Number of iMos in the LNs of uninfected mice and at 2.5 dpi in the dLNs of mice treated intravenously with liposomes filled with PBS as a control or with clodronate to deplete monocytes and macrophages. Data, displayed as individual mice and mean ± SEM, correspond to an experiment with four mice per group, which is representative of two similar experiments. (H) IFN-I expression at 2.5 dpi in the dLNs of mice receiving PBS or clodronate liposomes i.v. Data, shown as mean ± SEM, correspond to an experiment with five mice per group, which is representative of two similar experiments. (I) Representative flow cytometry plot depicting EGFP and CD11b expression in the iMo gate from the dLNs at 2.5 dpi with ECTV-EGFP. EGFPÀ (uninfected) and EGFP+ (infected). (J) IFN-I expression in sorted EGFPÀ or EGFP+ iMos gated as in (I). Data are displayed as mean ± SEM of pooled cells from five mice per group in one experiment, which is representative of two similar experiments. p values are not shown because the data correspond to two technical replicates. For all, *p < 0.05; **p < 0.01. several innate immune genes involved in IFN-I expression pressed significantly higher levels of only Tlr9 and Irf3 (Figure S2). including Tlr9, Myd88, M21d1 (which encodes cGAS), and Irf7 As compared to controls, significantly fewer iMos were recruited but not Tmem173, Irf3, and Nfkb1. On the other side, B cells ex- to the dLNs of mice that had been inoculated intravenously with

Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. 1151 liposomes filled with clodronate, known to deplete monocytes Alb-Cre Myd88fl/fl mice, whereas normal levels were detected and macrophages in vivo (Figure 3G; Seiler et al., 1997; Van in Lyz2-Cre Myd88fl/fl mice (Figure 5D). Rooijen, 1989). Moreover, mice treated with clodronate lipo- We next sought to understand why extrinsic TLR9 and MyD88 somes expressed significantly less IFN-I than those treated are required to recruit iMos to the dLNs. Because chemokine with PBS liposomes (Figure 3H). gradients regulate leukocyte migration (Griffith et al., 2014), we Next, we infected mice with ECTV-EGFP and sorted infected used RT-qPCR to determine which chemokines were upregu- (EGFP+) and uninfected (EGFPÀ) iMos from dLNs at 2.5 dpi lated in the dLNs at 1 dpi in a TLR9- and MyD88-dependent (Figure 3I). We found that EGFP+ iMos expressed significantly manner. We found that CCL2 and CCL7 (Figure 5E), which are li- more IFN-I than EGFPÀ iMos (Figure 3J). Thus, infected iMos gands for the chemokine receptor CCR2 and known to be are the major producers of IFN-I in the dLNs of mice infected involved in the recruitment of iMos to inflamed tissues (Griffith with ECTV. et al., 2014), were upregulated in B6 but not in Tlr9À/À, Vav1- Cre+ Myd88fl/fl, Itgax-Cre+ Myd88fl/fl, Vav1-Cre+ Tlr9fl/fl,and The Recruitment of iMos Needs Extrinsic MyD88 Itgax-Cre+ Tlr9fl/fl mice. This suggested that CCL2 and CCL7, whereas the Efficient Expression of IFN-I Requires induced by TLR9 and MyD88 signaling, might be involved in the Intrinsic STING recruitment of iMos to the dLNs. In agreement, mice deficient in We next sought to identify the molecular mechanisms respon- CCL7 (Ccl7À/À) recruited significantly fewer iMos to the dLNs sible for iMo recruitment. Compared to B6 mice, significantly than B6 mice upon ECTV infection. The impaired recruitment of fewer iMos were recruited into the dLNs of Tlr9À/À and iMos in Ccl7À/À mice was exacerbated by treatment with the Myd88À/À but not Tmem173gt mice (Figure 4A). Hence, CCR2 antagonist RS102895 (Figure 5F; Giunti et al., 2006). These the recruitment of iMos into the dLNs requires TLR9-MyD88 data suggest that the efficient recruitment of iMos to the dLNs re- but not STING. We next asked whether iMos require intrinsic quires expression of CCR2-binding chemokines and that this and/or extrinsic MyD88 to accumulate in the dLNs and/or ex- expression requires intrinsic TLR9 and MyD88 in CD11c+ cells. press IFN-I. For this, we used mixed bone marrow chimeras Of note, iMos were still the key cells that expressed IFN-I in made with a 1:1 mixture of bone marrow from B6 congenic RS102895-treated Ccl7À/À mice (Figure S3A). Yet, despite the CD45.1+ (WT) and CD45.2+ Myd88À/À bone marrow transferred reduced number of iMos, we did not detect significant differences into WT mice (henceforth, WT+Myd88À/À chimeras; Figure 4B). in the levels of IFN-I in the dLNs of B6 and RS102895-treated At 2.5 dpi, WT and Myd88À/À iMos accumulated in the dLNs of Ccl7À/À mice (not shown). Although we do not know the reason WT+Myd88À/À chimeras at similar frequencies (Figures 4Cand for the lack of significant differences, we have found that 4D). Of note, in WT+Myd88À/À chimeras infected with ECTV- RS102895-treated Ccl7À/À mice have significantly higher virus EGFP, EGFP+ Myd88À/À and EGFP+ WT iMos expressed similar loads than B6 mice in the dLNs at 2.5 dpi and iMos are still the ma- levels of IFN-I (Figure 4E). Conversely, in WT+Tmem173gt chi- jor producers of IFN-I (Figure S3B). Perhaps this increase in virus meras, EGFP+ Tmem173gt iMos expressed significantly less loads somehow compensates for the decrease in iMos. IFN-I than EGFP+ WT iMos (Figure 4F). Hence, to accumulate in the dLNs or produce IFN-I, iMos do not require intrinsic IRF7 Is Required for the Efficient Recruitment of iMos MyD88. However, they need intrinsic functional STING to effi- and IFN-I Expression in the dLNs ciently express IFN-I. Next, we sought to identify the transcription factors downstream of TLR9-MyD88 and STING necessary for the accumulation of The Accumulation of iMos in the dLNs Requires TLR9 iMos in the dLNs and for their expression of IFN-I. It is well estab- and MyD88 Expression in Chemokine-Producing lished that TLR9-MyD88 signaling activates the transcription CD11c+ Cells factors IRF7 and NF-kB(Orzalli and Knipe, 2014), whereas Next, we crossed mice carrying Cre recombinase in different STING mainly activates IRF3 and NF-kB(Wu and Chen, 2014) cell types with mice carrying floxed alleles of Myd88 but can also activate IRF7 (Ishikawa and Barber, 2008). As we (Myd88fl/fl)orTlr9 (Tlr9fl/fl) to specifically eliminate MyD88 previously reported, B6 mice that lack the transcription factor and TLR9 in cells of interest. Mice without Myd88 in hepato- IRF7 (Irf7À/À) are susceptible to mousepox whereas B6 mice cytes (Alb-Cre Myd88fl/fl, selected as controls because hepa- that lack IRF3 (Irf3À/À) are resistant (Rubio et al., 2013). Similar tocytes are late targets of ECTV infection) or in monocytes to Myd88À/À mice, Irf7À/À mice were more susceptible to and macrophages (Lyz2-Cre Myd88fl/fl) survived the infection. ECTV infection than STING-deficient mice because 100% of Conversely, most Vav1-Cre Tlr9fl/fl mice and all Vav1-Cre Irf7À/À and Myd88À/À but only 80% of Tmem173gt mice suc- Myd88fl/fl mice, which are deficient in TLR9 and Myd88, cumbed to mousepox (not shown). Also, the livers of Irf7À/À respectively, in all hematopoietic cells, succumbed to mouse- mice showed severe pathology as determined by histology pox. Similarly, most Itgax-Cre Tlr9fl/fl and all Itgax-Cre and immunohistochemistry (Figure 6A). Accordingly, the virus Myd88fl/fl, which lack TLR9 and MyD88, respectively, in titers in the livers of Irf7À/À mice were as high as those in CD11c+ cells, also died from mousepox (Figure 5A). Concor- Myd88À/À mice (Figure 6B). Consistent with being downstream dantly, iMos accumulated efficiently in the dLNs of Alb-Cre of MyD88, the virus titers in Irf7À/À mice were similar to those Myd88fl/fl and Lyz2-Cre Myd88fl/fl mice but poorly in the in Myd88À/À mice but significantly higher than in Tmem173gt dLNs of Vav1-Cre Myd88fl/fl, Itgax-Cre Myd88fl/fl, Vav1-Cre mice (p < 0.01). Also phenocopying MyD88 deficiency, the Tlr9fl/fl,andItgax-Cre Tlr9fl/fl mice (Figures 5B and 5C). More- recruitment of iMos to the dLNs of Irf7À/À mice was impaired over, Vav1-Cre Myd88fl/fl and Itgax-Cre Myd88fl/fl mice ex- (Figure 6C). Furthermore, the expression of IFN-I in the dLNs of pressed significantly lower levels of IFN-I in the dLNs than Irf7À/À mice was as low as in the dLNs of Myd88À/À mice and

1152 Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. A dLN at 2 .5 dpi 6 **** **** **** LN uninfected B6 B6 Myd88-/- Tlr9-/- Tmem173gt

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B C D 4 4 10 10 -/- 3 3 Myd88

iMo LN 10 10 55 1.5 -/- 2 2 dLN WT irradiated WT WT+mutant 10 10

(B6-CD45.1) (B6-CD45.1) Chimera 1 1 10 10 WT 43 1.0

2 months 2 dpi Myd88 4 4 10 10 0.5 3 3

bone 10 10 54 2 marrow ECTV-EGFP 2 0.0 10 10

Mutant (CD45.2) Ratio WT/ Non-dLN 1 1

10 10 44 dLN CD11b CD45.2 101 102 103 104 101 102 103 104 non-dLN Gr1 CD11b

E F IFNβ IFNα5 150 IFNβ IFNα5 150 28 4 4 25.1 ** ** 10 10

3 100 3 100 10 10 2 2 10 10 50 50 %ofWT CD45.2 1 1 CD45.2 10 10 25 %ofWT 15.8 0 0 101 102 103 104 101 102 103 104 -/- -/- gt gt EGFP T T EGFP 3 3 W W WT WT

Myd88 Myd88 Tmem17 Tmem17

Figure 4. The Recruitment of iMos Needs Extrinsic MyD88 whereas the Efficient Expression of IFN-I Requires Intrinsic STING (A) The indicated mice were infected with ECTV and at 2.5 dpi, their dLNs were analyzed for the presence of iMos. Representative flow cytometry plots (left) and the calculated numbers of iMos (right) are shown. Data, shown as individual mice and mean ± SEM, correspond to an experiment with five mice per group, which is representative of two similar experiments. (B) Diagram of the experiments in (C)–(F). (C) Representative flow cytometry plots of the dLNs and non-dLNs of WT+Myd88À/À chimeras at 2.5 dpi. The plots on the left show CD11b and Gr1 staining with the iMo gates marked. The plots on the right show the expression of CD45.2 and CD11c in the iMo gates. The gates for mutant (CD45.2+) and WT (CD45.2À) iMos are shown. (D) Ratio of WT/Myd88À/À iMos in the dLNs of WT+Myd88À/À chimeras. Data, shown as individual mice and mean ± SEM, correspond to an experiment with four mice per group, which is representative of two similar experiments. (E) Representative flow cytometry plot for EGFP and CD45.2 expression in gated iMos from dLNs of WT+Myd88À/À chimeras at 2.5 dpi with ECTV-EGFP (left) and IFN-I expression in the sorted EGFP+ WT and EGFP+ Myd88À/À iMos (right). Data are for pooled cells from five mice per group and representative of two similar experiments. Means ± SEM of three technical replicates are shown. (F) As in (E) but with cells from WT+Tmem173gt chimeras. For all, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. significantly lower than in the dLNs of Tmem173gt mice (Fig- Inflammatory Monocytes Require Intrinsic STING-IRF7 ure 6D). In addition, Irf7À/À mice did not upregulate the expres- and STING-NF-kB to Express IFN-a and IFN-b, sion of Ccl2 and Ccl7 in the dLNs (Figure 6E). Together, these Respectively data suggest that the expression of the chemokines that recruit We next determined whether iMos require intrinsic IRF7 to ex- iMos to the dLNs requires the transcription factor IRF7 down- press IFN-a and IFN-b. We found that infected iMos obtained stream of TLR9-MyD88. at 2.5 dpi with ECTV-EGFP from the dLNs of Tmem173gt mice

Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. 1153 A B C

100 5 ** * ** ** ** 10 ***

80 4 ) 8 ) 4 4 60 3 6 40 2 4 iMo (x10 iMo (x10 survival (%) 20 1 2 0 0 0 l l f fl /f 0 5 10 15 20 25 -/- fl/ fl/fl fl/ fl B6 fl/fl fl/fl dpi Tlr9 Tlr9 Alb-Cre Myd88fl/fl Lyz2-Cre Myd88fl/fl B6 naïveMyd88 Myd88 B6 naïve Cre -Cre fl/fl fl/fl Vav1-Cre Myd88 ** Itgax-Cre Tlr9 ** Alb-cre Myd88 Vav1- Itgax Vav1-Cre Tlr9fl/fl * Itgax-Cre Myd88fl/fl ** Vav1-CreLyz2-Cre-Myd88Itgax-Cre Myd88

D E F ** ** 20 5 **** 200 *** *** IFNβ **** ** *** *** IFNα5 *** 15 ) 4 150 CCL2 4 CCL7 3 100 10 2 5 iMo (x10 50 *** 1

0 Fold change over naïve 0 0 Fold change over naïve /fl /fl l l /- fl fl/fl fl/fl fl /0 fl/fl fl/f fl/fl fl/f - 0 -/- B6 B6 r 88 88 aïve 7 to Tlr9 n ibi 6 Ccl Myd88 Cre Tlr9 B inh 1- Vav v1-Cre MydItgax-Cre Tlr9 Alb-cre Myd88 +CCR2 Vav1-CreLyz2-Cre-Myd88 Myd88Itgax-Cre Myd88 Itgax-Cre Myd -/- Va 7 Ccl

Figure 5. The Accumulation of iMos in the dLNs Requires TLR9 and MyD88 in Chemokine-Producing CD11c+ Cells (A) Survival of the indicated mice to ECTV infection. Data correspond to one experiment with five mice per group and is representative of three similar experi- ments. p values are compared to Alb-Cre Myd88fl/fl mice. (B) Calculated number of iMos in the dLNs of the indicated mice at 2.5 dpi. Data, displayed as individual mice with mean ± SEM, correspond to one experiment with five mice per group, which is representative of three similar experiments. (C) As in (B), but for indicated mice. (D) Expression of IFN-I in the dLNs of the indicated mice at 2.5 dpi. p values are compared to Alb-Cre Myd88fl/fl mice. Data, displayed as mean ± SEM, correspond to one experiment with five mice per group, which is representative of three similar experiments. (E) Ccl2 and Ccl7 expression in the dLNs at 1 dpi. p values are compared to naive B6 mice (not shown) and are similar for Ccl2 and Ccl7. Data displayed as in (E). (F) As in (B), but for indicated mice. For all, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. expressed IFN-b inefficiently, whereas those from Irf3À/À and WT+Irf7À/À bone marrow chimeras (Figures 7B–7E). In both types Irf7À/À mice expressed as much IFN-b as iMos from WT B6 of chimeras, WT and mutant iMos accumulated in the dLNs at mice. In contrast, infected iMos from the dLNs of Irf3À/À mice similar frequencies (not shown). Hence, iMos do not need intrinsic expressed as much ‘‘early’’ IFN-a4 and ‘‘late’’ IFN-a5 as those NF-kB or IRF7 to accumulate in the dLNs. When tested for IFN-I from WT mice, but those from Tmem173gt and the few present expression, Irf7À/À iMos expressed significantly less early IFN- in Irf7À/À mice expressed significantly less (Figure 7A). There- a4 and late IFN-a5 than WT iMos but the levels of early IFN-b fore, the signaling pathways for IFN-a and IFN-b expression were similar. On the other hand, Nfkb1À/À iMos expressed as diverge downstream of STING, with IRF7 being required for effi- much early and late IFN-a but significantly less IFN-b than WT cient IFN-a but not IFN-b expression. iMos. Thus, in ECTV-infected iMos, efficient transcription of early Given our previous finding of a crosstalk between the NF-kBand and late IFN-a requires IRF7 whereas the expression of IFN-b is IFN-I pathways during ECTV infection (Rubio et al., 2013), we mostly dependent on NF-kBtranscription. speculated that in ECTV-infected iMos, NF-kB is the transcription factor downstream of STING that is responsible for the expression DISCUSSION of IFN-b. However, we were unable to directly test this idea in À À Nfkb1 / mice, because these mice lack popliteal lymph nodes We have studied how different pathways of pathogen sensing À À (Rubio et al., 2013). Thus, we produced WT+Nfkb1 / and also and cell types contribute to IFN-I expression and resistance to

1154 Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. AB C D E

Figure 6. IRF7 Is Required for the Efficient Recruitment of iMos and IFN-I Expression in the dLNs (A) Representative liver sections from Irf7À/À mice at 7 dpi stained with H&E (top) or with anti-ECTV Ab (bottom). The experiment was performed three times with four or five mice per group with similar results. (B) ECTV titers in the liver of the indicated mice at 7 dpi. Data, displayed as individual mice with mean ± SEM, correspond to one experiment with four or five mice per group, which is representative of three similar experiments. (C) Numbers of iMos in the dLNs of the indicated mice at 2.5 dpi. Data as in (B). (D) IFN-I in the dLNs of the indicated mice at 2.5 dpi expressed as percent of the values for the same interferon in infected B6 mice. Data, displayed as mean ± SEM, correspond to one experiment with five mice per group, which is representative of three similar experiments. (E) Expression of the indicated chemokines in the dLNs of the indicated mice at 1 dpi. Data as in (D). For all, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

a highly lethal infection caused by ECTV, a DNA virus of the to ECTV, produces RNA species that can activate these path- genus Orthopoxvirus that also includes variola virus (VARV, the ways and induce IFN-I expression (Myskiw et al., 2011; Pichlmair virus that causes smallpox) and vaccinia virus (VACV, the small- et al., 2009). pox vaccine). For this purpose, we have focused on the dLNs We also found that pDCs are not required for IFN-I expres- because of its critical role in restricting viral spread (Fang et al., sion or survival to ECTV infection. pDCs, which express RNA- 2008; Junt et al., 2007; Kastenmu¨ ller et al., 2012). and DNA-sensing TLR7, TLR8, and TLR9, have been touted The most important aspect of our work is the finding that the as professional IFN-I producers (Gilliet et al., 2008). However, DNA-sensing pathways TLR9-MyD88-IRF7 and STING-IRF7- they have been proven not essential for IFN-I expression and/ NF-kB are essential for efficient IFN-I production and resistance or resistance to infection in various infectious mouse models to mousepox but that their respective roles in IFN-I expression including vesicular stomatitis virus, influenza virus, mouse cyto- are very different: the TLR9-MyD88-IRF7 pathway is required megalovirus, and lymphocytic choriomeningitis virus (Reizis in CD11c+ cells for the expression in the dLNs of CCR2 li- et al., 2011). Furthermore, it has been shown that pDCs gands—and probably other pro-inflammatory molecules neces- contribute to IFN-I expression to systemic but not local infec- sary for the efficient recruitment of iMos to the dLNs—whereas tion with HSV-1, another large DNA virus (Swiecki et al., the STING-IRF7 and STING-NF-kB axes are needed for IFN-I 2013). That pDCs are not essential for resistance to ECTV is expression in infected iMos. That STING is necessary for IFN-I in contrast to the findings of Tahiliani et al. (2013), who recently expression is consistent with the recent finding that STING is reported that mice depleted of pDCs succumbed to mousepox. required for IFN-I expression in mouse cDCs infected with modi- Although that study did not examine IFN-I expression after fied VACV strain Ankara (MVA) (Dai et al., 2014). Although we infection, we attribute this discrepancy to differences in anti- have ruled out DAI as the critical DNA sensor, we have not yet BST2 mAb clones and doses between our study and theirs identified which is the receptor upstream of STING that senses and the possible depletion of other cell types by this type of ECTV to drive IFN-I expression. We hypothesize the key sensor mAbs (Swiecki and Colonna, 2010). is cGAS, because cGAS-STING is used by mouse cDCs (Dai The finding that the cells that exclusively express IFN-I in the et al., 2014; Li et al., 2013), mouse macrophages and fibroblasts dLNs are infected iMos is novel and unexpected because these (Li et al., 2013), and human embryonic kidney 293 cells (Ablasser cells are not considered professional IFN-I producers. Neverthe- et al., 2013) to produce IFN-I in vitro after infection with WT VACV less, it has been shown that Ly6C+ iMos use TLR2 to produce or MVA. IFN-I during VACV infection (Barbalat et al., 2009) and We have also ruled out TRIF and MAVS as key players in IFN-I that iMos produce IFN-b in response to Toxoplasma gondii expression and resistance to mousepox after ECTV infection. (Han et al., 2014). Moreover, other myeloid cells have been found That TRIF is not essential is not surprising because TLR9 uses to produce IFN-I. For example, conventional DCs (cDCs) pro- MyD88 but not TRIF as the adaptor, and because TLR9 is the duce IFN-I in response to lymphocytic choriomeningitis virus only TLR required for resistance to mousepox (Rubio et al., (Diebold et al., 2003), reovirus (Johansson et al., 2007), and rota- 2013; Samuelsson et al., 2008; Sutherland et al., 2011). How- virus (Lopez-Guerrero et al., 2010) in vivo. Also, cDCs and mac- ever, the finding that MAVS has no role is unexpected, because rophages express IFN-I in response to herpes simplex virus MAVS transduces signals from RNA-sensing RLRs, and it has (Rasmussen et al., 2007) and alveolar macrophages in response been shown that in cultured cells, VACV, which is very similar to Newcastle disease virus (Kumagai et al., 2007). In culture,

Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. 1155 A reason is that when compared to monocytes, they express low 200 levels of IRF7 and the cytosolic DNA sensors IFI16 (Ifi204) and * ** IFNβ cGAS (E330016A19Rik) as indicated in the Immgen database IFNα4 150 ** (http://www.immgen.org) and as suggested by our own RT- * ** IFNα5 qPCR analysis. 100 The identity and residence of the CD11c+ cells in which the % of B6 % of TLR9-MyD88-IRF7 pathway is needed for CCR2-ligand expres- 50 sion still needs to be elucidated. One possibility is that they are Langerhans cells or dermal DCs that reside in the footpad and 0 migrate to the dLNs in a TLR9-MyD88-dependent manner (Mar- B6 gt -/- -/- tı´n-Fontecha et al., 2009). It is also possible that these cells are Irf3 Irf7 DCs that reside in the subcapsular space that have recently shown to be the first to capture particulate antigens in the dLNs B Tmem173C Gated on iMo (Radtke et al., 2015). We speculate that CD11c+ cells need WT+Irf7-/- 150 B6 TLR9-MyD88 intrinsically to express the CCR2 ligands and prob- -/- chimeras Irf7 ** ably other inflammatory cytokines that are required for iMo migra- B6 *** 4 100 tion. However, it remains possible that the role of TLR9-MyD88 in 10 +

3 CD11c cells is also indirect for this function. Regardless of this, 10 iMos migrate to the dLNs in response to signals that depend on % of B6 % of 2

10 50 + CD45.1 TLR9-MyD88 in CD11c cells. After migrating to the dLNs, iMos 1

10 become targets of infection and, consequently, the major pro- Irf7-/- 101 102 103 104 0 ducers of IFN-I in the dLNs. Notably, iMos do not need intrinsic EGFP MyD88 to migrate to the dLNs or to produce IFN-I; instead, they IFNβ IFNα4 IFNα5 rely on intrinsic STING-IRF7 and STING-NF-kB signaling for D E expression of IFN-a and IFN-b, respectively. Gated on iMo 250 B6 The most frequently studied IRF downstream of STING is IRF3 -/- WT+Nfkb1 Nfkb1-/- (Wu and Chen, 2014). Therefore, it might be surprising that the 200 À À chimeras expression of IFN-I is not altered in Irf3 / mice. However, it

4 B6 150 has been shown that STING can directly activate IRF7 (Ishikawa

10 **

3 and Barber, 2008). Moreover, IRF7 is expressed at much higher

10 100 2 % of B6 % of levels than IRF3 in most myeloid cells (http://www.immgen.org), 10

CD45.1 50 and in our own experiments, IRF7 but not IRF3 was expressed at 1 -/-

10 Nfkb1 higher levels in iMos than in other cells. This suggests that in 0 101 102 103 104 iMos, STING-IRF7 signaling makes IRF3 redundant. EGFP IFNβ IFNα4 IFNα5 Our experiments suggest that iMos have additional pathways for IFN-I expression because infected iMos deficient in Irf7À/À gt Figure 7. Inflammatory Monocytes Require Intrinsic STING-IRF7 and Tmem173 expressed detectable (albeit significantly and STING-NF-kB to Express IFN-a and IFN-b, Respectively reduced) IFN-I. Although insufficient to protect most mice from (A) Expression of IFN-I in sorted EGFP+ iMos sorted from the dLNs of the lethal mousepox, these alternate pathways might explain why indicated mice at 2.5 dpi. Data, displayed as mean ± SEM from one experiment Tmem173gt mice, which recruit iMos to the dLNs, are less sus- representative of three, correspond to pooled cells from five mice per group ceptible than MyD88 and IRF7 mice, which do not recruit iMos and three technical replicates. to the dLNs. (B) Representative flow cytometry plots of gated iMos from the dLNs at 2.5 dpi À À We also found that the expression of IFN-b in iMos is strictly of WT+ Irf7 / chimeras showing CD45.1 and EGFP expression (left). (C) IFN-I expression in sorted infected (EGFP+) WT (CD45.1+) and Irf7À/À dependent on NF-kB, even though the ifnb1 enhancer has bind- (CD45.1À) iMos from WT+Irf7À/À chimeras as identified in (B). Data, displayed ing sites for NF-kB, IRF3, and IRF7 (Honda et al., 2005). Merika as mean ± SEM from one experiment representative of three, correspond to et al. (1998) have shown that NF-kB p65 is needed for the initial pooled cells from five mice per group and three technical replicates. capture and stabilization of CBP-p300 at the enhanceosome, so À/À (D) As in (B) but for WT+Nfkb1 chimeras. probably this role for NF-kB is more critical in driving IFN-b À/À (E) As in (C) but for WT+Nfkb1 chimeras. expression in iMos than it is in MEFs, where NF-kB appears For all, *p < 0.05; **p < 0.01; ***p < 0.001. largely dispensable for virus-driven IFN-b expression (Balachan- dran and Beg, 2011; Wang et al., 2007). influenza-virus-infected human monocyte-derived DCs also pro- In summary, we have identified iMos as the cell type critical for duce IFN-I (Cao et al., 2012). IFN-I production in the dLNs after infection with a poxvirus The discoveries that iMos express IFN-I only if they are in- through its biological route in its natural host. Moreover, we fected and that STING is the critical adaptor are internally consis- show that two DNA-sensing pathways, both traditionally associ- tent. STING is used by PRRs that recognize the presence of ated with a direct role in IFN-I expression, play distinct but PAMPs in the cytosol, which indicates an ongoing infection. sequentially roles in different cell types: the TLR9-MyD88-IRF7 Interestingly, although B cells constitute the vast majority of in- pathway functions in CD11c+ cells to recruit iMos to the dLNs fected cells in the dLNs, they failed to express IFN-I. A possible and the STING-IRF7 and STING-NF-kB pathways are directly

1156 Immunity 43, 1148–1159, December 15, 2015 ª2015 Elsevier Inc. responsible for IFN-I production after iMo infection. Together, Flow Cytometry these results provide important insights into how distinct path- Flow cytometry was performed as previously described (Xu et al., 2008). mAbs ogen-sensing mechanisms co-operate to recognize and limit to CD3 (145-2C11), CD4 (GK1.5), CD8a (53–6.7), CD11c (N418), IFN-g (XMG1.2), CD64 (X54-5/7.1), F4/80 (BM8), CD135 (Flt3, clone A2F10), pathogen spread in vivo. CD117 (c-Kit, clone 2B8), CD272 (6A6), CD26 (H194-112), IA/IE (M5/ 114.15.2), Ly-6G (IA8), Ly-6C (HK1.4), B220 (RA3-6B2), CD317 (BST2, EXPERIMENTAL PROCEDURES PDCA-1, Clone 927), and CXCL9 (MIG-2F5.5) were from Biolegend. mAbs to CD45.1 (A20), CD45.2 (104), Ly-6G and Ly-6C (Gr-1, Clone RB6-8C5), B220 Mice and Animal Experiments (RA3-6B2), and CD11b (H194-112) were from BD Biosciences. mAb to Si- All the procedures involving mice were carried out in strict accordance with the glec-H (eBio440c) was from eBioscience. recommendations in the Guide for the Care and Use of Laboratory Animals of To obtain single-cell suspensions, LNs were minced and dissociated in Lib- the NIH. All protocols were approved by Fox Chase Cancer Center’s (FCCC) erase TM (1.67 Wu¨ nsch units/ml) and DNase I (0.2 mg/ml; Roche Diagnostics) Institutional Animal Care and Use Committee. B6 (C57BL/6, CD45.2+; Taconic) in PBS with 25 mM of HEPES for 30 min at 37C. Liberase digestion was fol- and B6-LY5.1/Cr (B6-CD45.1, CD45.1+; NCI-Charles River) mice were pur- lowed by mechanical disruption of the tissue through a 70-mm filter. Cells chased at 6–8 weeks of age. All other mice, in a B6 background, were bred were washed once with complete RPMI medium before surface staining. For at FCCC from original breeders obtained from different sources and used at analysis, samples were acquired using a BD LSR II flow cytometer (BD Biosci- an age of 6–16 weeks. Vav1-Cre (B6.Cg-Tg(Vav1-cre)A2Kio/J), Alb-Cre ences), and data were analyzed with FlowJo software (TreeStar). For cell sort- (B6.Cg-Tg(Alb-cre)21Man/J), Lyz2-Cre (B6.129P2-Lyz2tm1(cre)Ifo/J), Itgax-Cre ing, samples were acquired with a BD FACSAria III sorter (BD Biosciences). (B6.Cg-Tg(Itgax-cre)1-1Reiz/J), Ccl7À/À (B6.129S4-Ccl7tm1ifc/J), Nfkb1À/À tm1Bal fl/fl tm1Defr gt (B6.Cg-Nfkb1 /J), Myd88 (B6.129P2(SJL)-Myd88 /J), Tmem173 Histopathology gt À/À tm1Kir À/À (C57BL/6J-Tmem173 /J), Tlr2 (B6.129-Tlr2 /J), and Il1r (B6.129S7- Livers were harvested and fixed with formalin and stained with H&E or with rab- tm1Imx Il1r1 /J) mice were originally purchased from Jackson Laboratories. bit anti-EVM135 as previously described (Xu et al., 2012). Sorted cells were tm1 À/À tm1 À/À B6.129-Tlr9 Aki/Obs (Tlr9 ) and B6.129-Myd88 Aki/Obs (Myd88 ) stained using a standard Wright-Giemsa protocol. mice were produced by Dr. S. Akira (Osaka University, Japan) (Adachi et al., 1998; Hemmi et al., 2000) and generously provided by Dr. Robert Finberg (Uni- RNA Preparation and RT-qPCR versity of Massachusetts, Worcester, MA). Irf7À/À (B6.129P2-Irf7tm1Ttg/ Total RNA from LNs was obtained with the RNeasy Mini Kit (QIAGEN) as pre- TtgRbrc), Irf3À/À (B6;129S6-Irf3tm1Ttg/TtgRbrc), and B6.129B6-Mavstm1Tsse viously described (Rubio et al., 2013; Xu et al., 2012). Total RNA from sorted were from Riken Bioresource Center (Tsukuba, Japan). Ifnar1À/À mice back- cells (104–105 cells) was extracted with Trizol reagent (Life Technologies) ac- crossed to B6 (Moltedo et al., 2011) were a gift from Dr. Thomas Moran (Mount cording to the manufacturer’s instructions. In brief, 104 cells were added Sinai School of Medicine, New York, NY), and Zbp1À/À mice (Ishii et al., 2008) into 1 ml of Trizol. When precipitating RNA, 10 mg of RNase-free glycogen (In- were a gift from Dr. S. Akira. Tlr9fl/fl mice were produced in the M.S. laboratory vitrogen) was added to the aqueous phase as a carrier. First-strand cDNA was and will be described in detail elsewhere. In brief, LoxP sites were inserted synthesized with High Capacity cDNA Reverse Transcription Kit (Lift Technol- flanking exon 2, which contains virtually all of the protein coding sequences, ogies). qPCR was performed as before (Rubio et al., 2013; Xu et al., 2012) of TLR9. The mutant allele was created in B6x129 ES cells (line BA1) and using probes from the Universal Library (Roche) and the oligonucleotides sug- proper targeting was confirmed by PCR, Southern blotting, and sequencing. gested by the manufacturer’s software. Subsequent to germline transmission, the floxed NeoR gene that was part of the original construct was deleted by breeding to a mouse line that expressed Cre-recombinase and the proper deletion again confirmed by Southern blot. Statistics The resulting mice were bred to B6 mice for ten generations before crossing Data were analyzed with Prism 6 software (GraphPad Software). For survival with the Cre-deleter mice obtained from Jackson. we used the Log-rank (Mantel-Cox). For other experiments, ANOVA with Tu- key correction for multiple comparisons or Student’s t test were used as appli- cable. In all figures, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Production of Bone Marrow Chimeric Mice Bone marrow chimeras were prepared as previously described (Sigal et al., 1999; Xu et al., 2010) using 5- to 7-week-old mice as donors and recipients. SUPPLEMENTAL INFORMATION For mixed bone marrow chimeras, bone marrow cells from the two donor types were mixed at 1:1 ratio. Chimeras were used in experiments 6–8 weeks Supplemental Information includes three figures and can be found with this after reconstitution. article online at http://dx.doi.org/10.1016/j.immuni.2015.11.015.

Viruses and Infection ACKNOWLEDGMENTS Virus stocks, including ECTV Moscow strain (ATCC VR-1374) and ECTV-EGFP (Fang et al., 2008), were propagated in tissue culture as previously described We thank Ms. Holly Gillin for assistance in the preparation of the manuscript (Xu et al., 2008). Mice were infected in the footpad with 3,000 plaque forming and Dr. Siddharth Balachandran for critical reading. We also thank the Fox units (PFUs) ECTV WT or ECTV-EGFP as indicated. For the determination of Chase Cancer Center (FCCC) Laboratory Animal, Flow Cytometry, and Tissue survival, the mice were monitored daily. To avoid unnecessary suffering, Culture Facilities for their services. This work was supported by NIH grants mice were euthanized and counted as dead when imminent death was certain R01AI065544, R01AI110457, and U19AI083008 to L.J.S. and P30CA006927 as determined by lack of activity and unresponsiveness to touch. For virus ti- to the FCCC. S.R. was supported by T32 CA-009035036 to FCCC. A generous ters, the entire spleen or portions of the liver were homogenized in PBS using a gift form the Kirby Foundation also contributed to funding this work. Tissue Lyser (QIAGEN). Virus titers were determined on BS-C-1 cells as before (Xu et al., 2008). Received: April 8, 2015 Revised: June 29, 2015 Cell Depletions Accepted: November 19, 2015 To deplete pDCs, B6 mice were injected with 500 mg rat mAb 927 or control rat Published: December 15, 2015 IgG (Blasius et al., 2006) 1 day before and 1 day after infection with ECTV. Effi- cient depletion of pDCs was confirmed by flow cytometry using mAbs B220 REFERENCES and 400c (anti-Siglec-H) on inguinal LNs and spleen at 2 days after the second depletion. To deplete monocytes/macrophages, mice received 200 ml clodro- Ablasser, A., Schmid-Burgk, J.L., Hemmerling, I., Horvath, G.L., Schmidt, T., nate-liposomes diluted to 5 mg/ml clodronate or control PBS-liposomes intra- Latz, E., and Hornung, V. (2013). Cell intrinsic immunity spreads to bystander venously 2 days before infection. cells via the intercellular transfer of cGAMP. Nature 503, 530–534.

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